Ramasamy Paulmurugan
Professor (Research) of Radiology (Molecular Imaging Program at Stanford)
Radiology - Rad/Molecular Imaging Program at Stanford
Academic Appointments
-
Professor (Research), Radiology - Rad/Molecular Imaging Program at Stanford
-
Member, Bio-X
-
Member, Stanford Cancer Institute
-
Member, Wu Tsai Neurosciences Institute
https://orcid.org/0000-0001-7155-47382024-25 Courses
- Biologics, advanced therapeutics and drug delivery methods
MED 211 (Aut) -
Independent Studies (6)
- Directed Reading in Radiology
RAD 299 (Aut, Win, Spr, Sum) - Early Clinical Experience in Radiology
RAD 280 (Aut, Win, Spr, Sum) - Graduate Research
RAD 399 (Aut, Win, Spr, Sum) - Medical Scholars Research
RAD 370 (Aut, Win, Spr, Sum) - Readings in Radiology Research
RAD 101 (Aut, Win, Spr, Sum) - Undergraduate Research
RAD 199 (Aut, Win, Spr, Sum)
- Directed Reading in Radiology
-
Prior Year Courses
2023-24 Courses
- Current Topics in Applied Medicine
MED 211 (Aut)
2022-23 Courses
- Current Topics in Applied Medicine
MED 211 (Aut)
- Current Topics in Applied Medicine
Stanford Advisees
-
Postdoctoral Faculty Sponsor
Sourabh Mehta, Farbod Tabesh, Suresh Thangudu, Goutham Vaidhyanathan Ganesh
All Publications
-
Luciferase-Based Reporter System for Investigating GPx4-Mediated Ferroptosis and Its Therapeutic Implications in Diabetes.
Analytical chemistry
2024
Abstract
Ferroptosis, a distinct form of regulated cell death, is characterized by iron-dependent lipid peroxide accumulation in cell membranes from dysregulated cellular iron homeostasis and compromised antioxidant defense mechanisms. Glutathione peroxidase 4 (GPx4) is crucial in the regulation of ferroptosis by controlling lipid peroxide accumulation. Recent research established the association of ferroptosis with several diseases, prompting investigation toward ferroptosis-targeted therapeutic approaches. However, there is a lack of sensor systems designed to evaluate ferroptosis modulation in intact cells. In this study, we developed a highly sensitive luciferase-based reporter system to study GPx4-mediated ferroptosis in cells. We constructed a novel vector flanking the GPx4 promoter driving luciferase gene expression, demonstrating ferroptosis-specific luciferase activity in transfected HEK293T cells. We established stable cells expressing the construct and optimized its suitability for high-throughput screening using well-established ferroptosis modulators. We identified eugenol, a phenolic compound, as a potent ferroptosis inhibitor using the developed reporter system. Eugenol demonstrated dose-dependent protection against ferroptosis-induced damage in pancreatic beta cells, as assessed by the expression of the key markers such as GPx4, SLC7A11, NRF2, and HO1. Further, we showed the regulation of iron levels and total iron-binding capacity of beta cells by eugenol in streptozotocin (STZ) -induced diabetic mice. Additionally, the diabetes-induced downregulation of GPx4 and antioxidant Nrf2 in pancreatic tissue was significantly mitigated by eugenol, as evidenced by both immunohistochemistry and gene expression analysis. This research validates the functionality of the ferroptosis sensor and offers an approach to develop antidiabetic therapy by targeting ferroptosis to protect beta-cell viability and function.
View details for DOI 10.1021/acs.analchem.4c03065
View details for PubMedID 39579117
-
Pulsed focused ultrasound alters the proteomic profile of the tumor microenvironment in a syngeneic mouse model of glioblastoma.
Journal of neuro-oncology
2024
Abstract
Glioblastoma (GBM), a lethal primary adult malignancy, is difficult to treat because of the restrictive nature of the blood-brain barrier (BBB), blood-tumor barrier (BTB), and the immunosuppressive tumor microenvironment (TME). Since pulsed focused ultrasound (pFUS) is currently used to improve therapeutic deliveries across these barriers, this study aims to characterize the impact of pFUS on the TME proteomics upon opening the BBB and BTB.We utilized MRI-guided, pFUS with ultrasound contrast microbubbles (termed 'pFUS' herein) to selectively and transiently open the BBB and BTB investigating proteomic modifications in the TME. Utilizing an orthotopically-allografted mouse GL26 GBM model (Ccr2RFP/wt - Cx3cr1GFP/wt), pFUS's effect on glioma proteomics was evaluated using a Luminex 48-plex assay.pFUS treated tumors exhibited increases in pro-inflammatory cytokines, chemokines, and trophic factors (CCTFs). Proteomic changes in tumors tend to peak at 24 h after single pFUS session (1x), with levels then plateauing or declining over the subsequent 24 h. Tumors receiving three pFUS sessions (3x) showed elevated CCTFs levels peaking as early as 6 h after the third session.pFUS together with microbubbles induces a sterile inflammatory response in the TME of a mouse GBM tumor. Moreover, this proinflammatory shift can be sustained and perhaps primed for more rapid responses upon multiple sessions of pFUS. These findings raise the intriguing potential that pFUS-induced BBB and BTB opening may not only be effective in facilitating the therapeutic agent delivery, but also be harnessed to modify the TME to assist immunotherapies in overcoming immune evasion in GBM.
View details for DOI 10.1007/s11060-024-04801-4
View details for PubMedID 39180641
View details for PubMedCentralID 4778014
-
Regiospecific Coelenterazine Analogs for Bioassays and Molecular Imaging.
Bioconjugate chemistry
2024
Abstract
Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1-6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue M2 was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure-activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λmax values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with M2-specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging.
View details for DOI 10.1021/acs.bioconjchem.4c00303
View details for PubMedID 39146513
-
Restoration of the Lost Human Beta Defensin-1 Protein in Cancer as a Strategy to Improve the Efficacy of Chemotherapy.
Journal of medicinal chemistry
2024
Abstract
Both innate and adaptive immunity are important components of the human defense system against various diseases including cancer. Human beta defensin-1 (hBD-1) is one such immunomodulatory peptide which is lost in malignant cancers, while high levels of expression are maintained in benign cells, making it a potential biomarker for the onset and metastasis of the disease. Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer for which no targeted therapy has been approved so far. That makes chemotherapy a first line of treatment despite high side effects. A priori Activation of Apoptosis Pathways of Tumor often referred to as AAAPT technology is a novel targeted tumor sensitizing technology that sensitizes low responsive and resistant tumor cells to evoke a better response from the current treatments for TNBC. Here, we show that hBD-1 is a targeted tumor sensitizer.
View details for DOI 10.1021/acs.jmedchem.4c01040
View details for PubMedID 39137365
-
Synthesis, characterization, and application of a biocompatible gene delivery nanocarrier constructed from gold nanostars and a chitosan-cyclodextrin-poly(ethylene imine) graft polymer
MATERIALS ADVANCES
2024
View details for DOI 10.1039/d4ma00433g
View details for Web of Science ID 001274030500001
-
Corrigendum to 'Molecular Identity Changes of Tumor-Associated Macrophages and Microglia After Magnetic Resonance Imaging-Guided Focused Ultrasound-Induced Blood-Brain Barrier Opening in a Mouse Glioblastoma Model' [Ultrasound in Med & Biol. 49 (2023) 1082-1090].
Ultrasound in medicine & biology
2023
View details for DOI 10.1016/j.ultrasmedbio.2023.12.016
View details for PubMedID 38161103
-
Single-chain multicolor-reporter templates for subcellular localization of molecular events in mammalian cells.
RSC chemical biology
2023; 4 (12): 1043-1049
Abstract
Single-chain multicolor-reporter imaging templates were developed for the subcellular localization of molecular events in mammalian cells. The templates were constructed by tandem linkage of fluorescent protein variants - fused with luciferases and the subcellular localization signal peptides. The templates simultaneously reported steroid hormonal activities at different optical spectra in the subcellular compartments. The templates contribute to the expansion of a toolbox of optical probes for subcellular localization of molecular events in intact cells.
View details for DOI 10.1039/d3cb00077j
View details for PubMedID 38033721
View details for PubMedCentralID PMC10685814
-
Protocol for in vitro sonoporation validation using non-targeted microbubbles for human studies of ultrasound-mediated gene delivery.
STAR protocols
2023; 4 (4): 102723
Abstract
Microbubbles are currently approved for diagnostic ultrasound imaging and are under evaluation in therapeutic protocols. Here, we present a protocol for in vitro sonoporation validation using non-targeted microbubbles for gene delivery. We describe steps for computational simulation, experimental calibration, reagent preparation, ultrasound treatment, validation, and gene expression analysis. This protocol uses approved diagnostic microbubbles and parameters that are applicable for human use. For complete details on the use and execution of this protocol, please refer to Bez et al. (2017).1.
View details for DOI 10.1016/j.xpro.2023.102723
View details for PubMedID 37976155
-
Single-chain multicolor-reporter templates for subcellular localization of molecular events in mammalian cells
RSC CHEMICAL BIOLOGY
2023
View details for DOI 10.1039/d3cb00077
View details for Web of Science ID 001078896300001
-
Correction to "Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents".
ACS nano
2023
View details for DOI 10.1021/acsnano.3c08363
View details for PubMedID 37721437
-
Correction to "SP94-Targeted Triblock Copolymer Nanoparticle Delivers Thymidine Kinase-p53-Nitroreductase Triple Therapeutic Gene and Restores Anticancer Function against Hepatocellular Carcinoma in Vivo".
ACS applied materials & interfaces
2023
View details for DOI 10.1021/acsami.3c09905
View details for PubMedID 37605509
-
Gold-Nanostar-Chitosan-Mediated Delivery of SARS-CoV-2 DNA Vaccine for Respiratory Mucosal Immunization: Development and Proof-of-Principle (vol 15, pg 17582, 2021)
ACS NANO
2023
View details for DOI 10.1021/acsnano.3c07103
View details for Web of Science ID 001049111100001
View details for PubMedID 37582219
-
Immune cell identity behind the Ktrans mapping of mouse glioblastoma.
Magnetic resonance imaging
2023
Abstract
Dynamic contrast-enhanced MR imaging (DCE-MRI) can assess the integrity of the blood brain barrier (BBB) and has been used in GBM patients to determine glioma grade, predict prognosis, evaluate treatment response, and differentiate treatment-induced effect from recurrence. The volume transfer constant Ktrans is the most frequently used metric in tumor assessment. Based on previous studies that a higher WHO grade of brain tumor was associated with greater impairments of immunity and that Ktrans value was associated with the pathological grading, the relationship between differential composition of immune cells in GBM tissue and dynamic changes in Ktrans mapping was anticipated in this study. The present study utilized an orthotopic allograft model of GBM in which mouse GL26 cells are implanted into Ccr2RFP/wtCx3cr1GFP/wt mice on a C57 background. The brain tumors exhibited heterogenous Ktrans values with the coefficients of variation (CV) above 75%, or relatively homogeneous Ktrans maps with CV values below 50%. The Ktrans values of homogeneous tumors ranged between 0.02/min-0.32/min with a median value of 0.10/min. The immune cell composition defined by quantitative immunohistochemistry and cell sorting was compared between the tumors with Ktrans values above 0.10/min (higher Ktrans) or below 0.10/min (lower Ktrans). Histological analysis showed that tumors with higher Ktrans values exhibited greater numbers of CCR2pos cells (257.60 ± 16.42/mm2 vs 203.23 ± 12.20/mm2, p = 0.04) and an increased ratio of CCR2pos cells to CX3CR1pos cells (1.20 ± 0.02 vs 0.38 ± 0.04, p = 0.001), the numbers of CX3CR1pos cells did not differ significantly based on Ktrans values (219.70 ± 16.20/mm2 vs 250.38 ± 21.20/mm2, p = 0.19). Flowcytometry analysis showed that tumors with higher Ktrans values (above 0.1/min) were associated with greater numbers of both overall monocytes (54.93 ± 6.81% vs 29.75 ± 3.54%, p = 0.01) and inflammatory monocytes (72.38 ± 1.49% vs 59.52 ± 2.44%, p = 0.001). In contrast, tumors with lower Ktrans values (below 0.1/min) exhibited greater numbers of patrolling monocytes (75.65 ± 4.14% vs 63 ± 6.94%, p = 0.05). In the tumors with lower Ktrans values, all three types of tumor associated cells, including patrolling monocytes, inflammatory monocytes, and microglia cells possessed a higher proportion of cells at pro-inflammatory status (41.77 ± 6.13% vs 25.06 ± 6.72%, p = 0.05; 27.50 ± 2.11% vs 20.62 ± 1.87%, p = 0.03; and 55.80 ± 9.88% vs 31.12 ± 7.31%, p = 0.05), inflammatory monocytes showed fewer anti-inflammatory cells (1.25 ± 0.62% vs 3.16 ± 3.56%, p = 0.04). Taken together, differences in Ktrans values were associated with differential immune cell phenotypes and polarizations. Ktrans mapping may therefore represent a novel approach for defining the immune status of GBM.
View details for DOI 10.1016/j.mri.2023.06.008
View details for PubMedID 37353182
-
Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.
Blood advances
2023
Abstract
Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34+ cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR by shRNA in CD34+ derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.
View details for DOI 10.1182/bloodadvances.2022008939
View details for PubMedID 37315179
-
Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.
International journal of molecular sciences
2023; 24 (10)
Abstract
The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MBB7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MBB7-H3 binding to MS1B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1B7-H3 and 28.3 ± 6.7/FOV for MS1WT cells). The fluorescently labeled MBB7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MBB7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MBB7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications.
View details for DOI 10.3390/ijms24109048
View details for PubMedID 37240396
-
Bright Molecular Strain Probe Templates for Reporting Protein-Protein Interactions.
Sensors (Basel, Switzerland)
2023; 23 (7)
Abstract
Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.
View details for DOI 10.3390/s23073498
View details for PubMedID 37050557
-
New Frontiers in Three-Dimensional Culture Platforms to Improve Diabetes Research.
Pharmaceutics
2023; 15 (3)
Abstract
Diabetes mellitus is associated with defects in islet β-cell functioning and consequent hyperglycemia resulting in multi-organ damage. Physiologically relevant models that mimic human diabetic progression are urgently needed to identify new drug targets. Three-dimensional (3D) cell-culture systems are gaining a considerable interest in diabetic disease modelling and are being utilized as platforms for diabetic drug discovery and pancreatic tissue engineering. Three-dimensional models offer a marked advantage in obtaining physiologically relevant information and improve drug selectivity over conventional 2D (two-dimensional) cultures and rodent models. Indeed, recent evidence persuasively supports the adoption of appropriate 3D cell technology in β-cell cultivation. This review article provides a considerably updated view of the benefits of employing 3D models in the experimental workflow compared to conventional animal and 2D models. We compile the latest innovations in this field and discuss the various strategies used to generate 3D culture models in diabetic research. We also critically review the advantages and the limitations of each 3D technology, with particular attention to the maintenance of β-cell morphology, functionality, and intercellular crosstalk. Furthermore, we emphasize the scope of improvement needed in the 3D culture systems employed in diabetes research and the promises they hold as excellent research platforms in managing diabetes.
View details for DOI 10.3390/pharmaceutics15030725
View details for PubMedID 36986591
-
Modulation of Macrophages by In Situ Ligand Bridging
ADVANCED FUNCTIONAL MATERIALS
2023
View details for DOI 10.1002/adfm.202215166
View details for Web of Science ID 000929290700001
-
Molecular Identity Changes of Tumor-Associated Macrophages and Microglia After Magnetic Resonance Imaging-Guided Focused Ultrasound-Induced Blood-Brain Barrier Opening in a Mouse Glioblastoma Model.
Ultrasound in medicine & biology
2023
Abstract
An orthotopically allografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the blood-brain barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). BBB opening was induced by magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) combined with microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors were quantified in microscopic images. Tumors in animals treated with a single session of MRgFUS did not exhibit significant changes in cell numbers when compared with tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS had significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells. The effect of MRgFUS on immune cell composition was also characterized and quantified using flow cytometry. Glioma implantation resulted in increased amounts of lymphocytes, monocytes and neutrophils in the brain parenchyma. Tumors administered MRgFUS exhibited increased numbers of monocytes and monocyte-derived TAMs. In addition, MRgFUS-treated tumors exhibited more CD80+ cells in monocytes and microglia. In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes and induce a shift toward a more pro-inflammatory status of the immune environment in glioblastoma.
View details for DOI 10.1016/j.ultrasmedbio.2022.12.006
View details for PubMedID 36717283
-
Ligand Coupling and Decoupling Modulates Stem Cell Fate
ADVANCED FUNCTIONAL MATERIALS
2023
View details for DOI 10.1002/adfm.202206673
View details for Web of Science ID 000916620600001
-
Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.
bioRxiv : the preprint server for biology
2023
Abstract
Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34 + cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin ( ENKUR ), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20 . Silencing of ENKUR by shRNA in CD34 + derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.VISUAL ABSTRACT: Key Points: Enkurin, a calcium adaptor protein, is identified as a novel marker of pathogenesis in MPNs.MPN megakaryocyte and platelet expression of enkurin at RNA and protein levels is inversely associated with a cell differentiation cycle gene, CDC20.Likely role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation.
View details for DOI 10.1101/2023.01.07.523111
View details for PubMedID 36712071
-
FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2023; 37 (1): e22652
Abstract
FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p<.05) and GRN TF gene expression (HNF4alpha, HEX, HNF1SS, TBX3) (p<.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p<.05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.
View details for DOI 10.1096/fj.202101506RRR
View details for PubMedID 36515690
-
Quantitative methods for molecular ultrasound imaging
SPIE-INT SOC OPTICAL ENGINEERING. 2023
View details for DOI 10.1117/12.2654230
View details for Web of Science ID 001011440800009
-
3117- ENKURIN: A NOVEL MARKER FOR MYELOPROLIFERATIVE NEOPLASMS FROM VALIDATED PLATELET, MEGAKARYOCYTE, AND WHOLE BLOOD SPECIMENS (vol 111, pg S103, 2022)
EXPERIMENTAL HEMATOLOGY
2023; 117: 69
View details for DOI 10.1016/j.exphem.2022.10.006
View details for Web of Science ID 000921127900001
-
Near-Infrared Imaging of Steroid Hormone Activities Using Bright BRET Templates.
International journal of molecular sciences
2022; 24 (1)
Abstract
Bioluminescence (BL) is an excellent optical readout for bioassays and molecular imaging. Herein, we accomplished new near infrared bioluminescence resonance energy transfer (NIR-BRET) templates for monitoring molecular events in cells with higher sensitivity. We first identified the best resonance energy donor for the NIR-BRET templates through the characterization of many coelenterazine (CTZ)-marine luciferase combinations. As a result, we found that NLuc-DBlueC and ALuc47-nCTZ combinations showed luminescence in the blue emission wavelength with excellent BL intensity and stability, for example, the NLuc-DBlueC and ALuc47-nCTZ combinations were 17-fold and 22-fold brighter than their second highest combinations, respectively, and were stably bright in living mammalian cells for at least 10 min. To harness the excellent BL properties to the NIR-BRET systems, NLuc and ALuc47 were genetically fused to fluorescent proteins (FPs), allowing large "blue-to-red" shifts, such as LSSmChe, LSSmKate2, and LSSmNep (where LSS means Large Stokes Shift). The excellent LSSmNep-NLuc combination showed approximately 170 nm large resonance energy shift from blue to red. The established templates were further utilized in the development of new NIR-BRET systems for imaging steroid hormone activities by sandwiching the ligand-binding domain of a nuclear receptor (NR-LBD) between the luciferase and the FP of the template. The NIR-BRET systems showed a specific luminescence signal upon exposure to steroid hormones, such as androgen, estrogen, and cortisol. The present NIR-BRET templates are important additions for utilizing their advantageous imaging of various molecular events with high efficiency and brightness in physiological samples.
View details for DOI 10.3390/ijms24010677
View details for PubMedID 36614119
-
Correction: Immunotheranostic microbubbles (iMBs) - a modular platform for dendritic cell vaccine delivery applied to breast cancer immunotherapy.
Journal of experimental & clinical cancer research : CR
2022; 41 (1): 357
View details for DOI 10.1186/s13046-022-02577-x
View details for PubMedID 36564801
-
Multimodal In Vivo Tracking of Chimeric Antigen Receptor T Cells in Preclinical Glioblastoma Models.
Investigative radiology
2022
Abstract
OBJECTIVES: Iron oxide nanoparticles have been used to track the accumulation of chimeric antigen receptor (CAR) T cells with magnetic resonance imaging (MRI). However, the only nanoparticle available for clinical applications to date, ferumoxytol, has caused rare but severe anaphylactic reactions. MegaPro nanoparticles (MegaPro-NPs) provide an improved safety profile. We evaluated whether MegaPro-NPs can be applied for in vivo tracking of CAR T cells in a mouse model of glioblastoma multiforme.MATERIALS AND METHODS: We labeled tumor-targeted CD70CAR (8R-70CAR) T cells and non-tumor-targeted controls with MegaPro-NPs, followed by inductively coupled plasma optical emission spectroscopy, Prussian blue staining, and cell viability assays. Next, we treated 42 NRG mice bearing U87-MG/eGFP-fLuc glioblastoma multiforme xenografts with MegaPro-NP-labeled/unlabeled CAR T cells or labeled untargeted T cells and performed serial MRI, magnetic particle imaging, and histology studies. The Kruskal-Wallis test was conducted to evaluate overall group differences, and the Mann-Whitney U test was applied to compare the pairs of groups.RESULTS: MegaPro-NP-labeled CAR T cells demonstrated significantly increased iron uptake compared with unlabeled controls (P < 0.01). Cell viability, activation, and exhaustion markers were not significantly different between the 2 groups (P > 0.05). In vivo, tumor T2* relaxation times were significantly lower after treatment with MegaPro-NP-labeled CAR T cells compared with untargeted T cells (P < 0.01). There is no significant difference in tumor growth inhibition between mice injected with labeled and unlabeled CAR T cells.CONCLUSIONS: MegaPro-NPs can be used for in vivo tracking of CAR T cells. Because MegaPro-NPs recently completed phase II clinical trial investigation as an MRI contrast agent, MegaPro-NP is expected to be applied to track CAR T cells in cancer immunotherapy trials in the near future.
View details for DOI 10.1097/RLI.0000000000000946
View details for PubMedID 36729074
-
A New Nrf2 Inhibitor Enhances Chemotherapeutic Effects in Glioblastoma Cells Carrying p53 Mutations.
Cancers
2022; 14 (24)
Abstract
TP53 tumor suppressor gene is a commonly mutated gene in cancer. p53 mediated senescence is critical in preventing oncogenesis in normal cells. Since p53 is a transcription factor, mutations in its DNA binding domain result in the functional loss of p53-mediated cellular pathways. Similarly, nuclear factor erythroid 2-related factor 2 (Nrf2) is another transcription factor that maintains cellular homeostasis by regulating redox and detoxification mechanisms. In glioblastoma (GBM), Nrf2-mediated antioxidant activity is upregulated while p53-mediated senescence is lost, both rendering GBM cells resistant to treatment. To address this, we identified novel Nrf2 inhibitors from bioactive compounds using a molecular imaging biosensor-based screening approach. We further evaluated the identified compounds for their in vitro and in vivo chemotherapy enhancement capabilities in GBM cells carrying different p53 mutations. We thus identified an Nrf2 inhibitor that is effective in GBM cells carrying the p53 (R175H) mutation, a frequent clinically observed hotspot structural mutation responsible for chemotherapeutic resistance in GBM. Combining this drug with low-dose chemotherapies can potentially reduce their toxicity and increase their efficacy by transiently suppressing Nrf2-mediated detoxification function in GBM cells carrying this important p53 missense mutation.
View details for DOI 10.3390/cancers14246120
View details for PubMedID 36551609
-
Photoswitchable Microgel for Dynamic Macrophage Modulation.
Advanced materials (Deerfield Beach, Fla.)
2022: e2205498
Abstract
Dynamic manipulation of supramolecular self-assembled structures has been achieved irreversibly or under non-physiological conditions, thereby limiting their biomedical, environmental, and catalysis applicability. In this study, microgels composed of azobenzene derivatives stacked via pi-cation and pi-pi interactions are developed that are electrostatically stabilized with Arg-Gly-Asp (RGD)-bearing anionic polymers. Lateral swelling of RGD-bearing microgels occurs via cis-azobenzene formation mediated by near-infrared light-upconverted-ultraviolet light, which disrupts intermolecular interactions on the visible light-absorbing upconversion nanoparticle-coated materials. Real-time imaging and molecular dynamics simulations demonstrate the deswelling of RGD-bearing microgels via visible light-mediated trans-azobenzene formation. Near-infrared light can induce in situ swelling of RGD-bearing microgels to increase RGD availability and trigger release of loaded interleukin-4 that facilitates the adhesion structure assembly linked with pro-regenerative polarization of host macrophages. In contrast, visible light can induce deswelling of RGD-bearing microgels to decrease RGD availability that suppresses macrophage adhesion that yields pro-inflammatory polarization. Our microgels exhibits high stability and non-toxicity. Versatile use of ligands and protein delivery can offer cytocompatible and photoswitchable manipulability of diverse host cells. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adma.202205498
View details for PubMedID 36268986
-
Immunotheranostic microbubbles (iMBs) - a modular platform for dendritic cell vaccine delivery applied to breast cancer immunotherapy.
Journal of experimental & clinical cancer research : CR
2022; 41 (1): 299
Abstract
BACKGROUND: Therapeutic strategies engaging the immune system against malignant cells have revolutionized the field of oncology. Proficiency of dendritic cells (DCs) for antigen presentation and immune response has spurred interest on DC-based vaccines for anti-cancer therapy. However, despite favorable safety profiles in patients, current DC-vaccines have not yet presented significant outcome due to technical barriers in active DC delivery, tumor progression, and immune dysfunction. To maximize the therapeutic response, we present here a unique cell-free DC-based vaccine capable of lymphoid organ targeting and eliciting T-cell-mediated anti-tumor effect.METHODS: We developed this novel immunotheranostic platform using plasma membranes derived from activated DCs incorporated into ultrasound contrast microbubbles (MBs), thereby offering real-time visualization of MBs' trafficking and homing in vivo. Human PBMC-derived DCs were cultured ex vivo for controlled maturation and activation using cell membrane antigens from breast cancer cells. Following DC membrane isolation, immunotheranostic microbubbles, called DC-iMBs, were formed for triple negative breast cancer treatment in a mouse model harboring a human reconstituted immune system.RESULTS: Our results demonstrated that DC-iMBs can accumulate in lymphoid organs and induce anti-tumor immune response, which significantly reduced tumor growth via apoptosis while increasing survival length of the treated animals. The phenotypic changes in immune cell populations upon DC-iMBs delivery further confirmed the T-cell-mediated anti-tumor effect.CONCLUSION: These early findings strongly support the potential of DC-iMBs as a novel immunotherapeutic cell-free vaccine for anti-cancer therapy.
View details for DOI 10.1186/s13046-022-02501-3
View details for PubMedID 36224614
-
Large-Scale Functionalized Metasurface-Based SARS-CoV-2 Detection and Quantification.
ACS nano
2022
Abstract
Plasmonic metasurfaces consist of metal-dielectric interfaces that are excitable at background and leakage resonant modes. The sharp and plasmonic excitation profile of metal-free electrons on metasurfaces at the nanoscale can be used for practical applications in diverse fields, including optoelectronics, energy harvesting, and biosensing. Currently, Fano resonant metasurface fabrication processes for biosensor applications are costly, need clean room access, and involve limited small-scale surface areas that are not easy for accurate sample placement. Here, we leverage the large-scale active area with uniform surface patterns present on optical disc-based metasurfaces as a cost-effective method to excite asymmetric plasmonic modes, enabling tunable optical Fano resonance interfacing with a microfluidic channel for multiple target detection in the visible wavelength range. We engineered plasmonic metasurfaces for biosensing through efficient layer-by-layer surface functionalization toward real-time measurement of target binding at the molecular scale. Further, we demonstrated the quantitative detection of antibodies, proteins, and the whole viral particles of SARS-CoV-2 with a high sensitivity and specificity, even distinguishing it from similar RNA viruses such as influenza and MERS. This cost-effective plasmonic metasurface platform offers a small-scale light-manipulation system, presenting considerable potential for fast, real-time detection of SARS-CoV-2 and pathogens in resource-limited settings.
View details for DOI 10.1021/acsnano.2c02500
View details for PubMedID 36125414
-
Evaluation of Immune Evasion in SARS-CoV-2 Delta and Omicron variants.
Computational and structural biotechnology journal
2022
Abstract
Emerging SARS-CoV-2 variants with higher transmissibility and immune escape remain a persistent threat across the globe. This is evident from the recent outbreaks of the Delta (B.1.617.2) and Omicron variants. These variants have originated from different continents and spread across the globe. In this study, we explored the genomic and structural basis of these variants for their lineage defining mutations of the spike protein through computational analysis, protein modeling, and molecular dynamic (MD) simulations. We further experimentally validated the importance of these deletion mutants for their immune escape using a pseudovirus-based neutralization assay, and an antibody (4A8) that binds directly to the spike protein's NTD. Delta variant with the deletion and mutations in the NTD revealed a better rigidity and reduced flexibility as compared to the wild-type spike protein (Wuhan isolate). Furthermore, computational studies of 4A8 monoclonal antibody (mAb) revealed a reduced binding of Delta variant compared to the wild-type strain. Similarly, the MD simulation data and virus neutralization assays revealed that the Omicron also exhibitsimmune escape, as antigenic beta-sheets appear to be disrupted. The results of the present study demonstrate the higher possibility of immune escape and thereby achieved better fitness advantages by the Delta and Omicron variants, which warrants further demonstrations through experimental evidences. Our study, based on in-silico computational modelling, simulations, and pseudovirus-based neutralization assay, highlighted and identified the probable mechanism through which the Delta and Omicron variants are more pathogenically evolved with higher transmissibility as compared to the wild-type strain.
View details for DOI 10.1016/j.csbj.2022.08.010
View details for PubMedID 35965661
-
Inhaled Gold Nano-star Carriers for Targeted Delivery of Triple Suicide Gene Therapy and Therapeutic MicroRNAs to Lung Metastases: Development and Validation in a Small Animal Model.
Advanced therapeutics
2022; 5 (8)
Abstract
Pulmonary metastases pose significant treatment challenges for many cancers, including triple-negative breast cancer (TNBC). We developed and tested a novel suicide gene and therapeutic microRNAs (miRs) combination therapy against lung metastases in vivo in mouse models after intranasal delivery using nontoxic gold nanoparticles (AuNPs) formulated to carry these molecular therapeutics. We used AuNPs coated with chitosan-β-cyclodextrin (CS-CD) and functionalized with a urokinase plasminogen activator (uPA) peptide to carry triple cancer suicide genes (thymidine kinase-p53-nitroreductase: TK-p53-NTR) plus therapeutic miRNAs (antimiR-21, antimiR-10b and miR-100). We synthesized three AuNPs: 20nm nanodots (AuND), and 20nm or 50nm nanostars (AuNS), then surface coated these with CS-CD using a microfluidic-optimized method. We sequentially coated the resulting positively charged AuNP-CS-CD core with synthetic miRNAs followed by TK-p53-NTR via electrostatic interactions, and added uPA peptide through CD-adamantane host-guest chemistry. A comparison of transfection efficiencies for different AuNPs showed that the 50nm AuNS allowed ∼4.16-fold higher gene transfection than other NPs. The intranasal delivery of uPA-AuNS-TK-p53-NTR-microRNAs NPs (pAuNS@TK-p53-NTR-miRs) in mice predominantly accumulated in lungs and facilitated ganciclovir and CB1954 prodrug-mediated gene therapy against TNBC lung metastases. This new nanosystem may serve as an adaptable-across-cancer-type, facile, and clinically scalable platform to allow future inhalational suicide gene-miR combination therapy for patients harboring pulmonary metastases.
View details for DOI 10.1002/adtp.202200018
View details for PubMedID 36212523
View details for PubMedCentralID PMC9543365
-
A rationally identified panel of microRNAs targets multiple oncogenic pathways to enhance chemotherapeutic effects in glioblastoma models.
Scientific reports
2022; 12 (1): 12017
Abstract
Glioblastoma (GBM) is the most common malignant brain tumor. Available treatments have limited success because most patients develop chemoresistance. Alternative strategies are required to improve anticancer effects of current chemotherapeutics while limiting resistance. Successful targeting of microRNAs (miRNAs) as regulators of gene expression can help reprogram GBM cells to better respond to chemotherapy. We aimed to identify a panel of miRNAs that target multiple oncogenic pathways to improve GBM therapy. We first identified differentially expressed miRNAs and tested if their target genes play central roles in GBM signaling pathways by analyzing data in the Gene Expression Omnibus and The Cancer Genome Atlas databases. We then studied the effects of different combinations of these miRNAs in GBM cells by delivering synthetic miRNAs using clinically compatible PLGA-PEG nanoparticles prior to treatment with temozolomide (TMZ) or doxorubicin (DOX). The successful miRNA panel was tested in mice bearing U87-MG cells co-treated with TMZ. We identified a panel of five miRNAs (miRNA-138, miRNA-139, miRNA-218, miRNA-490, and miRNA-21) and their oncogenic targets (CDK6, ZEB1, STAT3, TGIF2, and SMAD7) that cover four different signaling pathways (cell proliferation and apoptotic signaling, invasion and metastasis, cytokine signaling, and stemness) in GBM. We observed significant in vitro and in vivo enhancement of therapeutic efficiency of TMZ and DOX in GBM models. The proposed combination therapy using rationally selected miRNAs and chemotherapeutic drugs is effective owing to the ability of this specific miRNA panel to better target multiple genes associated with the hallmarks of cancer.
View details for DOI 10.1038/s41598-022-16219-x
View details for PubMedID 35835978
-
Inhaled Gold Nano-Star Carriers for Targeted Delivery of Triple Suicide Gene Therapy and Therapeutic MicroRNAs to Lung Metastases: Development and Validation in a Small Animal Model
ADVANCED THERAPEUTICS
2022
View details for DOI 10.1002/adtp.202200018
View details for Web of Science ID 000815596100001
-
CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells.
AMER ASSOC CANCER RESEARCH. 2022
View details for Web of Science ID 000892509502651
-
CD3 and CD8 targeting of ionizable lipid nanoparticles for in vivo mRNA delivery to T cells
AMER ASSOC CANCER RESEARCH. 2022
View details for Web of Science ID 000892509500599
-
Biomimetic nanobubbles for triple-negative breast cancer targeted ultrasound molecular imaging.
Journal of nanobiotechnology
2022; 20 (1): 267
Abstract
Triple-negative breast cancer (TNBC) is a highly heterogeneous breast cancer subtype with poor prognosis. Although anatomical imaging figures prominently for breast lesion screening, TNBC is often misdiagnosed, thus hindering early medical care. Ultrasound (US) molecular imaging using nanobubbles (NBs) capable of targeting tumor cells holds great promise for improved diagnosis and therapy. However, the lack of conventional biomarkers in TNBC impairs the development of current targeted agents. Here, we exploited the homotypic recognition of cancer cells to synthesize the first NBs based on TNBC cancer cell membrane (i.e., NBCCM) as a targeted diagnostic agent. We developed a microfluidic technology to synthesize NBCCM based on the self-assembly property of cell membranes in aqueous solutions. In vitro, optimal NBCCM had a hydrodynamic diameter of 683±162nm, showed long-lasting US contrast enhancements and homotypic affinity. In vivo, we demonstrated that NBCCM showed increased extravasation and retention in a TNBC mouse model compared to non-targeted NBs by US molecular imaging. Peak intensities and areas under the curves from time-intensity plots showed a significantly enhanced signal from NBCCM compared to non-targeted NBs (2.1-fold, P=0.004, and, 3.6-fold, P=0.0009, respectively). Immunofluorescence analysis further validated the presence of NBCCM in the tumor microenvironment. Circumventing the challenge for universal cancer biomarker identification, our approach could enable TNBC targeting regardless of tumor tissue heterogeneity, thus improving diagnosis and potentially gene/drug targeted delivery. Ultimately, our approach could be used to image many cancer types using biomimetic NBs prepared from their respective cancer cell membranes.
View details for DOI 10.1186/s12951-022-01484-9
View details for PubMedID 35689262
-
Correction to: The protean world of non-coding RNAs in glioblastoma.
Journal of molecular medicine (Berlin, Germany)
2022
View details for DOI 10.1007/s00109-022-02211-5
View details for PubMedID 35648188
-
Label-free discrimination of tumorigenesis stages using in vitro prostate cancer bone metastasis model by Raman imaging.
Scientific reports
2022; 12 (1): 8050
Abstract
Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654cm-1, which primarily contain proteins and lipids signals. Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis.
View details for DOI 10.1038/s41598-022-11800-w
View details for PubMedID 35577856
-
Submolecular Tuning of Ligand Size and Spacing for Dynamic Macrophage Modulation.
Advanced materials (Deerfield Beach, Fla.)
2022: e2110340
Abstract
Cell adhesion occurs when integrin recognizes and binds to Arg-Gly-Asp (RGD) ligands present in fibronectin. Herein, submolecular ligand size and spacing is tuned via the template-mediated in situ growth of nanoparticles for dynamic macrophage modulation. To tune liganded GNP size and spacing from 3 to 20nm, in situ localized assembly of gold nanoparticle (GNP) arrays on nano-magnetite templates are engineered. 3nm-spaced ligands stimulate the binding of integrin that mediates macrophage adhesion-assisted pro-regenerative polarization as compared to the 20nm-spaced ligands, which can be dynamically anchored to the substrate for stabilizing integrin binding and facilitating dynamic macrophage adhesion. Increasing the ligand size from 7 to 20nm only slightly promotes macrophage adhesion, not observed with 13nm-sized ligands. Increasing the ligand spacing from 3 to 17nm significantly hinders macrophage adhesion that induces inflammatory polarization. Submolecular tuning of ligand spacing can dominantly modulate host macrophages. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adma.202110340
View details for PubMedID 35476306
-
FN3 linked nanobubbles as a targeted contrast agent for US imaging of cancer-associated human PD-L1.
Journal of controlled release : official journal of the Controlled Release Society
2022
Abstract
PD-L1 (programmed death-ligand 1) targeted therapies may be useful for several cancers. The use of non-invasive diagnostic and prognostic molecular imaging platforms could improve clinical assessment of PD-L1 tumor status during these therapies. Contrast enhanced ultrasound molecular imaging (CE-USMI) techniques may offer versatile and cost-effective ways to detect and quantify the expression levels of cellular targets in vivo. However, conventional use of microbubbles as a blood pool contrast agent for CE-USMI is limited to accessing intravascular biomarkers rather than reflecting the tumor molecular status. Using a microfluidic based reconstruction process we therefore developed ultra-stable nanobubbles (NBs) as a contrast agent for molecular imaging of vascular and extravascular cell surface markers. We then functionalized these NBs by covalently linking to nanobody (FN3hPD-L1) targeting human (h)PD-L1 to measure the expression of human PD-L1 in the tumor microenvironment (TME) in vivo. We showed the specific binding of hPD-L1 targeted NBs in cell culture, and in xenografted mouse models of hPD-L1 expressing CT26 tumors. CE-USMI of hPD-L1 in the TME in vivo showed ~3-fold increase in contrast signal compared to non-targeted NBs. Overall, in vivo use of CE-USMI with hPD-L1 targeted NBs has the potential for clinical translation and imaging of human cancers during immunotherapy, and for prognostic evaluation of patient response to PD-L1 targeted immunotherapy.
View details for DOI 10.1016/j.jconrel.2022.04.030
View details for PubMedID 35469983
-
Receptor-Level Proximity and Fastening of Ligands Modulates Stem Cell Differentiation
ADVANCED FUNCTIONAL MATERIALS
2022
View details for DOI 10.1002/adfm.202200828
View details for Web of Science ID 000775729500001
-
Local Sound Speed Estimation for Pulse-Echo Ultrasound in Layered Media
IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL
2022; 69 (2): 500-511
Abstract
Our previous methodology in local sound speed estimation utilized time delays measured by the cross correlation of delayed full-synthetic aperture channel data to estimate the average speed of sound. However, focal distortions in this methodology lead to biased estimates of the average speed of sound, which, in turn, leads to biased estimates of the local speed of sound. Here, we demonstrate the bias in the previous methodology and introduce a coherence-based average sound speed estimator that eliminates this bias and is computationally much cheaper in practice. Because this coherence-based approach estimates the average sound speed in the medium over an equally spaced grid in depth rather than time, we derive a refined model that relates the local and average speeds of sound as a function of depth in layered media. A fast, closed-form inversion of this model yields highly accurate local sound speed estimates. The root-mean-square (rms) error of local sound speed reconstruction in simulations of two-layer media is 4.6 and 2.5 m/s at 4 and 8 MHz, respectively. This work examines the impact of frequency, f -number, aberration, and reverberation on sound speed estimation. Phantom and in vivo experiments in rats further validate the coherence-based sound speed estimator.
View details for DOI 10.1109/TUFFC.2021.3124479
View details for Web of Science ID 000748372800009
View details for PubMedID 34723801
-
Contrast Enhanced Ultrasound Molecular Imaging of Spontaneous Chronic Inflammatory Bowel Disease in an Interleukin-2 Receptor alpha-/- Transgenic Mouse Model Using Targeted Microbubbles.
Nanomaterials (Basel, Switzerland)
1800; 12 (2)
Abstract
Inflammatory bowel disease (IBD) is a lifelong inflammatory disorder with relapsing-remission cycles, which is currently diagnosed by clinical symptoms and signs, along with laboratory and imaging findings. However, such clinical findings are not parallel to the disease activity of IBD and are difficult to use in treatment monitoring. Therefore, non-invasive quantitative imaging tools are required for the multiple follow-up exams of IBD patients in order to monitor the disease activity and determine treatment regimens. In this study, we evaluated a dual P- and E-selectin-targeted microbubble (MBSelectin) in an interleukin-2 receptor alpha deficient (IL-2Ralpha-/-) spontaneous chronic IBD mouse model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI). We used IL-2Ralpha-/- (male and female on a C57BL/6 genetic background; n = 39) and C57BL/6 wild-type (negative control; n = 6) mice for the study. USMI of the proximal, middle, and distal colon was performed with MBSelectin using a small animal scanner (Vevo 2100) up to six times in each IL-2Ralpha-/- mouse between 6-30 weeks of age. USMI signals were compared between IL-2Ralpha-/- vs. wild-type mice, and sexes in three colonic locations. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E-stained sections and for selectin expression by immunofluorescence staining. We successfully detected spontaneous chronic colitis in IL-2Ralpha-/- mice between 6-30 weeks (onset at 6-14 weeks) compared to wild-type mice. Both male and female IL-2Ralpha-/- mice were equally (p = 0.996) affected with the disease, and there was no significant (p > 0.05) difference in USMI signals of colitis between the proximal, middle, and distal colon. We observed the fluctuating USMI signals in IL-2Ralpha-/- mice between 6-30 weeks, which might suggest a resemblance of the remission-flare pattern of human IBD. The ex vivo H&E and immunostaining further confirmed the inflammatory changes, and the high expression of P- and E-selectin in the colon. The results of this study highlight the IL-2Ralpha-/- mice as a chronic colitis model and are suitable for the long-term assessment of treatment response using a dual P- and E-selectin-targeted USMI.
View details for DOI 10.3390/nano12020280
View details for PubMedID 35055297
-
Editorial: Nanomaterials for targeted delivery of therapeutic and imaging agents.
Frontiers in cell and developmental biology
2022; 10: 978690
View details for DOI 10.3389/fcell.2022.978690
View details for PubMedID 36016653
-
One-Channel Microsliding Luminometer for Quantifying Low-Energy Bioluminescent Lights.
Methods in molecular biology (Clifton, N.J.)
2022; 2525: 365-375
Abstract
A high-throughput quantitative determination of multiple light-emitting samples is a virtue of many light determination systems. In this chapter, we introduce a compact and efficient light determination system with a microsliding platform, a single-channel photomultiplier tube (PMT), and the controlling software for quantitative imaging of low-energy lights. The microsliding platform is uniquely designed to hold a multichannel microslide or an 8-lane PCR tube strip. The platform supports consecutive measurement of the multiple light samples through sliding the microslide or the PCR tube strip like a conveyor belt. We exemplify determination of multiple alkaline phosphatase samples and single-chain bioluminescent probes using this system. We also outline the mechanical specification of the system. This unique luminometer is an important addition to compact on-site quantitative light determination systems that are useful in various research fields including analytical chemistry, biology, and basic science in medicine.
View details for DOI 10.1007/978-1-0716-2473-9_28
View details for PubMedID 35836083
-
Quantitative Imaging of Retinoic Acid Activities in Living Mammalian Cells.
Methods in molecular biology (Clifton, N.J.)
2022; 2525: 111-122
Abstract
Retinoic acid (RA) is an intriguing metabolite that is necessary for embryonic development and differentiation in vertebrates. The present protocol demonstrates how to image RA activities indirectly in mammalian cells with ligand-activatable single-chain bioluminescence (BL) probes. We introduce 13 different molecular designs for characterizing an efficient single-chain probe that quantitatively visualizes RA activities with significant sensitivity. The key components included in the probes are (i) the N- and C-terminal fragments of artificial luciferase 16 (ALuc16), (ii) the ligand-binding domain of human retinoic acid receptor alpha (RAR LBD), and (iii) an LXXLL motif derived from common coactivators of nuclear receptors. The probe is highly selective and sensitive to all-trans-RA (at-RA) in animal cells. This protocol exemplifies quantitative imaging of the RA levels in serum and cerebrospinal fluid with a linear range in two orders. The present protocol is an important addition to conventional techniques on quantitative imaging of endogenous at-RA levels in live mammalian cells.
View details for DOI 10.1007/978-1-0716-2473-9_8
View details for PubMedID 35836063
-
Ultrasound-Guided Microbubble-Mediated Locoregional Delivery of Multiple MicroRNAs Improves Chemotherapy in Hepatocellular Carcinoma.
Nanotheranostics
1800; 6 (1): 62-78
Abstract
Rationale: To assess treatment effects of 4 complementary miRNAs (miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21) encapsulated in a biodegradable PLGA-PEG nanoparticle, administered by an ultrasound-guided microbubble-mediated targeted delivery (UGMMTD) approach in mouse models of hepatocellular carcinoma (HCC). Methods: In vitro apoptotic index was measured in HepG2 and Hepa1-6 HCC cells treated with various combinations of the 4 miRNAs with doxorubicin. Three promising combinations were further tested in vivo by using UGMMTD. 63 HepG2 xenografts in mice were randomized into: group 1, miRNA-122/antimiRNA-10b/antimiRNA-21/US/doxorubicin; group 2, miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21/US/doxorubicin; group 3, miRNA-100/miRNA-122/antimiRNA-10b/US/doxorubicin; group 4, miRNA-122/anitmiRNA-10b/antimiRNA-21/doxorubicin; group 5, miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21/doxorubicin; group 6, miRNA-100/miRNA-122/antimiRNA-10b/doxorubicin; group 7, doxorubicin only treatment; and group 8, without any treatment. Tumor volumes were measured through 18 days. H&E staining, TUNEL assay, and qRT-PCR quantification for delivered miRNAs were performed. Results: In vivo results showed that UGMMTD of miRNAs with doxorubicin in groups 1-3 significantly (P<0.05) delayed tumor growth compared to control without any treatment, and doxorubicin only from day 7 to 18. On qRT-PCR, levels of delivered miRNAs were significantly (P<0.05) higher in groups 1-3 upon UGMMTD treatment compared to controls. TUNEL assay showed that upon UGMMTD, significantly higher levels of apoptotic cell populations were observed in groups 1-3 compared to controls. Toxicity was not observed in various organs of different groups. Conclusions: UGMMTD of miRNA-100/miRNA-122/antimiRNA-10b/antimiRNA-21 combination improved therapeutic outcome of doxorubicin chemotherapy in mouse models of HCC by substantial inhibition of tumor growth and significant increase in apoptotic index.
View details for DOI 10.7150/ntno.63320
View details for PubMedID 34976581
-
Manipulating Nanoparticle Aggregates Regulates Receptor-Ligand Binding in Macrophages.
Journal of the American Chemical Society
2022
Abstract
The receptor-ligand interactions in cells are dynamically regulated by modulation of the ligand accessibility. In this study, we utilize size-tunable magnetic nanoparticle aggregates ordered at both nanometer and atomic scales. We flexibly anchor magnetic nanoparticle aggregates of tunable sizes over the cell-adhesive RGD ligand (Arg-Gly-Asp)-active material surface while maintaining the density of dispersed ligands accessible to macrophages at constant. Lowering the accessible ligand dispersity by increasing the aggregate size at constant accessible ligand density facilitates the binding of integrin receptors to the accessible ligands, which promotes the adhesion of macrophages. In high ligand dispersity, distant magnetic manipulation to lift the aggregates (which increases ligand accessibility) stimulates the binding of integrin receptors to the accessible ligands available under the aggregates to augment macrophage adhesion-mediated pro-healing polarization both in vitro and in vivo. In low ligand dispersity, distant control to drop the aggregates (which decreases ligand accessibility) repels integrin receptors away from the aggregates, thereby suppressing integrin receptor-ligand binding and macrophage adhesion, which promotes inflammatory polarization. Here, we present "accessible ligand dispersity" as a novel fundamental parameter that regulates receptor-ligand binding, which can be reversibly manipulated by increasing and decreasing the ligand accessibility. Limitless tuning of nanoparticle aggregate dimensions and morphology can offer further insight into the regulation of receptor-ligand binding in host cells.
View details for DOI 10.1021/jacs.1c08861
View details for PubMedID 35275625
-
ENKURIN: A NOVEL MARKER FOR MYELOPROLIFERATIVE NEOPLASMS FROM VALIDATED PLATELET, MEGAKARYOCYTE, AND WHOLE BLOOD SPECIMENS
ELSEVIER SCIENCE INC. 2022: S103
View details for Web of Science ID 000890643400166
-
Compact Eight-Channel Light-Sensing System for Bioassays.
Methods in molecular biology (Clifton, N.J.)
2022; 2525: 377-386
Abstract
The present protocol introduces a new instrumental setup as a luminometer to simultaneously measure eight light samples with high sensitivity. The system consists of 8-channel photomultiplier tubes (8-PMTs) with different sensitivities to light. Therefore, it is critical to normalize the sensitivities of PMTs to light samples and integrate them as a system. We first introduce how to normalize the diverse light sensitivity among the PMTs using placental alkaline phosphatase (PLAP) as a model chemiluminescence light source. The normalized BBI system shows a statistically strong linear correlation graph to photon counts. The biomedical utility of this system is exemplified by (i) determining the alkaline phosphatase (AP) activities in mouse plasma samples as a cancer biomarker and (ii) diagnosing metastatic tissues during cancer progression using bioluminescent reporter.
View details for DOI 10.1007/978-1-0716-2473-9_29
View details for PubMedID 35836084
-
BRET Sensors for Imaging Membrane Integrity of Microfluidically Generated Extracellular Vesicles.
Methods in molecular biology (Clifton, N.J.)
2022; 2525: 227-238
Abstract
Extracellular vesicles (EVs) derived from various cell lines have been extensively used as natural nanodelivery vehicles for drug, protein, and nucleic acid deliveries in therapeutic applications for cancer. Recently, we developed a microfluidic-based reconstruction strategy as a novel method to generate microRNA-loaded membrane vesicles for cancer therapy in vivo. We used EVs and cell membranes isolated from different source of cells for this reconstruction process. The microfluidic system produced reconstructed vesicles of uniform sizes with high microRNA loading efficiency independent of input membrane sources (EVs or cell membranes). To address the functional integrity of the membrane structure and of proteins in the reconstructed EVs, we introduce a membrane-insertable bioluminescence resonance energy transfer (BRET) sensor system. This sensor, with its membrane-insertable palmitoylation signal peptide sequence derived from a growth-associated protein 43 (GAP43), helps in trafficking the fusion protein to the cell membrane upon its expression in cells and allows for imaging reconstructed membrane vesicles using optical imaging. In this chapter, we detail the stepwise methods used for the engineering of cells using this sensor, isolation of EVs from the engineered cells, preparation of reconstructed EVs by microfluidic processing, and BRET imaging of reconstructed EVs for membrane integrity evaluation.
View details for DOI 10.1007/978-1-0716-2473-9_17
View details for PubMedID 35836072
-
Noninvasive estimation of local speed of sound by pulse-echo ultrasound in a rat model of nonalcoholic fatty liver.
Physics in medicine and biology
1800
Abstract
Objective:Speed of sound has previously been demonstrated to correlate with fat concentration in the liver. However, estimating speed of sound in the liver noninvasively can be biased by the speed of sound of the tissue layers overlying the liver. Here, we demonstrate a noninvasive local speed of sound estimator, which is based on a layered media assumption, that can accurately capture the speed of sound in the liver. We validate the estimator using an obese Zucker rat model of non-alcoholic fatty liver disease and correlate the local speed of sound with liver steatosis.Approach:We estimated the local and global average speed of sound noninvasively in 4 lean Zucker rats fed a normal diet and 16 obese Zucker rats fed a high fat diet for up to 8 weeks. The ground truth speed of sound and fat concentration were measured from the excised liver using established techniques.Main Results:The noninvasive, local speed of sound estimates of the livers were similar in value to their corresponding "ground truth'' measurements, having a slope ± standard error of the regression of 0.82 ± 0.15 (R2= 0.74 and p < 0.001). Measurement of the noninvasive global average speed of sound did not reliably capture the ``ground truth'' speed of sound in the liver, having a slope of 0.35 ± 0.07 (R2= 0.74 and p < 0.001). Decreasing local speed of sound was observed with increasing hepatic fat accumulation (approximately -1.7 m/s per 1% increase in hepatic fat) and histopathology steatosis grading (approximately -10 to -13 m/s per unit increase in steatosis grade). Local speed of sound estimates were highly correlated with steatosis grade, having Pearson and Spearman correlation coefficients both ranging from -0.87 to -0.78. In addition, a lobe-dependent speed of sound in the liver was observed by theex vivomeasurements, with speed of sound differences of up to 25 m/s (p < 0.003) observed between lobes in the liver of the same animal.Significance:The findings of this study suggest that local speed of sound estimation has the potential to be used to predict or assist in the measurement of hepatic fat concentration and that the global average speed of sound should be avoided in hepatic fat estimation due to significant bias in the speed of sound estimate.
View details for DOI 10.1088/1361-6560/ac4562
View details for PubMedID 34933288
-
Engineered Cell-Derived Vesicles Displaying Targeting Peptide and Functionalized with Nanocarriers for Therapeutic microRNA Delivery to Triple-Negative Breast Cancer in Mice.
Advanced healthcare materials
2021: e2101387
Abstract
Polymeric nanocarriers (PNCs) can be used to deliver therapeutic microRNAs (miRNAs) to solid cancers. However, the ability of these nanocarriers to specifically target tumors remains a challenge. Alternatively, extracellular vesicles (EVs) derived from tumor cells show homotypic affinity to parent cells, but loading sufficient amounts of miRNAs into EVs is difficult. Here, we investigate whether uPAR-targeted delivery of nanococktails containing PNCs loaded with therapeutic antimiRNAs, and coated with uPA engineered extracellular vesicles (uPA-eEVs) can elicit synergistic antitumor responses. The uPA-eEVs coating on PNCs increases natural tumor targeting affinities, thereby enhancing the antitumor activity of antimiRNA nanococktails. The systemic administration of uPA-eEV-PNCs nanococktail showed a robust tumor tropism, which significantly enhanced the combinational antitumor effects of antimiRNA-21 and antimiRNA-10b, and led to significant tumor regression and extension of progression free survival for syngeneic 4T1 tumor-bearing mice. In addition, the uPA-eEV-PNCs-antimiRNAs nanococktail plus low dose doxorubicin resulted in a synergistic antitumor effect as evidenced by inhibition of tumor growth, reduction of lung metastases, and extension of survival of 4T1 tumor-bearing mice. Our targeted combinational nanococktail strategy could be readily translated to the clinical setting by using autologous cancer cells that have flexibility for ex vivo expansion and genetic engineering. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adhm.202101387
View details for PubMedID 34879180
-
Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging.
Scientific reports
2021; 11 (1): 23026
Abstract
Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3*hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7±2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1±1.2 a.u.) compared to non-targeted control (0.4±0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.
View details for DOI 10.1038/s41598-021-02445-2
View details for PubMedID 34845270
-
Detection and Characterization of Sentinel Lymph Node by Ultrasound Molecular Imaging with B7-H3-Targeted Microbubbles in Orthotopic Breast Cancer Model in Mice.
Molecular imaging and biology
2021
Abstract
PURPOSE: Accurate identification and assessment of sentinel lymph node (SLN) using noninvasive imaging methods can play a vital role in tumor staging, surgical planning, and prognostic evaluation. In this study, we assessed the efficacy of B7-H3-targeted molecular-ultrasound imaging for the early SLN detection, and characterization in a mouse model of orthotopic breast cancer.PROCEDURES: We established a mouse breast cancer model with lymph node metastasis by injecting MAD-MB 231 cells which were engineered to express firefly luciferase reporter gene into the fat pad of the right 4th mammary gland in female BALB/c nude mice. The sole lymph node (LN) close to the tumor was regarded as the SLN for imaging investigation, which included metastatic and non-metastatic SLNs. The LN in the right 4th mammary gland from normal mice was used as normal control (normal mice LN). The commercially available preclinical streptavidin-coated, perfluorocarbon-containing lipid-shelled microbubbles (VisualSonics, Toronto, Canada) were used to generate B7-H3-targeted microbubbles (MBB7-H3) and control microbubbles (MBControl). Then, ultrasound molecular imaging (USMI) was performed using a high-resolution transducer (MS250; center frequency, 21MHz; Vevo 2100; VisualSonics, Toronto, Canada) after intravenous injection of microbubbles.RESULTS: The SLN was clearly detected and located under conventional (B-mode) and contrast-enhanced ultrasonography with microbubble injection. The metastatic SLNs showed a markedly higher signal from B7-H3-targeted microbubbles (MBB7-H3) compared to the non-metastatic SLNs and normal LNs. The metastatic SLN was further confirmed by ex vivo bioluminescence imaging and eventually verified by histological analysis.CONCLUSIONS: Our findings suggest the potential value of USMI using B7-H3 targeted microbubbles in breast cancer and establish an effective imaging method for the non-invasive detection and characterization of SLN.
View details for DOI 10.1007/s11307-021-01680-3
View details for PubMedID 34787812
-
Gold-Nanostar-Chitosan-Mediated Delivery of SARS-CoV-2 DNA Vaccine for Respiratory Mucosal Immunization: Development and Proof-of-Principle.
ACS nano
2021
Abstract
The COVID-19 pandemic is caused by the coronavirus SARS-CoV-2 (SC2). A variety of anti-SC2 vaccines have been approved for human applications, including those using messenger RNA (mRNA), adenoviruses expressing SC2 spike (S) protein, and inactivated virus. The protective periods of immunization afforded by these intramuscularly administered vaccines are currently unknown. An alternative self-administrable vaccine capable of mounting long-lasting immunity via sterilizing neutralizing antibodies would be hugely advantageous in tackling emerging mutant SC2 variants. This could also diminish the possibility of vaccinated individuals acting as passive carriers of COVID-19. Here, we investigate the potential of an intranasal (IN)-delivered DNA vaccine encoding the S protein of SC2 in BALB/c and C57BL/6J immunocompetent mouse models. The immune response to IN delivery of this SC2-spike DNA vaccine transported on a modified gold-chitosan nanocarrier shows a strong and consistent surge in antibodies (IgG, IgA, and IgM) and effective neutralization of pseudoviruses expressing S proteins of different SC2 variants (Wuhan, beta, and D614G). Immunophenotyping and histological analyses reveal chronological events involved in the recognition of SC2 S antigen by resident dendritic cells and alveolar macrophages, which prime the draining lymph nodes and spleen for peak SC2-specific cellular and humoral immune responses. The attainable high levels of anti-SC2 IgA in lung mucosa and tissue-resident memory T cells can efficiently inhibit SC2 and its variants at the site of entry and also provide long-lasting immunity.
View details for DOI 10.1021/acsnano.1c05002
View details for PubMedID 34705425
-
Dynamic Ligand Screening by Magnetic Nanoassembly Modulates Stem Cell Differentiation.
Advanced materials (Deerfield Beach, Fla.)
2021: e2105460
Abstract
In native microenvironment, diverse physical barriers exist to dynamically modulate stem cell recruitment and differentiation for tissue repair. In this study, we utilize nanoassembly-based magnetic screens of various sizes and elastically tethered them over RGD ligand (cell-adhesive motif)-presenting material surface to generate various nano-gaps between the screens and the RGDs without modulating the RGD density. Large screens exhibiting low RGD distribution stimulate integrin clustering to facilitate focal adhesion, mechanotransduction, and differentiation of stem cells, which were not observed with small screens. Magnetic downward pulling of the large screens decreases nano-gaps, which dynamically suppress the focal adhesion, mechanotransduction, and differentiation of stem cells. Conversely, magnetic upward pulling of the small screens increases the nano-gaps, which dynamically activate focal adhesion, mechanotransduction, and differentiation of stem cells. This regulation mechanism was also shown to be effective in the microenvironment in vivo. Further diversifying geometries of the physical screens could further enable diverse modalities of multifaceted and safe unscreening of the distributed RGDs to unravel and modulate stem cell differentiation for tissue repair. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adma.202105460
View details for PubMedID 34655440
-
Camouflaged Hybrid Cancer Cell-Platelet Fusion Membrane Nanovesicles Deliver Therapeutic MicroRNAs to Presensitize Triple-Negative Breast Cancer to Doxorubicin.
Advanced functional materials
2021; 31 (41)
Abstract
Camouflaged cell-membrane-based nanoparticles have been gaining increasing attention owing to their improved biocompatibility and immunomodulatory properties. Using nanoparticles prepared from the membranes of specific cell types, or fusions derived from different cells membranes, can improve their functional performance in several aspects. Here, we used cell membranes extracted from breast cancer cells and platelets to fabricate a hybrid-membrane vesicle fusion (cancer cell-platelet-fusion-membrane vesicle, CPMV) in which we loaded therapeutic microRNAs (miRNAs) for the treatment of triple-negative breast cancer (TNBC). We used a clinically scalable microfluidic platform for the fusion of cell membranes. The reconstitution process during synthesis allows for efficient loading of miRNAs into CPMVs. We systematically optimized the conditions for preparation of miRNA-loaded CPMVs and demonstrated their property of homing to source cells using in vitro experiments, and by therapeutic evaluation in vivo. In vitro, the CPMVs exhibited significant recognition of their source cells and avoided engulfment by macrophages. After systemic delivery in mice, the CPMVs showed a prolonged circulation time and site-specific accumulation at implanted TNBC-xenografts. The delivered antimiRNAs sensitized TNBCs to doxorubicin, resulting in an improved therapeutic response and survival rate. This strategy has considerable potential for clinical translation to improve personalized therapy for breast cancer and other malignancies.
View details for DOI 10.1002/adfm.202103600
View details for PubMedID 34899115
View details for PubMedCentralID PMC8664068
-
Magnetic Control and Real-Time Monitoring of Stem Cell Differentiation by the Ligand Nanoassembly.
Small (Weinheim an der Bergstrasse, Germany)
2021: e2102892
Abstract
Native extracellular matrix (ECM) exhibits dynamic change in the ligand position. Herein, the ECM-emulating control and real-time monitoring of stem cell differentiation are demonstrated by ligand nanoassembly. The density of gold nanoassembly presenting cell-adhesive Arg-Gly-Asp (RGD) ligand on Fe3 O4 (magnetite) nanoparticle in nanostructures flexibly grafted to material is changed while keeping macroscale ligand density invariant. The ligand nanoassembly on the Fe3 O4 can be magnetically attracted to mediate rising and falling ligand movements via linker stretching and compression, respectively. High ligand nanoassembly density stimulates integrin ligation to activate the mechanosensing-assisted stem cell differentiation, which is monitored via in situ real-time electrochemical sensing. Magnetic control of rising and falling ligand movements hinders and promotes the adhesion-mediated mechanotransduction and differentiation of stem cells, respectively. These rising and falling ligand states yield the difference in the farthest distance (34.6 nm) of the RGD from material surface, thereby dynamically mimicking static long and short flexible linkers, which hinder and promote cell adhesion, respectively. Design of cytocompatible ligand nanoassemblies can be made with combinations of dimensions, shapes, and biomimetic ligands for remotely regulating stem cells for offering novel methodologies to advance regenerative therapies.
View details for DOI 10.1002/smll.202102892
View details for PubMedID 34515417
-
Advances in Engineered Polymer Nanoparticle Tracking Platforms towards Cancer Immunotherapy-Current Status and Future Perspectives.
Vaccines
2021; 9 (8)
Abstract
Engineering polymeric nanoparticles for their shape, size, surface chemistry, and functionalization using various targeting molecules has shown improved biomedical applications for nanoparticles. Polymeric nanoparticles have created tremendous therapeutic platforms, particularly applications related to chemo- and immunotherapies in cancer. Recently advancements in immunotherapies have broadened this field in immunology and biomedical engineering, where "immunoengineering" creates solutions to target translational science. In this regard, the nanoengineering field has offered the various techniques necessary to manufacture and assemble multifunctional polymeric nanomaterial systems. These include nanoparticles functionalized using antibodies, small molecule ligands, targeted peptides, proteins, and other novel agents that trigger and encourage biological systems to accept the engineered materials as immune enhancers or as vaccines to elevate therapeutic functions. Strategies to engineer polymeric nanoparticles with therapeutic and targeting molecules can provide solutions for developing immune vaccines via maintaining the receptor storage in T- and B cells. Furthermore, cancer immunotherapy using polymeric nanomaterials can serve as a gold standard approach for treating primary and metastasized tumors. The current status of the limited availability of immuno-therapeutic drugs highlights the importance of polymeric nanomaterial platforms to improve the outcomes via delivering anticancer agents at localized sites, thereby enhancing the host immune response in cancer therapy. This review mainly focuses on the potential scientific enhancements and recent developments in cancer immunotherapies by explicitly discussing the role of polymeric nanocarriers as nano-vaccines. We also briefly discuss the role of multifunctional nanomaterials for their therapeutic impacts on translational clinical applications.
View details for DOI 10.3390/vaccines9080935
View details for PubMedID 34452059
-
Camouflaged Hybrid Cancer Cell-Platelet Fusion Membrane Nanovesicles Deliver Therapeutic MicroRNAs to Presensitize Triple-Negative Breast Cancer to Doxorubicin
ADVANCED FUNCTIONAL MATERIALS
2021
View details for DOI 10.1002/adfm.202103600
View details for Web of Science ID 000673272900001
-
Immunoregulation of Macrophages by Controlling Winding and Unwinding of Nanohelical Ligands
ADVANCED FUNCTIONAL MATERIALS
2021
View details for DOI 10.1002/adfm.202103409
View details for Web of Science ID 000663240400001
-
Management of COVID-19: Current Status and Future Prospects.
Microbes and infection
2021: 104832
Abstract
COVID-19, a highly transmissible pandemic disease, affecting millions of lives around the world. Severely infected patients show acute respiratory distress symptoms. Sustainable management strategies are required to save the lives of the infected people and further preventing spread of the virus. Diagnosis, treatment, and vaccination development initiatives are already exhibited from the scientific community to fight against this virus. In this review, we primarily discuss the management strategies including Prevention of spread, prophylaxis, vaccinations, and treatment for COVID-19. Further, analysis of vaccine development status and performance are also briefly discussed. Global social and economic impact of COVID-19 are also analyzed as part of this review.
View details for DOI 10.1016/j.micinf.2021.104832
View details for PubMedID 33872807
-
Passive Cavitation Mapping by Cavitation Source Localization From Aperture-Domain Signals-Part II: Phantom and In Vivo Experiments
IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL
2021; 68 (4): 1198–1212
Abstract
Passive cavitation mapping (PCM) techniques typically utilize a time-exposure acoustic (TEA) approach, where the received radio frequency data are beamformed, squared, and integrated over time. Such PCM-TEA cavitation maps typically suffer from long-tail artifacts and poor axial resolution with pulse-echo diagnostic arrays. Here, we utilize a recently developed PCM technique based on cavitation source localization (CSL), which fits a hyperbolic function to the received cavitation wavefront. A filtering method based on the root-mean-square error (rmse) of the hyperbolic fit is utilized to filter out spurious signals. We apply a wavefront correction technique to the signals with poor fit quality to recover additional cavitation signals and improve cavitation localization. Validation of the PCM-CSL technique with rmse filtering and wavefront correction was conducted in experiments with a tissue-mimicking flow phantom and an in vivo mouse model of cancer. It is shown that the quality of the hyperbolic fit, necessary for the PCM-CSL, requires an rmse < 0.05 mm2 in order to accurately localize the cavitation sources. A detailed study of the wavefront correction technique was carried out, and it was shown that, when applied to experiments with high noise and interference from multiple cavitating microbubbles, it was capable of effectively correcting noisy wavefronts without introducing spurious cavitation sources, thereby improving the quality of the PCM-CSL images. In phantom experiments, the PCM-CSL was capable of precisely localizing sources on the therapy beam axis and off-axis sources. In vivo cavitation experiments showed that PMC-CSL showed a significant improvement over PCM-TEA and yielded acceptable localization of cavitation signals in mice.
View details for DOI 10.1109/TUFFC.2020.3035709
View details for Web of Science ID 000634502600024
View details for PubMedID 33141666
-
Ultrasound Triggered Co-Delivery of Therapeutic MicroRNAs and a Triple Suicide Gene Therapy Vector by Using Biocompatible Polymer Nanoparticles for Improved Cancer Therapy in Mouse Models
ADVANCED THERAPEUTICS
2021
View details for DOI 10.1002/adtp.202000197
View details for Web of Science ID 000625355200001
-
Diagnosis For COVID-19: Current Status and Future Prospects.
Expert review of molecular diagnostics
2021
Abstract
INTRODUCTION: Coronavirus disease 2019 (COVID-19), a respiratory illness caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had its first detection in December 2019 in Wuhan (China) and spread across the world. In March 2020, the World Health Organization (WHO) declared COVID-19 a pandemic disease. The utilization of prompt and accurate molecular diagnosis of SARS-CoV-2 virus, isolating the infected patients, and treating them are the keys to managing this unprecedented pandemic. International travel acted as a catalyst for the widespread transmission of the virus.Areas Covered:This review discusses phenotype, structural, and molecular evolution of recognition elements and primers, its detection in the laboratory, and at point of care. Further, market analysis of commercial products and their performance is also evaluated, providing new ways to confront the ongoing global public health emergency.Expert Commentary:The outbreak for COVID-19 created mammoth chaos to the healthcare sector, and still, day by day, new epicenters for the outbreak are being reported. Emphasis should be placed on developing more effective, rapid, and early diagnostic devices. The testing laboratories should invest more in clinically relevant multiplexed and scalable detection tools to fight against a pandemic like this where massive demand for testing exists.
View details for DOI 10.1080/14737159.2021.1894930
View details for PubMedID 33621145
-
Remote Switching of Elastic Movement of Decorated Ligand Nanostructures Controls the Adhesion-Regulated Polarization of Host Macrophages
ADVANCED FUNCTIONAL MATERIALS
2021
View details for DOI 10.1002/adfm.202008698
View details for Web of Science ID 000618955600001
-
Remote Control of Time-Regulated Stretching of Ligand-Presenting Nanocoils In Situ Regulates the Cyclic Adhesion and Differentiation of Stem Cells.
Advanced materials (Deerfield Beach, Fla.)
2021: e2008353
Abstract
Native extracellular matrix (ECM) can exhibit cyclic nanoscale stretching and shrinking of ligands to regulate complex cell-material interactions. Designing materials that allow cyclic control of changes in intrinsic ligand-presenting nanostructures in situ can emulate ECM dynamicity to regulate cellular adhesion. Unprecedented remote control of rapid, cyclic, and mechanical stretching ("ON") and shrinking ("OFF") of cell-adhesive RGD ligand-presenting magnetic nanocoils on a material surface in five repeated cycles are reported, thereby independently increasing and decreasing ligand pitch in nanocoils, respectively, without modulating ligand-presenting surface area per nanocoil. It is demonstrated that cyclic switching "ON" (ligand nanostretching) facilitates time-regulated integrin ligation, focal adhesion, spreading, YAP/TAZ mechanosensing, and differentiation of viable stem cells, both in vitro and in vivo. Fluorescence resonance energy transfer (FRET) imaging reveals magnetic switching "ON" (stretching) and "OFF" (shrinking) of the nanocoils inside animals. Versatile tuning of physical dimensions and elements of nanocoils by regulating electrodeposition conditions is also demonstrated. The study sheds novel insight into designing materials with connected ligand nanostructures that exhibit nanocoil-specific nano-spaced declustering, which is ineffective in nanowires, to facilitate cell adhesion. This unprecedented, independent, remote, and cytocompatible control of ligand nanopitch is promising for regulating the mechanosensing-mediated differentiation of stem cells in vivo.
View details for DOI 10.1002/adma.202008353
View details for PubMedID 33527502
-
Ferroptosis inducers are a novel therapeutic approach for advanced prostate cancer.
Cancer research
2021
Abstract
Ferroptosis is a type of programmed cell death induced by the accumulation of lipid peroxidation and lipid reactive oxygen species (ROS) in cells. It has been recently demonstrated that cancer cells are vulnerable to ferroptosis inducers (FIN). However, the therapeutic potential of ferroptosis inducers in prostate cancer in pre-clinical settings has not been explored. In this study, we demonstrate that mediators of ferroptosis SLC7A11, SLC3A2 and GPX4 are expressed in treatment-resistant prostate cancer. We further demonstrate that treatment-resistant prostate cancer cells are sensitive to two ferroptosis inducers, erastin and RSL3. Treatment with erastin and RSL3 led to a significant decrease in prostate cancer cell growth and migration in vitro and significantly delayed the tumor growth of treatment-resistant prostate cancer in vivo, with no measurable side effects. Combination of erastin or RSL3 with standard-of-care second-generation anti-androgens for advanced prostate cancer halted prostate cancer cell growth and migration in vitro and tumor growth in vivo. These results demonstrate the potential of erastin or RSL3 independently and in combination with standard-of-care second-generation anti-androgens as novel therapeutic strategies for advanced prostate cancer.
View details for DOI 10.1158/0008-5472.CAN-20-3477
View details for PubMedID 33483372
-
Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells.
Methods in molecular biology (Clifton, N.J.)
2021; 2274: 261-270
Abstract
Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR-BL signals in mammalian cells.
View details for DOI 10.1007/978-1-0716-1258-3_22
View details for PubMedID 34050478
-
Highly Bright NIR-BRET System for Imaging Molecular Events in Live Cells.
Methods in molecular biology (Clifton, N.J.)
2021; 2274: 247-259
Abstract
The present protocol demonstrates a novel mammalian cell imaging platform exerting a bioluminescence resonance energy transfer (BRET) system. This platform achieves a ~300nm blue-to-near infrared shift of the emission (NIR-BRET) with the development of a unique coelenterazine (CTZ) derivative named BBlue2.3 and a fusion reporter protein probe named iRFP-RLuc8.6-535SG. The best NIR-BRET shift was achieved by tuning the blue emission peak of BBlue2.3 to a Soret band of the iRFP. In mammalian cells, BBlue2.3 emits light that is ~50-fold brighter than DeepBlueC in cell imaging when combined with RLuc8.6-535SG. This NIR-BRET platform is sufficiently brighter to be used for imaging live mammalian cells at single-cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. This unique optical platform provides the brightest NIR-BLI template that can be used for imaging a diverse group of cellular events in living subjects.
View details for DOI 10.1007/978-1-0716-1258-3_21
View details for PubMedID 34050477
-
Wearable Collector for Noninvasive Sampling of SARS-CoV-2 from Exhaled Breath for Rapid Detection.
ACS applied materials & interfaces
2021
Abstract
Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.
View details for DOI 10.1021/acsami.1c09309
View details for PubMedID 34428374
-
Functionalized Nanomaterials as Tailored Theranostic Agents in Brain Imaging.
Nanomaterials (Basel, Switzerland)
2021; 12 (1)
Abstract
Functionalized nanomaterials of various categories are essential for developing cancer nano-theranostics for brain diseases; however, some limitations exist in their effectiveness and clinical translation, such as toxicity, limited tumor penetration, and inability to cross blood-brain and blood-tumor barriers. Metal nanomaterials with functional fluorescent tags possess unique properties in improving their functional properties, including surface plasmon resonance (SPR), superparamagnetism, and photo/bioluminescence, which facilitates imaging applications in addition to their deliveries. Moreover, these multifunctional nanomaterials could be synthesized through various chemical modifications on their physical surfaces via attaching targeting peptides, fluorophores, and quantum dots (QD), which could improve the application of these nanomaterials by facilitating theranostic modalities. In addition to their inherent CT (Computed Tomography), MRI (Magnetic Resonance Imaging), PAI (Photo-acoustic imaging), and X-ray contrast imaging, various multifunctional nanoparticles with imaging probes serve as brain-targeted imaging candidates in several imaging modalities. The primary criteria of these functional nanomaterials for translational application to the brain must be zero toxicity. Moreover, the beneficial aspects of nano-theranostics of nanoparticles are their multifunctional systems proportioned towards personalized disease management via comprising diagnostic and therapeutic abilities in a single biodegradable nanomaterial. This review highlights the emerging aspects of engineered nanomaterials to reach and deliver therapeutics to the brain and how to improve this by adopting the imaging modalities for theranostic applications.
View details for DOI 10.3390/nano12010018
View details for PubMedID 35009968
-
A Priori Activation of Apoptosis Pathways of Tumor (AAAPT) technology: Development of targeted apoptosis initiators for cancer treatment.
PloS one
2021; 16 (2): e0225869
Abstract
Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. Amongst many, the principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept "A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously and selectively in cancer cells sparing normal cells. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (lung cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro when used as a combination. At higher doses, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AAAPT leading molecules as neoadjuvant to chemotherapy to achieve better efficacy and reduced off-target toxicity compared to the current treatments.
View details for DOI 10.1371/journal.pone.0225869
View details for PubMedID 33556062
-
Current status of targeted microbubbles in diagnostic molecular imaging of pancreatic cancer.
Bioengineering & translational medicine
2021; 6 (1): e10183
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is often associated with a poor prognosis due to silent onset, resistance to therapies, and rapid spreading. Most patients are ineligible for curable surgery as they present with advanced disease at the time of diagnosis. Present diagnostic methods relying on anatomical changes have various limitations including difficulty to discriminate between benign and malignant conditions, invasiveness, the ambiguity of imaging results, or the inability to detect molecular biomarkers of PDAC initiation and progression. Therefore, new imaging technologies with high sensitivity and specificity are critically needed for accurately detecting PDAC and noninvasively characterizing molecular features driving its pathogenesis. Contrast enhanced targeted ultrasound (CETUS) is an upcoming molecular imaging modality that specifically addresses these issues. Unlike anatomical imaging modalities such as CT and MRI, molecular imaging using CETUS is promising for early and accurate detection of PDAC. The use of molecularly targeted microbubbles that bind to neovascular targets can enhance the ultrasound signal specifically from malignant PDAC tissues. This review discusses the current state of diagnostic imaging modalities for pancreatic cancer and places a special focus on ultrasound targeted-microbubble technology together with its clinical translatability for PDAC detection.
View details for DOI 10.1002/btm2.10183
View details for PubMedID 33532585
View details for PubMedCentralID PMC7823123
-
Cardio Phenotypic Potential of Mesenchymal Stem Cells.
Current protocols
2021; 1 (3): e62
Abstract
Cell therapy is being investigated as a powerful intervention to ameliorate the consequences of coronary artery disease. Among the different stem cell options, mesenchymal stem cells (MSCs) are particularly attractive due to their high availability, as well as immune-privileged status. However, it is still unclear whether mesenchymal stem cells can acquire cardiomyogenic characteristics after they are transplanted to the myocardium. In this article, we outline protocols that illustrate the plasticity of MSCs and their ability to acquire cardiogenic characteristics when they are in an ischemic-like environment, as typically encountered after transplantation into the ischemic heart. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mesenchymal stem cells (MSCs) Support Protocol 1: Characterization of MSCs by flow cytometry Basic Protocol 2: Isolation of neonatal cardiomyoctes (NCMs) Support Protocol 2: Characterization of NCMs Basic Protocol 3: Cardiogenic plasticity of MSCs under ischemic-like conditions Support Protocol 3: Characterization of the cardiomyogenic potential of MSCs.
View details for DOI 10.1002/cpz1.62
View details for PubMedID 33661576
-
High-Throughput Whole-Plate Imaging of Cells for Multiple Biological Applications.
Methods in molecular biology (Clifton, N.J.)
2021; 2274: 367-384
Abstract
Advanced multipurpose cell imaging systems along with integrated rapid quantitation software can enhance and expedite cancer cell culture studies in a variety of applications. Though accurate cell culture studies are an important and necessary component of nearly all cancer biomarker detection and therapy studies, the methods we currently use are of low-throughput, time consuming, and lack accuracy. Hence, it is important to improve several features of the assays to increase the accuracy of their quantitative outputs in most studies. In general, we perform cell culture analysis semimanually by counting a small aliquot of suspended cells using a hemocytometer or viewing a small area of cells on a plate using a bright-field microscope, and then extrapolate the counts or observations to estimate the values for the total numbers of cells. The fundamental problem with this process lies in using techniques, such as extrapolation, which inherently introduces intrasample variability while collecting the cells by enzymatic trypsinization for these assays that are affecting cell growth and other downstream assessments. Fluorescence (FL) microscopy-based assays are also used to image and count cells for various applications, including cell viability, proliferation, apoptosis, cell death, transfection efficiency, protein expression, stem cell properties, colony formation, cytotoxicity, drug dose-response, and treatment efficacy studies. These methods are not optimal for many researches, as they require real-time visualization under a microscope plus manual analysis to determine the final results. Owing to long exposure times for cells under fluorescent light of a microscope, the cells may be exposed to suboptimal conditions that affect cell growth, and with occasional photobleaching of the expressed FL probes. Alternatively, the use of cell imaging systems that integrate both advanced bright-field and FL imaging for cell counting and quantification can be useful. In this protocol, we discuss the advantages of a high-throughput cell imaging system using a whole-plate imaging format when used in various bioimaging studies by highlighting a few applications of the system. The system is designed to fundamentally improve the accuracy and time of cell culture analysis while also allowing us to perform the assay without trypsinization, thus avoiding the need to replicate multiple wells for monitoring cell growth over time.
View details for DOI 10.1007/978-1-0716-1258-3_30
View details for PubMedID 34050486
-
Minicircles for a two-step blood biomarker and PET imaging early cancer detection strategy.
Journal of controlled release : official journal of the Controlled Release Society
2021
Abstract
Early cancer detection can dramatically increase treatment options and survival rates for patients, yet detection of early-stage tumors remains difficult. Here, we demonstrate a two-step strategy to detect and locate cancerous lesions by delivering tumor-activatable minicircle (MC) plasmids encoding a combination of blood-based and imaging reporter genes to tumor cells. We genetically engineered the MCs, under the control of the pan-tumor-specific Survivin promoter, to encode: 1) Gaussia Luciferase (GLuc), a secreted biomarker that can be easily assayed in blood samples; and 2) Herpes Simplex Virus Type 1 Thymidine Kinase mutant (HSV-1 sr39TK), a PET reporter gene that can be used for highly sensitive and quantitative imaging of the tumor location. We evaluated two methods of MC delivery, complexing the MCs with the chemical transfection agent jetPEI or encapsulating the MCs in extracellular vesicles (EVs) derived from a human cervical cancer HeLa cell line. MCs delivered by EVs or jetPEI yielded significant expression of the reporter genes in cell culture versus MCs delivered without a transfection agent. Secreted GLuc correlated with HSV-1 sr39TK expression with R2 = 0.9676. MC complexation with jetPEI delivered a larger mass of MC for enhanced transfection, which was crucial for in vivo animal studies, where delivery of MCs via jetPEI resulted in GLuc and HSV-1 sr39TK expression at significantly higher levels than controls. To the best of our knowledge, this is the first report of the PET reporter gene HSV-1 sr39TK delivered via a tumor-activatable MC to tumor cells for an early cancer detection strategy. This work explores solutions to endogenous blood-based biomarker and molecular imaging limitations of early cancer detection strategies and elucidates the delivery capabilities and limitations of EVs.
View details for DOI 10.1016/j.jconrel.2021.05.026
View details for PubMedID 34029631
-
In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.
Biomaterials
2021; 281: 121339
Abstract
Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.
View details for DOI 10.1016/j.biomaterials.2021.121339
View details for PubMedID 35078042
-
A Microfluidics-Based Scalable Approach to Generate Extracellular Vesicles with Enhanced Therapeutic MicroRNA Loading for Intranasal Delivery to Mouse Glioblastomas.
ACS nano
2021
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles derived from different cell sources, are used as promising nanovesicles for delivering therapeutic microRNAs (miRNAs) and drugs in cancer therapy. However, their clinical translation is limited by the quantity, size heterogeneity, and drug or small RNA loading efficiency. Herein, we developed a scalable microfluidic platform that can load therapeutic miRNAs (antimiRNA-21 and miRNA-100) and drugs while controlling the size of microfluidically processed EVs (mpEVs) using a pressure-based disruption and reconstitution process. We prepared mpEVs of optimal size using microvesicles isolated from neural stem cells engineered to overexpress CXCR4 receptor and characterized them for charge and miRNA loading efficiency. Since the delivery of therapeutic miRNAs to brain cancer is limited by the blood-brain barrier (BBB), we adopted intranasal administration of miRNA-loaded CXCR4-engineered mpEVs in orthotopic GBM mouse models and observed a consistent pattern of mpEVs trafficking across the nasal epithelia, bypassing the BBB into the intracranial compartment. In addition, the CXCR4-engineered mpEVs manifested selective tropism toward GBMs by stromal-derived factor-1 chemotaxis to deliver their miRNA cargo. The delivered miRNAs sensitized GBM cells to temozolomide, resulting in prominent tumor regression, and improved the overall survival of mice. A simple and efficient approach of packaging miRNAs in mpEVs using microfluidics, combined with a noninvasive nose-to-brain delivery route presents far-reaching potential opportunities to improve GBM therapy in clinical practice.
View details for DOI 10.1021/acsnano.1c07587
View details for PubMedID 34723509
-
Acoustically Driven Microbubbles Enable Targeted Delivery of microRNA-Loaded Nanoparticles to Spontaneous Hepatocellular Neoplasia in Canines.
Advanced therapeutics
2020; 3 (12)
Abstract
Spatially localized microbubble cavitation by ultrasound offers an effective means of altering permeability of natural barriers (i.e. blood vessel and cell membrane) in favor of nanomaterials accumulation in the target site. In this study, a clinically relevant, minimally invasive ultrasound guided therapeutic approach is investigated for targeted delivery of anticancer microRNA loaded PLGA-b-PEG nanoparticles to spontaneous hepatocellular neoplasia in a canine model. Quantitative assessment of the delivered microRNAs revealed prominent and consistent increase in miRNAs levels (1.5-to 2.3-fold increase (p<0.001)) in ultrasound treated tumor regions compared to untreated control regions. Immunohistology of ultrasound treated tumor tissue presented a clear evidence for higher amount of nanoparticles extravasation from the blood vessels. A distinct pattern of cytokine expression supporting CD8+ T cells mediated "cold-to-hot" tumor transition was evident in all patients. On the outset, proposed platform can enhance delivery of miRNA-loaded nanoparticles to deep seated tumors in large animals to enhance chemotherapy.
View details for DOI 10.1002/adtp.202000120
View details for PubMedID 33415184
View details for PubMedCentralID PMC7784952
-
Acoustically Driven Microbubbles Enable Targeted Delivery of microRNA-Loaded Nanoparticles to Spontaneous Hepatocellular Neoplasia in Canines
ADVANCED THERAPEUTICS
2020
View details for DOI 10.1002/adtp.202000120
View details for Web of Science ID 000592655800001
-
Therapeutic Ultrasound Parameter Optimization for Drug Delivery Applied to a Murine Model of Hepatocellular Carcinoma.
Ultrasound in medicine & biology
2020
Abstract
Ultrasound and microbubble (USMB)-mediated drug delivery is a valuable tool for increasing the efficiency of the delivery of therapeutic agents to cancer while maintaining low systemic toxicity. Typically, selection of USMB drug delivery parameters used in current research settings are either based on previous studies described in the literature or optimized using tissue-mimicking phantoms. However, phantoms rarely mimic in vivo tumor environments, and the selection of parameters should be based on the application or experiment. In the following study, we optimized the therapeutic parameters of the ultrasound drug delivery system to achieve the most efficient in vivo drug delivery using fluorescent semiconducting polymer nanoparticles as a model nanocarrier. We illustrate that voltage, pulse repetition frequency and treatment time (i.e., number of ultrasound pulses per therapy area) delivered to the tumor can successfully be optimized in vivo to ensure effective delivery of the semiconducting polymer nanoparticles to models of hepatocellular carcinoma. The optimal in vivo parameters for USMB drug delivery in this study were 70 V (peak negative pressure = 3.4 MPa, mechanical index = 1.22), 1-Hz pulse repetition frequency and 100-s therapy time. USMB-mediated drug delivery using in vivo optimized ultrasound parameters caused an up to 2.2-fold (p < 0.01) increase in drug delivery to solid tumors compared with that using phantom-optimized ultrasound parameters.
View details for DOI 10.1016/j.ultrasmedbio.2020.09.009
View details for PubMedID 33153807
-
SARS-CoV-2 Vaccine Development: An Overview and Perspectives.
ACS pharmacology & translational science
2020; 3 (5): 844–58
Abstract
Coronavirus disease 2019, abbreviated as COVID-19, is caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It started in late December 2019 in Wuhan, China, and by mid-March 2020, the disease had spread globally. As of July 17, 2020, this pandemic virus has infected 13.9 million people and claimed the life of approximately 593 000 people globally, and the numbers continue to climb. An unprecedented effort is underway to develop therapeutic and prophylactic strategies against this disease. Various drugs and vaccines are undergoing rapid development, and some of these are already in phase III clinical trials. Although Russia was the first to release a vaccine by skipping phase III clinical trials, there is no evidence of large-scale clinical trials, and the safety and efficacy of the vaccine are still a concern. Nevertheless, critical lessons can be learned and data garnered for developing promising vaccines against this rapidly emerging virus or other similar pathogens in the future. In this overview, we cover the available information on the various vaccine development initiatives by different companies, the potential strategies adopted for vaccine design, and the challenges and clinical impact expected from these vaccines. We also briefly discuss the possible role of these vaccines and the specific concerns for their use in patients with pre-existing disease conditions such as cardiovascular, lung, kidney, and liver diseases, cancer patients who are receiving immunosuppressive medications, including anticancer chemotherapies, and many other sensitive populations, such as children and the elderly.
View details for DOI 10.1021/acsptsci.0c00109
View details for PubMedID 33062951
-
Bioluminescent Imaging Systems for Assay Developments.
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry
2020
View details for DOI 10.2116/analsci.20R003
View details for PubMedID 32963202
-
Current status of targeted microbubbles in diagnostic molecular imaging of pancreatic cancer
BIOENGINEERING & TRANSLATIONAL MEDICINE
2020
View details for DOI 10.1002/btm2.10183
View details for Web of Science ID 000569340100001
-
Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model.
Journal of visualized experiments : JoVE
2020
Abstract
There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.
View details for DOI 10.3791/61546
View details for PubMedID 32955500
-
Structural and Electronic Transport Properties of Fluorographene Directly Grown on Silicates for Possible Biosensor Applications
ACS APPLIED NANO MATERIALS
2020; 3 (6): 5399–5409
View details for DOI 10.1021/acsanm.0c00768
View details for Web of Science ID 000545689000050
-
Highly sensitive eight-channel light sensing system for biomedical applications.
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology
2020
Abstract
We demonstrate the potential of an eight-channel light sensing platform system, named Black Box I (BBI), for rapid and highly sensitive measurement of low-level light using a nonradioactive optical readout. We developed, normalized, and characterized the photon sensitivities of the eight channels of the BBI using placental alkaline phosphatase (PLAP) as a model imaging reporter. We found that the BBI system had a statistically strong linear correlation with the reference IVIS Lumina II system. When we applied normalization constants, we were able to optimize the photomultiplier tubes (PMT) of all eight channels of the BBI (up to r2 = 0.998). We investigated the biomedical utilities of BBI by: (i) determining alkaline phosphatase activities in mouse plasma samples as a diagnostic secretory biomarker of cancer, and (ii) diagnosing cancer metastases in the organs of mice bearing triple negative breast cancer. We provide an important new addition to low-cost biomedical instruments intended for pre-clinical diagnostic imaging with high sensitivity, high sample throughput, portability, and rapid on-site analysis of low-level light.
View details for DOI 10.1039/d0pp00017e
View details for PubMedID 32159572
-
SP94-Targeted Triblock Copolymer Nanoparticle Delivers Thymidine Kinase-p53-Nitroreductase Triple Therapeutic Gene and Restores Anticancer Function against Hepatocellular Carcinoma in Vivo.
ACS applied materials & interfaces
2020
Abstract
Gene-directed enzyme-prodrug therapy (GDEPT) is a promising approach for cancer therapy, but it suffers from poor targeted delivery in vivo. Polyethylenimine (PEI) is a cationic polymer efficient in delivering negatively charged nucleic acids across cell membranes; however, it is highly toxic in vivo. Hence, we efficiently reduced PEI toxicity without compromising its transfection efficiency by conjugating it with poly(d,l-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) as triblock copolymers through a multistep synthetic process. The synthesized nanoparticles showed efficient delivery of loaded nucleic acids to tumor cells in vitro and in vivo in mice. We used this nanoparticle to deliver a rationally engineered thymidine kinase (TK)-p53-nitroreductase (NTR) triple therapeutic gene against hepatocellular carcinoma (HCC), where p53 tumor suppressor gene is mutated in more than 85% of cancers. TK-p53-NTR triple gene therapy restores p53 function and potentiates cancer cell response to delivered prodrugs (ganciclovir (GCV) and CB1954). We used SP94 peptide-functionalized PLGA-PEG-PEI nanoparticles for the optimal delivery of TK-p53-NTR therapeutic gene in vivo. The nanoparticles prepared from the conjugated polymer showed high loading efficiency for the DNA and markedly enhanced TK-NTR-mediated gene therapy upon the simultaneous coexpression of p53 by the concurrent rescue of the endogenous apoptotic pathway in HCC cells of both p53-mutant and wild-type phenotypes in vitro. In vivo delivery of TK-p53-NTR genes by SP94-targeted PLGA-PEG-PEI NP in mice resulted in a strong expression of suicide genes selectively in tumors, and subsequent administration of GCV and CB1954 led to a decline in tumor growth, and established a superior therapeutic outcome against HCC. We demonstrate a highly efficient approach that exogenously supplements p53 to enable synergy with the outcome of TK-NTR suicide gene therapy against HCC.
View details for DOI 10.1021/acsami.9b20071
View details for PubMedID 32048820
-
Prostate Cancer Phenotype Influences Bone Mineralization at Metastasis: A Study Using an In Vitro Prostate Cancer Metastasis Testbed.
JBMR plus
2020; 4 (2): e10256
Abstract
In this study, two types of prostate cancer cell lines, highly metastatic PC-3 and low metastatic MDA PCa 2b (PCa) were cultured on bone mimetic scaffolds to recapitulate metastasis to bone. A unique in vitro 3D tumor model that uses a sequential culture (SC) of human mesenchymal stem cells followed by seeding with cancer cells after bone formation was initiated to study the phenotype-specific interaction between prostate cancer cells and bone microenvironment. The PCa cells were observed to be less prolific and less metastatic, and to form multicellular tumoroids in the bone microenvironment, whereas PC-3 cells were more prolific and were highly metastatic, and did not form multicellular tumoroids in the bone microenvironment. The metastatic process exhibited by these two prostate cancer cell lines showed a significant and different effect on bone mineralization and extracellular matrix formation. Excessive bone formation in the presence of PC-3 and significant osteolysis in the presence of PCa were observed, which was also indicated by osteocalcin and MMP-9 expression as measured by ELISA and qRT-PCR. The field emission scanning electron microscopy images revealed that the structure of mineralized collagen in the presence of PC-3 is different than the one observed in healthy bone. All experimental results indicated that both osteolytic and osteoblastic bone lesions can be recapitulated in our tumor testbed model and that different cancer phenotypes have a very different influence on bone at metastasis. The 3D in vitro model presented in this study provides an improved, reproducible, and controllable system that is a useful tool to elucidate osteotropism of prostate cancer cells. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
View details for DOI 10.1002/jbm4.10256
View details for PubMedID 32083238
-
The Myocardial Microenvironment Modulates the Biology of Transplanted Mesenchymal Stem Cells.
Molecular imaging and biology
2020
Abstract
PURPOSE: The maximal efficacy of cell therapy depends on the survival of stem cells, as well as on the phenotypic and biologic changes that may occur on these cells after transplantation. It has been hypothesized that the post-ischemic myocardial microenvironment can play a critical role in these changes, potentially affecting the survival and reparative potential of mesenchymal stem cells (MSCs). Here, we use a dual reporter gene sensor for the in vivo monitoring of the phenotype of MSCs and study their therapeutic effect on cardiac function.PROCEDURES: The mitochondrial sensor was tested in cell culture in response to different mitochondrial stressors. For in vivo testing, MSCs (3*105) were delivered in a murine ischemia-reperfusion (IR) model. Bioluminescence imaging was used to assess the mitochondrial biology and the viability of transplanted MSCs, while high-resolution ultrasound provided a non-invasive analysis of cardiac contractility and dyssynchrony.RESULTS: The mitochondrial sensor showed increased activity in response to mitochondrial stressors. Furthermore, when tested in the living subject, it showed a significant increase in mitochondrial dysfunction in MSCs delivered in IR, compared with those delivered under sham conditions. Importantly, MSCs delivered to ischemic hearts, despite their mitochondrial stress and poor survival, were able to induce a significant improvement in cardiac function, through decreased collagen deposition and resynchronization/contractility of left ventricular wall motion.CONCLUSIONS: The ischemic myocardium induces changes in the phenotype of transplanted MSCs. Despite their limited survival, MSCs still elicit a certain therapeutic response, as evidenced by improvement in myocardial remodeling and cardiac function. Maximization of the survival and reparative efficacy of stem cells remains a key for the success of stem cell therapies.
View details for DOI 10.1007/s11307-019-01470-y
View details for PubMedID 31907845
-
Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma.
Investigative radiology
2020
Abstract
Early detection of pancreatic ductal adenocarcinoma (PDAC) represents the most significant step toward the treatment of this aggressive lethal disease. Previously, we engineered a preclinical Thy1-targeted microbubble (MBThy1) contrast agent that specifically recognizes Thy1 antigen overexpressed in the vasculature of murine PDAC tissues by ultrasound (US) imaging. In this study, we adopted a single-chain variable fragment (scFv) site-specific bioconjugation approach to construct clinically translatable MBThy1-scFv and test for its efficacy in vivo in murine PDAC imaging, and functionally evaluated the binding specificity of scFv ligand to human Thy1 in patient PDAC tissues ex vivo.We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MBThy1-scFv to MS1 cells overexpressing human Thy1 (MS1Thy1) under liquid shear stress conditions in vitro using a flow chamber setup at 0.6 mL/min flow rate, corresponding to a wall shear stress rate of 100 seconds, similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MBThy1-scFv was tested in the transgenic mouse model (C57BL/6J - Pdx1-Cre; KRas; Ink4a/Arf) of PDAC and in control mice (C57BL/6J) with L-arginine-induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and in patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining.Under shear stress flow conditions in vitro, MBThy1-scFv bound to MS1Thy1 cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P < 0.01) compared with MBNontargeted (0.5 ± 0.5 MB/cell). In vivo, MBThy1-scFv (5.3 ± 1.9 arbitrary units [a.u.]) but not the MBNontargeted (1.2 ± 1.0 a.u.) produced high US molecular imaging signal (4.4-fold vs MBNontargeted; n = 8; P < 0.01) in the transgenic mice with spontaneous PDAC tumors (2-6 mm). Imaging signal from mice with L-arginine-induced pancreatitis (n = 8) or normal pancreas (n = 3) were not significantly different between the two MB constructs and were significantly lower than PDAC Thy1 molecular signal. Clinical-grade scFv conjugated to Alexa Fluor 647 dye recognized MS1Thy1 cells but not the parental wild-type cells as evaluated by flow cytometry. More importantly, scFv showed highly specific binding to VEGFR2-positive vasculature and fibroblast-like stromal components surrounding the ducts of human PDAC tissues as evaluated by confocal microscopy.Our findings summarize the development and validation of a clinically relevant Thy1-targeted US contrast agent for the early detection of human PDAC by US molecular imaging.
View details for DOI 10.1097/RLI.0000000000000697
View details for PubMedID 32569010
-
Sound Speed Estimation in Layered Media Using the Angular Coherence of Plane Waves
SPIE-INT SOC OPTICAL ENGINEERING. 2020
View details for DOI 10.1117/12.2548878
View details for Web of Science ID 000558363700011
-
Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.
Analytical chemistry
2020
Abstract
MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm-1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.
View details for DOI 10.1021/acs.analchem.0c02401
View details for PubMedID 32985868
-
Pathway-specific reporter genes to study stem cell biology.
Stem cells (Dayton, Ohio)
2020
Abstract
Little is known on the phenotypic characteristics of stem cells after they are transplanted to the myocardium, in part due to lack of non-invasive platforms to study stem cells directly in the living subject. Reporter gene imaging has played a valuable role in the non-invasive assessment of cell fate in vivo. In this study, we validated a pathway-specific reporter gene that can be used to non-invasively image the phenotype of stem cells transplanted to the myocardium.Rat mesenchymal stem cells (MSCs) were studied for phenotypic evidence of myogenic characteristics under in vitro conditions. After markers of myogenic characteristics were identified, we constructed a reporter gene sensor, comprising the firefly luciferase (Fluc) reporter gene driven by the TroponinT promoter (cardio MSCs had 3-fold PCR expression compared to control MSCs) using a two-step signal amplification strategy. MSCs transfected with TroponinT-Fluc were studied and validated under in vitro conditions, showing a strong signal after MSCs acquired myogenic characteristics. Lastly, we cardio MSCs had higher expression of the reporter sensor compared to control cells (0.005 ± 0.0005 vs 0.0025 ± 0.0008 Tnt-Fluc/Ubi-Fluc, P < 0.05) that this novel sensor can detect the change in the phenotype of MSCs directly in the living subject.Pathway-specific reporter gene imaging allows assessment of changes in the phenotype of MSCs after delivery to the ischemic myocardium, providing important information on the phenotype of these cells. Imaging sensors like the one described here are critical to better understand the changes that stem cells undergo after transplantation. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: There is increasing awareness in the cell therapy community of the need for a better understanding of the biology of transplanted cells after their transplantation to the myocardium. We have previously shown the use of reporter gene labeling of stem cells to track the kinetics of cell viability. In this report, we extend these studies and report the development and validation of a pathway-specific reporter gene to study the changing phenotype of mesenchymal stromal cells after transplantation to the ischemic myocardium. In addition, we propose an algorithm for the development of these monitoring strategies for the entire scientific community to adopt.
View details for DOI 10.1002/stem.3167
View details for PubMedID 32129537
-
Combating Intracellular Pathogens with Nanohybrid-Facilitated Antibiotic Delivery.
International journal of nanomedicine
2020; 15: 8437–49
Abstract
Background: Lipid polymer hybrid nanoparticles (LPHNPs) have been widely investigated in drug and gene delivery as well as in medical imaging. A knowledge of lipid-based surface engineering and its effects on how the physicochemical properties of LPHNPs affect the cell-nanoparticle interactions, and consequently how it influences the cytological response, is in high demand.Methods: Herein, we have engineered antibiotic-loaded (doxycycline or vancomycin) LPHNPs with cationic and zwitterionic lipids and examined the effects on their physicochemical characteristics (size and charge), antibiotic entrapment efficiency, and the in vitro intracellular bacterial killing efficiency against Mycobacterium smegmatis or Staphylococcus aureus infected macrophages.Results: The incorporation of cationic or zwitterionic lipids in the LPHNP formulation resulted in a size reduction in LPHNPs formulations and shifted the surface charge of bare NPs towards positive or neutral values. Also observed were influences on the drug incorporation efficiency and modulation of the drug release from the biodegradable polymeric core. The therapeutic efficacy of LPHNPs loaded with vancomycin was improved as its minimum inhibitory concentration (MIC) (2 g/mL) versus free vancomycin (4 g/mL). Importantly, our results show a direct relationship between the cationic surface nature of LPHNPs and its intracellular bacterial killing efficiency as the cationic doxycycline or vancomycin loaded LPHNPs reduced 4 or 3 log CFU respectively versus the untreated controls.Conclusion: In our study, modulation of surface charge in the nanomaterial formulation increased macrophage uptake and intracellular bacterial killing efficiency of LPHNPs loaded with antibiotics, suggesting alternate way for optimizing their use in biomedical applications.
View details for DOI 10.2147/IJN.S271850
View details for PubMedID 33162754
-
Ultrasound-mediated delivery of miRNA-122 and anti-miRNA-21 therapeutically immunomodulates murine hepatocellular carcinoma in vivo.
Journal of controlled release : official journal of the Controlled Release Society
2020
Abstract
Hepatocellular carcinoma (HCC) is the most common cause of cancer-related mortality, and patients with HCC show poor response to currently available treatments, which demands new therapies. We recently developed a synthetic microRNA-based molecularly targeted therapy for improving HCC response to chemotherapy by eliminating drug resistance. We used ultrasound-targeted microbubble destruction (UTMD) to locally deliver microRNA-loaded nanoparticles to HCC. Since the immune microenvironment plays a crucial role in HCC disease development and response to treatment, and UTMD and microRNAs have the potential to interfere with the immune system, in this study we analyzed the immunomodulatory effects of UTMD and miRNAs in HCC. We used an immunocompetent syngeneic HCC mouse model for the study. We conducted cytokine profiling in tumor, lymph nodes, and serum of animals within the first 24 h of treatment to analyze changes in the level of pro- and antitumoral cytokines. The results showed: (1) Hepa1-6 syngeneic tumors expressed HCC-related cytokines, (2) UTMD-microRNA combination therapy triggered transient cytokine storms, and (3) delivery of microRNA-122 and anti-microRNA-21 affected the immune microenvironment by decreasing the level of GM-CSF in tumors while modulating protumoral IL-1α, IL-1β, IL-5, IL-6 and IL-17 and antitumoral IL-2 and IL-12 in tumor-proximal lymph nodes, and increasing IL-2 in the serum of tumor-bearing mice. Local delivery of targeted therapy by UTMD significantly reduced the concentration of IL-12 and IL-17 in lymph nodes of treated and contralateral tumors suggesting a systemic response. CONCLUSION: UTMD-mediated delivery of microRNA-122 and anti-microRNA-21 modulated the immune microenvironment of Hepa1-6 tumors at the level of cytokine expressions. Exploiting antitumoral immune effects could enhance the therapeutic efficacy of the proposed combination therapy for HCC.
View details for DOI 10.1016/j.jconrel.2020.01.051
View details for PubMedID 32004588
-
Assessment of Metastatic and Reactive Sentinel Lymph Nodes with B7-H3-Targeted Ultrasound Molecular Imaging: A Longitudinal Study in Mouse Models.
Molecular imaging and biology
2020
Abstract
To explore the potential of B7-H3-targeted ultrasound molecular imaging (USMI) for longitudinal assessment and differentiation of metastatic and reactive sentinel lymph nodes (SLNs) in mouse models.Metastatic and reactive SLN models were established by injection of 4T1 breast cancer cells and complete Freund's adjuvant (CFA) respectively to the 4th mammary fat pad of female BALB/c mice. At day 21, 28, and 35 after inoculation, USMI was performed following intravenous injection of B7-H3-targeted microbubbles (MBB7-H3) or IgG-control microbubbles (MBcontrol). All SLNs were histopathologically examined after the last imaging session.A total of 20 SLNs from tumor-bearing mice (T-SLNs) and five SLNs from CFA-injected mice (C-SLNs) were examined by USMI. Nine T-SLNs were histopathologically positive for metastasis (MT-SLNs). From day 21 to 35, T-SLNs showed a rising trend in MBB7-H3 signal with a steep increase in MT-SLNs at day 35 (213.5 ± 80.8 a.u.) as compared to day 28 (87.6 ± 77.2 a.u., P = 0.002) and day 21 (55.7 ± 35.5 a.u., P < 0.001). At day 35, MT-SLNs had significantly higher MBB7-H3 signal than non-metastatic T-SLNs (NMT-SLNs) (101.9 ± 48.0 a.u., P = 0.001) and C-SLNs (38.5 ± 34.0 a.u., P = 0.001); MBB7-H3 signal was significantly higher than MBcontrol in MT-SLNs (P = 0.001), but not in NMT-SLNs or C-SLNs (both P > 0.05). A significant correlation was detected between MBB7-H3 signal and volume fraction of metastasis in MT-SLNs (r = 0.76, P = 0.017).B7-H3-targeted USMI allows differentiation of MT-SLNs from NMT-SLNs and C-SLNs in mouse models and has great potential to evaluate tumor burden in SLNs of breast cancer.
View details for DOI 10.1007/s11307-020-01478-9
View details for PubMedID 32034623
-
Efficacy of affibody-based ultrasound molecular imaging of vascular B7-H3 for breast cancer detection.
Clinical cancer research : an official journal of the American Association for Cancer Research
2020
Abstract
Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for pre-clinical ultrasound (US) imaging with anti-B7-H3-antibody functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted-MB.Binding of ABYB7-H3 was confirmed with soluble and cell-surface B7-H3 by flow-cytometry. MB were functionalized with ABYB7-H3 or anti-B7-H3-antibody (AbB7-H3). Control and targeted-MB were tested for binding to hB7-H3-expressing cells (MS1hB7-H3) under shear stress conditions. US imaging was performed with MBABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 co-implanted with MS1hB7-H3 or control MS1WT cells and a transgenic mouse model of breast cancer development.ABYB7-H3 specifically binds to MS1hB7-H3 and murine-B7-H3-expressing monocytes. MBABY-B7-H3 (8.5 ± 1.4 MB/cell) and MBAb-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (p<0.0001) binding to the MS1hB7-H3 cells compared to control MBNon-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. In vivo, MBABY-B7-H3 produced significantly higher (p<0.04) imaging signal in orthotopic tumors co-engrafted with MS1hB7-H3 (8.4 ± 3.3 a.u.) compared to tumors with MS1WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MBABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (p<0.0002) imaging signal compared to MBNon-targeted (1.3 ± 0.3 a.u.), while MBABY-B7-H3 signal in normal mammary glands and tumors with B7-H3-blocking significantly reduced (p<0.02) imaging signal.MBABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.
View details for DOI 10.1158/1078-0432.CCR-19-1655
View details for PubMedID 31924738
-
Reconstructed Apoptotic Bodies as Targeted "Nano Decoys" to Treat Intracellular Bacterial Infections within Macrophages and Cancer Cells.
ACS nano
2020
Abstract
Staphylococcus aureus (S. aureus) is a highly pathogenic facultative anaerobe that in some instances resides as an intracellular bacterium within macrophages and cancer cells. This pathogen can establish secondary infection foci, resulting in recurrent systemic infections that are difficult to treat using systemic antibiotics. Here, we use reconstructed apoptotic bodies (ReApoBds) derived from cancer cells as "nano decoys" to deliver vancomycin intracellularly to kill S. aureus by targeting inherent "eat me" signaling of ApoBds. We prepared ReApoBds from different cancer cells (SKBR3, MDA-MB-231, HepG2, U87-MG, and LN229) and used them for vancomycin delivery. Physicochemical characterization showed ReApoBds size ranges from 80 to 150 nm and vancomycin encapsulation efficiency of 60 ± 2.56%. We demonstrate that the loaded vancomycin was able to kill intracellular S. aureus efficiently in an in vitro model of S. aureus infected RAW-264.7 macrophage cells, and U87-MG (p53-wt) and LN229 (p53-mt) cancer cells, compared to free-vancomycin treatment (P < 0.001). The vancomycin loaded ReApoBds treatment in S. aureus infected macrophages showed a two-log-order higher CFU reduction than the free-vancomycin treatment group. In vivo studies revealed that ReApoBds can specifically target macrophages and cancer cells. Vancomycin loaded ReApoBds have the potential to kill intracellular S. aureus infection in vivo in macrophages and cancer cells.
View details for DOI 10.1021/acsnano.0c00921
View details for PubMedID 32347709
-
TARGETED POLYGIONS ENGINEERED WITH SURFACE miRNAs FOR COMBINED MULTIMODALITY IMAGING AND ENHANCEMENT OF TEMOZOLOMIDE TREATMENT: A NOVEL INTRANASALLY-DELIVERED THERANOSTIC STRATEGY AGAINST GLIOBLASTOMA
OXFORD UNIV PRESS INC. 2019: 87–88
View details for Web of Science ID 000509478702023
-
PRECEDING p53 STABILIZATION USING DOXORUBICIN AUGMENTS PRIMA-1-MEDIATED p53 REFOLDING AND INCREASED CELLULAR APOPTOSIS: EVALUATION OF A SEQUENTIAL COMBINATION THERAPY AGAINST GLIOBLASTOMA
OXFORD UNIV PRESS INC. 2019: 88
View details for Web of Science ID 000509478702025
-
Structural determination of Enzyme-Graphene Nanocomposite Sensor Material.
Scientific reports
2019; 9 (1): 15519
Abstract
State-of-the-art ultra-sensitive blood glucose-monitoring biosensors, based on glucose oxidase (GOx) covalently linked to a single layer graphene (SLG), will be a valuable next generation diagnostic tool for personal glycemic level management. We report here our observations of sensor matrix structure obtained using a multi-physics approach towards analysis of small-angle neutron scattering (SANS) on graphene-based biosensor functionalized with GOx under different pH conditions for various hierarchical GOx assemblies within SLG. We developed a methodology to separately extract the average shape of GOx molecules within the hierarchical assemblies. The modeling is able to resolve differences in the average GOx dimer structure and shows that treatment under different pH conditions lead to differences within the GOx at the dimer contact region with SLG. The coupling of different analysis methods and modeling approaches we developed in this study provides a universal approach to obtain detailed structural quantifications, for establishing robust structure-property relationships. This is an essential step to obtain an insight into the structure and function of the GOx-SLG interface for optimizing sensor performance.
View details for DOI 10.1038/s41598-019-51882-7
View details for PubMedID 31664095
-
Evaluation of integrin alphavbeta6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.
Nature communications
2019; 10 (1): 4673
Abstract
Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin alphavbeta6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin alphavbeta6.
View details for DOI 10.1038/s41467-019-11863-w
View details for PubMedID 31611594
-
Biodegradable polymers for modern vaccine development
JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY
2019; 77: 12–24
View details for DOI 10.1016/j.jiec.2019.04.044
View details for Web of Science ID 000473376600002
-
Biodegradable polymers for modern vaccine development.
Journal of industrial and engineering chemistry (Seoul, Korea)
2019; 77: 12-24
Abstract
Most traditional vaccines are composed either of a whole pathogen or its parts; these vaccines, however, are not always effective and can even be harmful. As such, additional agents known as adjuvants are necessary to increase vaccine safety and efficacy. This review summarizes the potential of biodegradable materials, including synthetic and natural polymers, for vaccine delivery. These materials are highly biocompatible and have minimal toxicity, and most biomaterial-based vaccines delivering antigens or adjuvants have been shown to improve immune response, compared to formulations consisting of the antigen alone. Therefore, these materials can be applied in modern vaccine development.
View details for DOI 10.1016/j.jiec.2019.04.044
View details for PubMedID 32288512
View details for PubMedCentralID PMC7129903
-
Microvesicle-mediated delivery of minicircle DNA results in effective gene-directed enzyme prodrug cancer therapy.
Molecular cancer therapeutics
2019
Abstract
An emerging approach for cancer treatment employs the use of extracellular vesicles (EVs), specifically exosomes and microvesicles, as delivery vehicles. We previously demonstrated that microvesicles can functionally deliver plasmid DNA to cells and showed that plasmid size and sequence, in part, determine the delivery efficiency. In this study, delivery vehicles comprised of microvesicles loaded with engineered minicircle (MC) DNA that encodes prodrug converting enzymes were developed as a cancer therapy in mammary carcinoma models. We demonstrated that MCs can be loaded into shed microvesicles with greater efficiency than their parental plasmid counterparts and that microvesicle-mediated MC delivery led to significantly higher and more prolonged transgene expression in recipient cells than microvesicles loaded with the parental plasmid. Microvesicles loaded with MCs encoding a thymidine kinase (TK)/nitroreductase (NTR) fusion protein produced prolonged TK-NTR expression in mammary carcinoma cells. In vivo delivery of TK-NTR and administration of prodrugs led to the effective killing of both targeted cells and surrounding tumor cells via TK-NTR-mediated conversion of co-delivered prodrugs into active cytotoxic agents. In vivo evaluation of the bystander effect in mouse models demonstrated that for effective therapy, at least 1% of tumor cells need to be delivered with TK-NTR-encoding MCs. These results suggest that MC delivery via microvesicles can mediate gene transfer to an extent that enables effective prodrug conversion and tumor cell death such that it comprises a promising approach to cancer therapy.
View details for DOI 10.1158/1535-7163.MCT-19-0299
View details for PubMedID 31451563
-
Chronic Model of Inflammatory Bowel Disease in IL-10-/- Transgenic Mice: Evaluation with Ultrasound Molecular Imaging.
Theranostics
2019; 9 (21): 6031-6046
Abstract
Objective: Acute mouse models of inflammatory bowel disease (IBD) fail to mirror the chronic nature of IBD in patients. We sought to develop a chronic mouse IBD model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI) by using dual P- and E-selectin targeted microbubbles (MBSelectin). Materials and Methods: Interleukin 10 deficient (IL-10-/- on a C57BL/6 genetic background; n=55) and FVB (n=16) mice were used. In IL-10-/-mice, various experimental regimens including piroxicam, 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sulfate sodium (DSS), respectively were used for promoting colitis; colitis was induced with DSS in FVB mice. Using clinical and small animal ultrasound scanners, evolution of inflammation in proximal, middle and distal colon, was monitored with USMI by using MBSelectin at multiple time points. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E staining and for P-selectin expression on immunofluorescence staining. Results: Sustained colitis was not detected with USMI in IL-10-/- or FVB mice with various experimental regimens. USMI signals either gradually decreased after the colitis enhancing/inducing drug/agents were discontinued, or the mortality rate of mice was high. Inflammation was observed on H&E staining in IL-10-/- mice with piroxicam promotion, while stable overexpression of P-selectin was not found on immunofluorescence staining in the same mice. Conclusion: Sustained colitis in IL-10-/- mice induced with piroxicam, TNBS or DSS, and in FVB mice induced with DSS, was not detected with USMI using MBSelectin, and this was verified by immunofluorescence staining for inflammation marker P-selectin. Thus, these models may not be appropriate for long-term monitoring of chronic colitis and subsequent treatment response with dual-selectin targeted USMI.
View details for DOI 10.7150/thno.37397
View details for PubMedID 31534535
View details for PubMedCentralID PMC6735517
-
Ultrasound/microbubble-mediated targeted delivery of anticancer microRNA-loaded nanoparticles to deep tissues in pigs.
Journal of controlled release : official journal of the Controlled Release Society
2019
Abstract
In this study, we designed and validated a platform for ultrasound and microbubble-mediated delivery of FDA-approved pegylated poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with anticancer microRNAs (miRNAs) to deep tissues in a pig model. Small RNAs have been shown to reprogram tumor cells and sensitize them to clinically used chemotherapy. To overcome their short intravascular circulation half-life and achieve controlled and sustained release into tumor cells, anticancer miRNAs need to be encapsulated into nanocarriers. Focused ultrasound combined with gas-filled microbubbles provides a noninvasive way to improve the permeability of tumor vasculature and increase the delivery efficiency of drug-loaded particles. A single handheld, curvilinear ultrasound array was used in this study for image-guided therapy with clinical-grade SonoVue contrast agent. First, we validated the platform on phantoms to optimize the microbubble cavitation dose based on acoustic parameters, including peak negative pressure, pulse length, and pulse repetition frequency. We then tested the system in vivo by delivering PLGA nanoparticles co-loaded with antisense-miRNA-21 and antisense-miRNA-10b to pig liver and kidney. Enhanced miRNA delivery was observed (1.9- to 3.7-fold increase) as a result of the ultrasound treatment compared to untreated control regions. Additionally, we used highly fluorescent semiconducting polymer nanoparticles to visually assess nanoparticle extravasation. Fluorescent microscopy suggested the presence of nanoparticles in the extravascular compartment. Hematoxylin and eosin staining of treated tissues did not reveal tissue damage. The results presented in this manuscript suggest that the proposed platform may be used to safely and noninvasively enhance the delivery of miRNA-loaded nanoparticles to target regions in deep organs in large animal models.
View details for DOI 10.1016/j.jconrel.2019.07.024
View details for PubMedID 31326463
-
Intranasal delivery of targeted polyfunctional gold-iron oxide nanoparticles loaded with therapeutic microRNAs for combined theranostic multimodality imaging and presensitization of glioblastoma to temozolomide.
Biomaterials
2019; 218: 119342
Abstract
The prognosis for glioblastoma (GBM) remains depressingly low. The biological barriers of the brain present a major challenge to achieving adequate drug concentrations for GBM therapy. To address this, we explore the potential of the nose-to-brain direct transport pathway to bypass the blood-brain barrier, and to enable targeted delivery of theranostic polyfunctional gold-iron oxide nanoparticles (polyGIONs) surface loaded with therapeutic miRNAs (miR-100 and antimiR-21) to GBMs in mice. These nanoformulations would thus allow presensitization of GBM cells to the systemically delivered chemotherapy drug temozolomide (TMZ), as well as in vivo multimodality molecular and anatomic imaging of nanoparticle delivery, trafficking, and treatment effects. First, we synthesized GIONs coated with beta-cyclodextrin-chitosan (CD-CS) hybrid polymer, and co-loaded with miR-100 and antimiR-21. Then we decorated their surface with PEG-T7 peptide using CD-adamantane host-guest chemistry. The resultant polyGIONs showed efficient miRNA loading with enhanced serum stability. We characterized them for particle size, PDI, polymer functionalization, charge and release using dynamic light scattering analysis, TEM and qRT-PCR. For in vivo intranasal delivery, we used U87-MG GBM cell-derived orthotopic xenograft models in mice. Intranasal delivery resulted in efficient accumulation of Cy5-miRNAs in mice treated with T7-targeted polyGIONs, as demonstrated by in vivo optical fluorescence and MR imaging. We measured the therapeutic response of these FLUC-EGFP labelled U87-MG GBMs using bioluminescence imaging. Overall, there was a significant increase in survival of mice co-treated with T7-polyGIONs loaded with miR-100/antimiR-21 plus systemic TMZ, compared to the untreated control group, or the animals receiving non-targeted polyGIONs-miR-100/antimiR-21, or TMZ alone. Once translated clinically, this novel theranostic nanoformulation and its associated intranasal delivery strategy will have a strong potential to potentiate the effects of TMZ treatment in GBM patients.
View details for DOI 10.1016/j.biomaterials.2019.119342
View details for PubMedID 31326657
-
Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes
ACS COMBINATORIAL SCIENCE
2019; 21 (6): 473–81
View details for DOI 10.1021/acscombsci.9b00035
View details for Web of Science ID 000471211900005
-
The protean world of non-coding RNAs in glioblastoma.
Journal of molecular medicine (Berlin, Germany)
2019
Abstract
Non-coding ribonucleic acids (ncRNAs) are a diverse group of RNA molecules that are mostly not translated into proteins following transcription. We review the role of ncRNAs in the pathobiology of glioblastoma (GBM), and their potential applications for GBM therapy. Significant advances in our understanding of the protean manifestations of ncRNAs have been made, allowing us to better decipher the molecular complexity of GBM. A large number of regulatory ncRNAs appear to have a greater influence on the molecular pathology of GBM than thought previously. Importantly, also, a range of therapeutic approaches are emerging whereby ncRNA-based systems may be used to molecularly target GBM. The most successful of these is RNA interference, and some of these strategies are being evaluated in ongoing clinical trials. However, a number of limitations exist in the clinical translation of ncRNA-based therapeutic systems, such as delivery mechanisms and cytotoxicity; concerted research endeavors are currently underway in an attempt to overcome these. Ongoing and future studies will determine the potential practical role for ncRNA-based therapeutic systems in the clinical management of GBM. These applications may be especially promising, given that current treatment options are limited and prognosis remains poor for this challenging malignancy.
View details for DOI 10.1007/s00109-019-01798-6
View details for PubMedID 31129756
-
Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo.
Theranostics
2019; 9 (9): 2646-2661
Abstract
Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named "Bottle Blue (BBlue)" and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ~50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.
View details for DOI 10.7150/thno.32219
View details for PubMedID 31131059
View details for PubMedCentralID PMC6525985
-
Ligand-activated BRET9 imaging for measuring protein-protein interactions in living mice.
Chemical communications (Cambridge, England)
2019
Abstract
Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.
View details for DOI 10.1039/c9cc07634d
View details for PubMedID 31807738
-
In vitro Determination of Rapamycin-triggered FKBP-FRB Interactions Using a Molecular Tension Probe
ANALYTICAL SCIENCES
2019; 35 (1): 71–78
View details for DOI 10.2116/analsci.18SDP08
View details for Web of Science ID 000457511100010
-
Cell-based biosensors: Recent trends, challenges and future perspectives.
Biosensors & bioelectronics
2019; 141: 111435
Abstract
Existing at the interface of biology and electronics, living cells have been in use as biorecognition elements (bioreceptors) in biosensors since the early 1970s. They are an interesting choice of bioreceptors as they allow flexibility in determining the sensing strategy, are cheaper than purified enzymes and antibodies and make the fabrication relatively simple and cost-effective. And with advances in the field of synthetic biology, microfluidics and lithography, many exciting developments have been made in the design of cell-based biosensors in the last about five years. 3D cell culture systems integrated with electrodes are now providing new insights into disease pathogenesis and physiology, while cardiomyocyte-integrated microelectrode array (MEA) technology is set to be standardized for the assessment of drug-induced cardiac toxicity. From cell microarrays for high-throughput applications to plasmonic devices for anti-microbial susceptibility testing and advent of microbial fuel cell biosensors, cell-based biosensors have evolved from being mere tools for detection of specific analytes to multi-parametric devices for real time monitoring and assessment. However, despite these advancements, challenges such as regeneration and storage life, heterogeneity in cell populations, high interference and high costs due to accessory instrumentation need to be addressed before the full potential of cell-based biosensors can be realized at a larger scale. This review summarizes results of the studies that have been conducted in the last five years toward the fabrication of cell-based biosensors for different applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them.
View details for DOI 10.1016/j.bios.2019.111435
View details for PubMedID 31238280
-
Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo
THERANOSTICS
2019; 9 (9): 2646–61
View details for DOI 10.7150/thno.32219
View details for Web of Science ID 000465285400015
-
Chronic Model of Inflammatory Bowel Disease in IL-10(-/-) Transgenic Mice: Evaluation with Ultrasound Molecular Imaging
THERANOSTICS
2019; 9 (21): 6031–46
View details for DOI 10.7150/thno.37397
View details for Web of Science ID 000481603700001
-
Near-Infrared Bioluminescence Imaging with a through-Bond Energy Transfer Cassette.
Chembiochem : a European journal of chemical biology
2019
Abstract
A coelenterazine (CTZ) analogue emitting near-infrared (NIR) bioluminescence was synthesized for through-bond energy transfer (TBET)-based imaging modalities. The analogue, named Cy5-CTZ, was prepared by conjugating cyanine-5 (Cy5) dye to CTZ through an acetylene linker. This novel derivative is intrinsically fluorescent and emits NIR-shifted luminescence upon reacting with an appropriate luciferase, the Renilla luciferase. This Cy5-CTZ substrate is optically stable in physiological samples and rapidly permeabilize through the plasma membrane into the cytosolic compartment of live cells.
View details for DOI 10.1002/cbic.201900149
View details for PubMedID 30957352
-
Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.
ACS combinatorial science
2019
Abstract
Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was ∼10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.
View details for PubMedID 31034200
-
Restoring guardianship of the genome: Anticancer drug strategies to reverse oncogenic mutant p53 misfolding
CANCER TREATMENT REVIEWS
2018; 71: 19–31
View details for DOI 10.1016/j.ctrv.2018.09.004
View details for Web of Science ID 000452934900003
-
Bioengineered stem cell membrane functionalized nanocarriers for therapeutic targeting of severe hindlimb ischemia
BIOMATERIALS
2018; 185: 360–70
View details for DOI 10.1016/j.biomaterials.2018.08.018
View details for Web of Science ID 000448494500028
-
in vitro determination of rapamycin-triggered FKBP-FRB interactions using a molecular tension probe.
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry
2018
View details for PubMedID 30504653
-
Comparison of cell-based assays to quantify treatment effects of anticancer drugs identifies a new application for Bodipy-L-cystine to measure apoptosis
SCIENTIFIC REPORTS
2018; 8
View details for DOI 10.1038/s41598-018-34696-x
View details for Web of Science ID 000449272100057
-
Comparison of cell-based assays to quantify treatment effects of anticancer drugs identifies a new application for Bodipy-L-cystine to measure apoptosis.
Scientific reports
2018; 8 (1): 16363
Abstract
Cell-based assays that measure anticancer drug effects are essential for evaluating chemotherapeutic agents. Many assays targeting various cellular mechanisms are available, leading to inconsistent results when using different techniques. We critically compared six common assays, as well as a new assay using Bodipy.FL.L-cystine (BFC), to identify the most accurate and reproducible in measuring anticancer drug effects. We tested three common chemotherapies (methotrexate, paclitaxel, and etoposide) in two cell lines (Ln229 and MDA-MB231). Spectroscopic assays such as Cell Titer Blue, and 2',7'-dichlorofluorescin diacetate (DCFDA) yielded a strong drug dose response, especially for paclitaxel and etoposide (R2=0.9). MTT and Calcein-AM fluorescent dye-based assays were less consistent in that regard. Among three flow cytometry assays, Propidium Iodide (PI)-based DNA content analysis and a new BFC-based glutathione-redox (GSH) assay produced drug dose dependent results. Compared to PI, BFC showed a better correlation (R2=0.7-0.9) in depicting live and apoptotic cells. We found that the combination of Cell Titer Blue spectroscopy and BFC flow cytometry assays were most accurate in assessing anticancer drug effects by clear distinction between live and apoptotic cells, independent of drug mechanism of action. We present a new application of BFC as an agent for measuring cellular apoptosis.
View details for PubMedID 30397244
-
Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents
ACS NANO
2018; 12 (11): 10817–32
View details for DOI 10.1021/acsnano.8b02587
View details for Web of Science ID 000451789200022
-
Intracellular microRNA quantification in intact cells: a novel strategy based on reduced graphene oxide-based fluorescence quenching
MRS COMMUNICATIONS
2018; 8 (3): 642–51
View details for DOI 10.1557/mrc.2018.120
View details for Web of Science ID 000448887900003
-
Multi-organ on a chip for personalized precision medicine
MRS COMMUNICATIONS
2018; 8 (3): 652–67
View details for DOI 10.1557/mrc.2018.148
View details for Web of Science ID 000448887900004
-
Intracellular MicroRNA Quantification in Intact Cells: A Novel Strategy based on Reduced Graphene Oxide Based Fluorescence Quenching.
MRS communications
2018; 8 (3): 642-651
Abstract
Nanomaterials have been proposed as key components in biosensing, imaging, and drug-delivery since they offer distinctive advantages over conventional approaches. The unique chemical and physical properties of graphene make it possible to functionalize and develop protein transducers, therapeutic delivery vehicles, and microbial diagnostics. In this study we evaluate reduced graphene oxide (rGO) as a potential nanomaterial for quantification of microRNAs including their structural differentiation in vitro in solution and inside intact cells. Our results provide evidence for the potential use of graphene nanomaterials as a platform for developing devices that can be used for microRNA quantitation as biomarkers for clinical applications.
View details for DOI 10.1557/mrc.2018.120
View details for PubMedID 30705781
View details for PubMedCentralID PMC6349379
-
Bioengineered stem cell membrane functionalized nanocarriers for therapeutic targeting of severe hindlimb ischemia.
Biomaterials
2018; 185: 360–70
Abstract
Bioengineering strategies to enhance the natural targeting function of nanocarriers would expand their therapeutic applications. Here, we designed bioengineered stem cell membrane-functionalized nanocarriers (BSMNCs) harboring C-X-C chemokine receptor type 4 (CXCR4) to achieve robust targeting and also to increase their retention time in ischemic tissue. Stem cell membrane coated nanocarrier (SMNCs) or poly (lactic-co-glycolic acid) (PLGA) nanocarriers (PNCs) and BSMNCs were prepared by functionalizing PNCs with human adipose-derived stem cells (hASCs) membranes and hASCs engineered to overexpress CXCR4-receptor, respectively. The functionalization of PNCs with stem cell membranes derived from hASCs significantly enhance the nanocarrier penetration across endothelial cell barrier compare to PNCs. In addition, stem cell membrane functionalization on PNCs also significantly decreased the nanoparticles uptake in J774 (murine) and THP (human) macrophages respectively from 84% to 76%-29% and 24%. Interestingly, BSMNCs showed much higher level of accumulation in ischemic tissue than SMNCs. Systemic retro-orbital injection of BSMNCs loaded with VEGF into mice with hindlimb ischemia resulted substantially enhancement of blood reperfusion, muscle repair, and limb salvage compared to animals treated with SMNCs loaded with similar concentration of VEGF. The reported strategy could be used to create biocompatible and custom-tailored biomimetic nanoparticles with various hybrid functionalities, which may overcome the limitations of current nanoparticle-based therapeutic and imaging platforms.
View details for PubMedID 30273835
-
Longitudinal assessment of ultrasound-guided complementary microRNA therapy of hepatocellular carcinoma
JOURNAL OF CONTROLLED RELEASE
2018; 281: 19–28
View details for DOI 10.1016/j.jconrel.2018.05.009
View details for Web of Science ID 000436466100003
-
Longitudinal assessment of ultrasound-guided complementary microRNA therapy of hepatocellular carcinoma.
Journal of controlled release : official journal of the Controlled Release Society
2018
Abstract
Hepatocellular carcinoma (HCC) is the second-leading cause of cancer related deaths worldwide and new strategies to efficiently treat HCC are critically needed. The aim of this study was to assess the longitudinal treatment effects of two complementary miRNAs (miRNA-122 and antimiR-21) encapsulated in biodegradable poly lactic-co-glycolic acid (PLGA) - poly ethylene glycol (PEG) nanoparticles (PLGA-PEG-NPs), administered by an ultrasound-guided and microbubble-mediated delivery approach in doxorubicin-resistant and non-resistant human HCC xenografts. Using in vitro assays, we show that repeated miRNA treatments resulted in gradual reduction of HCC cell proliferation and reversal of doxorubicin resistance. Optimized US parameters resulted in a 9-16 fold increase (p = 0.03) in miRNA delivery in vivo in HCC tumors after two US treatments compared to tumors without US treatment. Furthermore, when combined with doxorubicin (10 mg/kg), longitudinal miRNA delivery showed a significant inhibition of tumor growth in both resistant and non-resistant tumors compared to non-treated, and doxorubicin treated controls. We also found that ultrasound-guided miRNA therapy was not only effective in inhibiting HCC tumor growth but also allowed lowering the dose of doxorubicin needed to induce apoptosis. In conclusion, the results of this study suggest that ultrasound-guided and MB-mediated delivery of miRNA-122 and antimiR-21, when combined with doxorubicin, is a highly effective approach to treat resistant HCC while reducing doxorubicin doses needed for treating non-resistant HCC in longitudinal treatment experiments. Further refinement of this strategy could potentially lead to better treatment outcomes for patients with HCC.
View details for PubMedID 29758233
-
Ultrasound-guided delivery of thymidine kinase-nitroreductase dual therapeutic genes by PEGylated-PLGA/PIE nanoparticles for enhanced triple negative breast cancer therapy.
Nanomedicine (London, England)
2018; 13 (9): 1051–66
Abstract
AIM: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. Since no targeted therapy is available, gene-directed enzyme prodrug therapy (GDEPT) could be an attractive strategy for treating TNBC.MATERIALS & METHODS: Polyethylene glycol (PEG)ylated-poly(lactic-co-glycolic acid)/polyethyleneimine nanoparticles (PLGA/PEI NPs) were synthesized and complexed with TK-NTR fusion gene. Ultrasound (US)and microbubble (MB) mediated sonoporation was used for efficient delivery of the TK-NTR-DNA-NP complex to TNBC tumor in vivo for cancer therapy. Therapeutic effect was evaluated by treating TNBC cells in vitro and tumor xenograft in vivo by using prodrugs ganciclovir (GCV) and CB1954.RESULTS: TNBC cells treated with GCV/CB1954 prodrugs after transfection of TK-NTR-DNA by PEGylated-PLGA/PEI NP resulted in high apoptotic-index. US-MB image-guided delivery of TK-NTR-DNA-NP complex displayed significant expression level of TK-NTR protein and showed tumor reduction when treated with GCV/CB1954 prodrugs in TNBC xenograft in vivo.CONCLUSION: US-MB image-guided delivery of TK-NTR gene by PEGylated-PLGA/PEI NPs could be a potential prodrug therapy for TNBC in the clinic.
View details for PubMedID 29790803
-
Ultrasound-guided delivery of thymidine kinase-nitroreductase dual therapeutic genes by PEGylated-PLGA/PEI nanoparticles for enhanced triple negative breast cancer therapy
NANOMEDICINE
2018; 13 (9): 1051–66
View details for DOI 10.2217/nnm-2017-0328
View details for Web of Science ID 000435412500008
-
A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells.
Oncotarget
2018; 9 (30): 21495–511
Abstract
Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.
View details for PubMedID 29765555
-
Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide in vitro and in a mouse model.
Oncotarget
2018; 9 (30): 21478–94
Abstract
Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ. We synthesized both targeted and non-targeted nanoparticles, and characterized them for size, surface charge and encapsulation efficiency of antagomiRs. When using targeted nanoparticles in U87MG and Ln229 GBM cells, we showed higher uptake-associated improvement in sensitivity of these cells to lower concentrations of TMZ in medium. Co-inhibition of microRNA-21 and microRNA-10b reduced the number of viable cells and increased cell cycle arrest at G2/M phase upon TMZ treatment. We found a significant increase in expression of key target genes for microRNA-21 and microRNA-10b upon using targeted versus non-targeted nanoparticles. There was also significant reduction in tumor volume when using TMZ after pre-treatment with loaded nanoparticles in human GBM cell xenografts in mice. In vivo targeted nanoparticles plus different doses of TMZ showed a significant therapeutic response even at the lowest dose of TMZ, indicating that preloading cells with antagomiR-21 and antagomiR-10b increases cellular chemosensitivity towards lower TMZ doses. Future clinical applications of this combination therapy may result in improved GBM response by using lower doses of TMZ and reducing nonspecific treatment side effects.
View details for PubMedID 29765554
-
Cell membrane-coated nanocarriers: the emerging targeted delivery system for cancer theranostics
DRUG DISCOVERY TODAY
2018; 23 (4): 891–99
Abstract
Cancer is a leading cause of death worldwide. The use of nanocarriers (NCs) has generated significant interest to improve cancer therapy by targeted delivery. However, conventional NCs in general lack specificity and have poor biodistribution, resulting in low efficacy in cancer therapy. To circumvent this problem, there has been an increasing focus on cancer cell membrane-coated NCs (CCMCNCs), which can deliver therapeutics directly to tumor cells. CCMCNCs comprise active cancer cell surface adhesive molecules combined with other functional proteins, and offer extended blood circulation with robust cell-specific targeting, ensuring enhanced intratumoral penetration and higher tumor-specific accumulation of NCs. In this review, we discuss the preparation, homologous targeting mechanisms, and application of CCMCNCs in targeted cancer therapy.
View details for PubMedID 29426004
-
Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection.
Nanotheranostics
2018; 2 (4): 306–19
Abstract
Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence "light-up" feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence "light-up" signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.
View details for DOI 10.7150/ntno.26389
View details for PubMedID 29977742
View details for PubMedCentralID PMC6030766
-
Ultrasound and microbubble-mediated targeted delivery of drug loaded nanoparticles to porcine liver
IEEE. 2018
View details for Web of Science ID 000610818700093
-
Restoring guardianship of the genome: Anticancer drug strategies to reverse oncogenic mutant p53 misfolding.
Cancer treatment reviews
2018; 71: 19–31
Abstract
p53 is a transcription factor that activates numerous genes involved in essential maintenance of genetic stability. P53 is the most frequently mutated gene in human cancer. One third of these mutations are structural, resulting in mutant p53 with a disrupted protein conformation. Here we review current progress in a relatively underexplored aspect of p53-targeted drug development, that is, strategies to reactivate wild-type function of misfolded mutant p53. Unfortunately, most p53-targeted drugs are still at early stages of development and many of them are progressing slowly toward clinical implementation. Significant challenges need to be addressed before clinical translation of new anti-misfolding p53-targeted drugs.
View details for PubMedID 30336366
-
Tumor Cell-Derived Extracellular Vesicle-Coated Nanocarriers: An Efficient Theranostic Platform for the Cancer-Specific Delivery of Anti-miR-21 and Imaging Agents.
ACS nano
2018
Abstract
MicroRNAs are critical regulators of cancer initiation, progression, and dissemination. Extensive evidence suggests that the inhibition of over-expressed oncogenic miRNA function can be a robust strategy for anticancer therapy. However, in vivo targeted delivery of miRNA therapeutics to various types of cancers remains a major challenge. Inspired by their natural synthesis and cargo delivery capabilities, researchers have exploited tumor cell-derived extracellular vesicles (TEVs) for the cancer-targeted delivery of therapeutics and theranostics. Here, we investigate a TEV-based nanoplatform for multimodal miRNA delivery and phototherapy treatments as well as the magnetic resonance imaging of cancer. We demonstrated loading of anti-miR-21 that blocks the function of endogenous oncogenic miR-21 over-expressed in cancer cells into and subsequent delivery by TEVs derived from 4T1 cells. We also produced Cy5-anti-miR-21-loaded TEVs from two other cancer cell lines (HepG2 and SKBR3) and confirmed their robust homologous and heterologous transfection efficiency and intracellular Cy5-anti-miR-21 delivery. Additionally, TEV-mediated anti-miR-21 delivery attenuated doxorubicin (DOX) resistance in breast cancer cells with a 3-fold higher cell kill efficiency than in cells treated with DOX alone. We then investigated TEVs as a biomimetic source for the functionalization of gold-iron oxide nanoparticles (GIONs) and demonstrated nanotheranostic properties of TEV-GIONs in vitro. TEV-GIONs demonstrated excellent T2 contrast in in vitro magnetic resonance (MR) imaging and resulted in efficient photothermal effect in 4T1 cells. We also evaluated the biodistribution and theranostic property of anti-miR-21 loaded TEV-GIONs in vivo by labeling with indocyanine green near-infrared dye. We further validated the tumor specific accumulation of TEV-GIONs using MR imaging. Our findings demonstrate that the distribution pattern of the TEV-anti-miR-21-GIONs correlated well with the tumor-targeting capability as well as the activity and efficacy obtained in response to doxorubicin combination treatments. TEVs and TEV-GIONs are promising nanotheranostics for future applications in cancer molecular imaging and therapy.
View details for PubMedID 30346694
-
TARGETED NANOPARTICLE DELIVERY OF THERAPEUTIC ANTIMIR-21 AND ANTIMIR-10B PRESENSITIZES GLIOBLASTOMA TO LOWER EFFECTIVE DOSES OF TEMOZOLOMIDE IN CELLS AND XENOGRAFTS
OXFORD UNIV PRESS INC. 2017: 86
View details for Web of Science ID 000415152501170
-
A MOLECULAR IMAGING BIOSENSOR MONITORS THE EFFECTS OF ANTI-MISFOLDING DRUGS THAT RESTORE MUTANT p53 FUNCTION AND ENHANCE COMBINATION CHEMOTHERAPY FOR GLIOBLASTOMA
OXFORD UNIV PRESS INC. 2017: 62
View details for Web of Science ID 000415152501068
-
A Model-Based Personalized Cancer Screening Strategy for Detecting Early-Stage Tumors Using Blood-Borne Biomarkers
CANCER RESEARCH
2017; 77 (10): 2570-2584
Abstract
An effective cancer blood biomarker screening strategy must distinguish aggressive from non-aggressive tumors at an early, intervenable time. However, for blood-based strategies to be useful, the quality and quantiy of the biomarker shed into the blood and its relationship to tumor growth or progression must be validated. To study how blood biomarker levels correlate with early-stage viable tumor growth in an mouse model of human cancer, we monitored early tumor growth of engineered human ovarian cancer cells (A2780) implanted orthotopically into nude mice. Biomarker shedding was monitored by serial blood sampling, while tumor viability and volume was monitored by bioluminescence imaging and ultrasound imaging. From these metrics we developed a mathematical model of cancer biomarker kinetics in different compartments that accounts for biomarker shedding from tumor and healthy cells, biomarker entry into vasculature, biomarker elimination from plasma and subject-specific tumor growth. We validated the model in a separate set of mice where subject-specific tumor growth rates were accurately predicted. To illustrate clinical translation of this strategy, we allometrically scaled model parameters from mouse to human and used parameters for PSA shedding and prostate cancer. In this manner, we found that blood biomarker sampling data alone was capable of enabling the detection and discrimination of simulated aggressive (2-month tumor doubling time) and non-aggressive (18-month tumor doubling time) tumors as early as 7.2 months and 8.9 years before clinical imaging, respectively. Our model and screening strategy offer broad impact in their applicability to any solid cancer and the biomarkers they shed, thereby allowing a distinction between aggressive vs. non-aggressive tumors using blood biomarker sampling data alone.
View details for DOI 10.1158/0008-5472.CAN-16-2904
View details for Web of Science ID 000401252900003
View details for PubMedID 28283654
-
RRx-001: a systemically non-toxic M2-to-M1 macrophage stimulating and prosensitizing agent in Phase II clinical trials
EXPERT OPINION ON INVESTIGATIONAL DRUGS
2017; 26 (1): 109-119
Abstract
According to Hanahan and Weinberg, cancer manifests as six essential physiologic hallmarks: (1) self-sufficiency in growth signals, (2) insensitivity to growth-inhibitory signals, (3) evasion of programmed cell death, (4) limitless replicative potential, (5) sustained angiogenesis, and (6) invasion and metastasis. As a facilitator of these traits as well as immunosuppression and chemoresistance, the presence of tumor-associated macrophages (TAMs) may serve as the seventh hallmark of cancer. Anticancer agents that successfully reprogram TAMs to target rather than support tumor cells may hold the key to better therapeutic outcomes. Areas covered: This article summarizes the characteristics of the macrophage-stimulating agent RRx-001, a molecular iconoclast, sourced from the aerospace industry, with a particular emphasis on the cell-to-cell transfer mechanism of action (RBCs to TAMs) underlying its antitumor activity as well as its chemo and radioprotective properties, consolidated from various preclinical and clinical studies. Expert opinion: RRx-001 is macrophage-stimulating agent with the potential to synergize with chemotherapy, radiotherapy and immunotherapy while simultaneously protecting normal tissues from their cytotoxic effects. Given the promising indications of activity in multiple tumor types and these normal tissue protective properties, RRx-001 may be used to treat a broad spectrum of malignancies, if it is approved in the future.
View details for DOI 10.1080/13543784.2017.1268600
View details for PubMedID 27935336
-
Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications.
ACS chemical biology
2017
Abstract
Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention both by modifying the N-terminal secretory signal peptide and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.
View details for PubMedID 28767220
-
Gemcitabine and Antisense-microRNA Co-encapsulated PLGA-PEG Polymer Nanoparticles for Hepatocellular Carcinoma Therapy
ACS APPLIED MATERIALS & INTERFACES
2016; 8 (49): 33412-33422
Abstract
Hepatocellular carcinoma (HCC) is highly prevalent, and the third most common cause of cancer-associated deaths worldwide. HCC tumors respond poorly to chemotherapeutic anticancer agents due to inherent and acquired drug resistance, and low drug permeability. Targeted drug delivery systems with significant improvement in therapeutic efficiency are needed for successful HCC therapy. Here, we report the results of a technique optimized for the synthesis and formulation of antisense-miRNA-21 and gemcitabine (GEM) co-encapsulated PEGylated-PLGA nanoparticles (NPs) and their in vitro therapeutic efficacy in human HCC (Hep3B and HepG2) cells. Water-in-oil-in-water (w/o/w) double emulsion method was used to coload antisense-miRNA-21 and GEM in PEGylated-PLGA-NPs. The cellular uptake of NPs displayed time dependent increase of NPs concentration inside the cells. Cell viability analyses in HCC (Hep3B and HepG2) cells treated with antisense-miRNA-21 and GEM co-encapsulated NPs demonstrated a nanoparticle concentration dependent decrease in cell proliferation, and the maximum therapeutic efficiency was attained in cells treated with nanoparticles co-encapsulated with antisense-miRNA-21 and GEM. Flow cytometry analysis showed that control NPs and antisense-miRNA-21-loaded NPs are not cytotoxic to both HCC cell lines, whereas treatment with free GEM and GEM-loaded NPs resulted in ∼9% and ∼15% apoptosis, respectively. Cell cycle status analysis of both cell lines treated with free GEM or NPs loaded with GEM or antisense-miRNA-21 displayed a significant cell cycle arrest at the S-phase. Cellular pathway analysis indicated that Bcl2 expression was significantly upregulated in GEM treated cells, and as expected, PTEN expression was noticeably upregulated in cells treated with antisense-miRNA-21. In summary, we successfully synthesized PEGylated-PLGA nanoparticles co- encapsulated with antisense-miRNA-21 and GEM. These co-encapsulated nanoparticles revealed increased treatment efficacy in HCC cells, compared to cells treated with either antisense-miRNA-21- or GEM-loaded NPs at equal concentration, indicating that down-regulation of endogenous miRNA-21 function can reduce HCC cell viability and proliferation in response to GEM treatment.
View details for DOI 10.1021/acsami.6b08153
View details for Web of Science ID 000389963300008
View details for PubMedID 27960411
View details for PubMedCentralID PMC5206908
-
Molecular Imaging Biosensor Monitors p53 Sumoylation in Cells and Living Mice
ANALYTICAL CHEMISTRY
2016; 88 (23): 11420-11428
Abstract
Small molecule mediated stabilization of p53 tumor suppressor protein through sumoylation is a promising new strategy for improving cancer chemotherapy. A molecular tool that monitors p53 sumoylation status and expedites screening for drugs that enhance p53 sumoylation would be beneficial. We report a molecularly engineered reporter fragment complementation biosensor based on optical imaging of Firefly luciferase (FLuc), to quantitatively image p53 sumoylation and desumoylation in cells and living mice. We initially characterized this biosensor by successfully imaging sumoylation of several target proteins, achieving significant FLuc complementation for ERα (p < 0.01), p53 (p < 0.005), FKBP12 (p < 0.03), ID (p < 0.03), and HDAC1 (p < 0.002). We then rigorously tested the sensitivity and specificity of the biosensor using several variants of p53 and SUMO1, including deletion mutants, and those with modified sequences containing the SUMO-acceptor site of target proteins. Next we evaluated the performance of the biosensor in HepG2 cells by treatment with ginkgolic acid, a drug that reduces p53 sumoylation, as well as trichostatin A, a potential inducer of p53 sumoylation by enhancement of its nuclear export. Lastly, we demonstrated the in vivo utility of this biosensor in monitoring and quantifying the effects of these drugs on p53 sumoylation in living mice using bioluminescence imaging. Adoption of this biosensor in future high throughput drug screening has the important potential to help identify new and repurposed small molecules that alter p53 sumoylation, and to preclinically evaluate candidate anticancer drugs in living animals.
View details for DOI 10.1021/acs.analchem.6b02048
View details for PubMedID 27934110
-
Dynamic Microenvironment Induces Phenotypic Plasticity of Esophageal Cancer Cells Under Flow
SCIENTIFIC REPORTS
2016; 6
Abstract
Cancer microenvironment is a remarkably heterogeneous composition of cellular and non-cellular components, regulated by both external and intrinsic physical and chemical stimuli. Physical alterations driven by increased proliferation of neoplastic cells and angiogenesis in the cancer microenvironment result in the exposure of the cancer cells to elevated levels of flow-based shear stress. We developed a dynamic microfluidic cell culture platform utilizing eshopagael cancer cells as model cells to investigate the phenotypic changes of cancer cells upon exposure to fluid shear stress. We report the epithelial to hybrid epithelial/mesenchymal transition as a result of decreasing E-Cadherin and increasing N-Cadherin and vimentin expressions, higher clonogenicity and ALDH positive expression of cancer cells cultured in a dynamic microfluidic chip under laminar flow compared to the static culture condition. We also sought regulation of chemotherapeutics in cancer microenvironment towards phenotypic control of cancer cells. Such in vitro microfluidic system could potentially be used to monitor how the interstitial fluid dynamics affect cancer microenvironment and plasticity on a simple, highly controllable and inexpensive bioengineered platform.
View details for DOI 10.1038/srep38221
View details for Web of Science ID 000389301200001
View details for PubMedID 27910892
View details for PubMedCentralID PMC5133540
-
A transgenic mouse model expressing an ER alpha folding biosensor reveals the effects of Bisphenol A on estrogen receptor signaling
SCIENTIFIC REPORTS
2016; 6
Abstract
Estrogen receptor-α (ERα) plays an important role in normal and abnormal physiology of the human reproductive system by interacting with the endogenous ligand estradiol (E2). However, other ligands, either analogous or dissimilar to E2, also bind to ERα. This may create unintentional activation of ER signaling in reproductive tissues that can lead to cancer development. We developed a transgenic mouse model that constitutively expresses a firefly luciferase (FLuc) split reporter complementation biosensor (NFLuc-ER-LBDG521T-CFLuc) to simultaneously evaluate the dynamics and potency of ligands that bind to ERα. We first validated this model using various ER ligands, including Raloxifene, Diethylstilbestrol, E2, and 4-hydroxytamoxifen, by employing FLuc-based optical bioluminescence imaging of living mice. We then used the model to investigate the carcinogenic property of Bisphenol A (BPA), an environmental estrogen, by long-term exposure at full and half environmental doses. We showed significant carcinogenic effects on female animals while revealing activated downstream ER signaling as measured by bioluminescence imaging. BPA induced tumor-like outgrowths in female transgenic mice, histopathologically confirmed to be neoplastic and epithelial in origin. This transgenic mouse model expressing an ERα folding-biosensor is useful in evaluation of estrogenic ligands and their downstream effects, and in studying environmental estrogen induced carcinogenesis in vivo.
View details for DOI 10.1038/srep34788
View details for PubMedID 27721470
-
Ultrasound-guided therapeutic modulation of hepatocellular carcinoma using complementary microRNAs.
Journal of controlled release
2016; 238: 272-280
Abstract
Treatment options for patients with hepatocellular carcinoma (HCC) are limited, in particular in advanced and drug resistant HCC. MicroRNAs (miRNA) are non-coding small RNAs that are emerging as novel drugs for the treatment of cancer. The aim of this study was to assess treatment effects of two complementary miRNAs (sense miRNA-122, and antisense antimiR-21) encapsulated in biodegradable poly (lactic-co-glycolic acid) nanoparticles (PLGA-NP), administered by an ultrasound-guided and microbubble-enhanced delivery approach in doxorubicin-resistant and non-resistant human HCC xenografts. Proliferation and invasiveness of human HCC cells after miRNA-122/antimiR-21 and doxorubicin treatment were assessed in vitro. Confocal microscopy and qRT-PCR were used to visualize and quantitate successful intracellular miRNA-loaded PLGA-NP delivery. Up and down-regulation of miRNA downstream targets and multidrug resistance proteins and extent of apoptosis were assessed in vivo in treated human HCC xenografts in mice. Compared to single miRNA therapy, combination therapy with the two complementary miRNAs resulted in significantly (P<0.05) stronger decrease in cell proliferation, invasion, and migration of HCC cells as well as higher resensitization to doxorubicin. Ultrasound-guided delivery significantly increased in vivo miRNA-loaded PLGA-NP delivery in human HCC xenografts compared to control conditions by 5-9 fold (P<0.001). miRNA-loaded PLGA-NP were internalized in HCC cells and anti-apoptotic proteins were down regulated with apoptosis in ~27% of the tumor volume of doxorubicin-resistant human HCC after a single treatment with complementary miRNAs and doxorubicin. Thus, ultrasound-guided delivery of complementary miRNAs is highly efficient in the treatment of doxorubicin- resistant and non-resistant HCC. Further development of this new treatment approach could aid in better treatment of patients with HCC.
View details for DOI 10.1016/j.jconrel.2016.08.005
View details for PubMedID 27503707
View details for PubMedCentralID PMC5185600
-
Tailored Nanoparticle Codelivery of antimiR-21 and antimiR-10b Augments Glioblastoma Cell Kill by Temozolomide: Toward a "Personalized" Anti-microRNA Therapy.
Molecular pharmaceutics
2016; 13 (9): 3164-3175
Abstract
Glioblastoma remains an aggressive brain malignancy with poor prognosis despite advances in multimodal therapy that include standard use of Temozolomide. MicroRNA-21 (miR-21) and microRNA-10b (miR-10b) are oncomiRs overexpressed in glioblastoma, promoting many aspects of cancer biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 (antimiR-21) and antisense miR-10b (antimiR-10b) might beneficially knockdown endogenous miR-21 and miR-10b function and reprogram cells prior to Temozolomide treatment. PLGA nanoparticles were effective in intracellular delivery of encapsulated oligonucleotides. Concentrations of delivered antimiR-21 and antimiR-10b were optimized and specifically tailored to copy numbers of intracellular endogenous microRNAs. Coinhibition of miR-21 and miR-10b significantly reduced the number of viable cells (by 24%; p < 0.01) and increased (2.9-fold) cell cycle arrest at G2/M phase upon Temozolomide treatment in U87 MG cells. Cell-tailored nanoparticle-assisted concurrent silencing of miR-21 and miR-10b prior to Temozolomide treatment is an effective molecular therapeutic strategy in cell culture, warranting the need for further studies prior to future in vivo "personalized" medicine applications.
View details for DOI 10.1021/acs.molpharmaceut.6b00388
View details for PubMedID 27508339
-
Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.
Radiology
2016; 280 (3): 826-836
Abstract
Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.
View details for DOI 10.1148/radiol.2016151150
View details for PubMedID 27332865
-
Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.
Radiology
2016; 280 (3): 815-825
Abstract
Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.
View details for DOI 10.1148/radiol.2016140049
View details for PubMedID 27308957
-
Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.
Bioorganic & medicinal chemistry
2016; 24 (16): 3378-3386
Abstract
The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS.
View details for DOI 10.1016/j.bmc.2016.05.011
View details for PubMedID 27312421
-
Noninvasive Monitoring of the Mitochondrial Function in Mesenchymal Stromal Cells
MOLECULAR IMAGING AND BIOLOGY
2016; 18 (4): 510-518
Abstract
Mitochondria are a gatekeeper of cell survival and mitochondrial function can be used to monitor cell stress. Here we validate a pathway-specific reporter gene to noninvasively image the mitochondrial function of stem cells.We constructed a mitochondrial sensor with the firefly luciferase (Fluc) reporter gene driven by the NQO1 enzyme promoter. The sensor was introduced in stem cells and validated in vitro and in vivo, in a mouse model of myocardial ischemia/reperfusion (IR).The sensor activity showed an inverse relationship with mitochondrial function (R (2) = -0.975, p = 0.025) and showed specificity and sensitivity for mitochondrial dysfunction. In vivo, NQO1-Fluc activity was significantly higher in IR animals vs. controls, indicative of mitochondrial dysfunction, and was corroborated by ex vivo luminometry.Reporter gene imaging allows assessment of the biology of transplanted mesenchymal stromal cells (MSCs), providing important information that can be used to improve the phenotype and survival of transplanted stem cells.
View details for DOI 10.1007/s11307-016-0929-x
View details for Web of Science ID 000379191800006
View details for PubMedID 26865378
View details for PubMedCentralID PMC4966544
-
A molecular imaging biosensor detects in vivo protein folding and misfolding
JOURNAL OF MOLECULAR MEDICINE-JMM
2016; 94 (7): 799-808
Abstract
Aberrant protein folding represents the molecular basis of many important human diseases. Although the discovery of new anti-misfolding drugs is a major priority in molecular therapeutics, there is currently no generalizable protein folding assay for use in cell-based high throughput screening (HTS) of chemical libraries, or for in vivo imaging. We molecularly engineered a bioluminescence-based biosensor composed of rationally split Firefly luciferase reporter fragments flanking a test protein, and used this in a protein-fragment complementation assay to quantitatively measure folding of the test protein. We comprehensively validated this biosensor in vitro, in cells, and by optically imaging protein folding and misfolding in living mice using several test proteins including enhanced green fluorescent protein, Renilla luciferase, Gaussia luciferase, and SIRT1. Applications of this novel biosensor are potentially far-reaching in both cell-based HTS approaches to discover new anti-misfolding drugs, and when using the same biosensor in validation studies of drug candidates in small animal models.Novel anti-misfolding drugs are needed as molecular therapeutics for many diseases. We developed first in vivo imaging protein folding biosensor to aid drug discovery. Biosensor created by flanking a test protein with rationally split Firefly luciferase. Biosensor validated by detecting folding of test proteins EGFP, Rluc, Gluc, and SIRT1. Generalizable molecular biosensor for translational applications in drug screening.
View details for DOI 10.1007/s00109-016-1437-9
View details for PubMedID 27277823
-
GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers
MOLECULAR ENDOCRINOLOGY
2016; 30 (5): 504-517
Abstract
GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.
View details for DOI 10.1210/me.2015-1319
View details for Web of Science ID 000377209900003
View details for PubMedID 27003442
-
Folate Receptor-Targeted Polymeric Micellar Nanocarriers for Delivery of Orlistat as a Repurposed Drug against Triple-Negative Breast Cancer.
Molecular cancer therapeutics
2016; 15 (2): 221-231
Abstract
Triple negative breast cancer (TNBC) is a recalcitrant malignancy with no available targeted therapy. Off target effects and poor bioavailability of the FDA approved anti-obesity drug orlistat hinder its clinical translation as a repurposed new drug against TNBC. Here we demonstrate a newly engineered drug formulation for packaging orlistat tailored to TNBC treatment. We synthesized TNBC-specific folate receptor targeted micellar nanoparticles (NPs) carrying orlistat, which improved the solubility (70-80 μg/ml) of this water insoluble drug. The targeted NPs also improved the delivery and bioavailability of orlistat to MDA-MB-231 cells in culture and to tumor xenografts in nude mouse model. We prepared HEA-EHA copolymer micellar NPs by copolymerization of 2-hydroxyethylacrylate (HEA) and 2-ethylhexylacrylate (EHA), and functionalized them with folic acid and an imaging dye. Fluorescence activated cell sorting (FACS) analysis of TNBC cells indicated a dose dependent increase in apoptotic populations in cells treated with free orlistat, orlistat NPs, and folate-receptor targeted Fol-HEA-EHA-orlistat NPs in which Fol-HEA-EHA-orlistat NPs showed significantly higher cytotoxicity than free orlistat. In vitro analysis data demonstrated significant apoptosis at nanomolar concentrations in cells activated through caspase 3 and PARP inhibition. In vivo analysis demonstrated significant antitumor effects in living mice after targeted treatment of tumors, and confirmed by fluorescence imaging. Moreover, Folate receptor targeted Fol-DyLight747-orlistat NPs treated mice exhibited significantly higher reduction in tumor volume compared to control group. Taken together, these results indicate that orlistat packaged in HEA-b-EHA micellar NPs is a highly promising new drug formulation for TNBC therapy.
View details for DOI 10.1158/1535-7163.MCT-15-0579
View details for PubMedID 26553061
-
Orlistat and antisense-miRNA-loaded PLGA-PEG nanoparticles for enhanced triple negative breast cancer therapy.
Nanomedicine
2016; 11 (3): 235-247
Abstract
This study explores the use of hydrophilic poly(ethylene glycol)-conjugated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NPs) as delivery system to improve the antitumor effect of antiobesity drug orlistat for triple-negative breast cancer (TNBC) therapy by improving its bioavailability.PLGA-PEG-NPs were synthesized by emulsion-diffusion-evaporation method, and the experiments were conducted in vitro in MDA-MB-231 and SKBr3 TNBC and normal breast fibroblast cells.Delivery of orlistat via PLGA-PEG-NPs reduced its IC50 compared with free orlistat. Combined treatment of orlistat-loaded NPs and doxorubicin or antisense-miR-21-loaded NPs significantly enhanced apoptotic effect compared with independent doxorubicin, anti-miR-21-loaded NPs, orlistat-loaded NPs or free orlistat treatments.We demonstrate that orlistat in combination with antisense-miR-21 or current chemotherapy holds great promise as a novel and versatile treatment agent for TNBC.
View details for DOI 10.2217/nnm.15.193
View details for PubMedID 26787319
-
Pterostilbene Ameliorates Streptozotocin-Induced Diabetes through Enhancing Antioxidant Signaling Pathways Mediated by Nrf2.
Chemical research in toxicology
2016; 29 (1): 47-57
Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) remains a master regulator of cytoprotective and antioxidant genes. In this study, we investigated the antidiabetic role of pterostilbene (PTS) in streptozotocin (STZ)-induced diabetic model through Nrf2-mediated antioxidant mechanisms. The ability of PTS to activate Nrf2 in MIN6 cells was assessed by dissociation of the Nrf2-Keap1 complex at different time points and by expression of ARE-driven downstream target genes of Nrf2. Immunoblot experiments examining Nrf2 activation and phosphorylation indicated that it conferred cytoprotection against STZ-induced cellular damage. In STZ-induced diabetic mice, PTS administration significantly decreased blood glucose levels through the improvement of insulin secretion. In addition, we also observed insulin-positive cells with recovered islet architecture in the pancreas of STZ-induced diabetic mice after treatment with PTS. The activation of Nrf2 and expression of its downstream target genes were observed upon PTS treatment, thereby reducing oxidative damage to pancreas. Furthermore, PTS treatment significantly reverted the abundance of key glucose metabolism enzymes, such as hexokinase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and fructose-1,6-bisphosphatase, to near-normal levels in liver tissue of STZ-induced diabetic mice. These results clearly indicate that PTS maintains glucose homeostasis, suggesting the possibility that it is a future candidate for use in diabetes management.
View details for DOI 10.1021/acs.chemrestox.5b00378
View details for PubMedID 26700463
-
Temozolomide-loaded PLGA nanoparticles to treat glioblastoma cells: a biophysical and cell culture evaluation
NEUROLOGICAL RESEARCH
2016; 38 (1): 51-59
Abstract
Current chemotherapies for brain glioblastoma do not achieve sufficient drug concentrations within tumors. Polymeric nanoparticles have useful physicochemical properties that make them promising as nanoparticle platforms for glioblastoma drug delivery. Poly[lactic-co-glycolic acid] (PLGA) nanoparticles encapsulating temozolomide (TMZ) could improve localized delivery and sustained drug release to glioblastomas.We investigated three different procedures to encapsulate TMZ within PLGA nanoparticles. We studied the biophysical features of optimized nanocarriers, including their size, shape, surface properties, and release characteristics of TMZ. We evaluated the antiproliferative and cytotoxic effects of TMZ-loaded PLGA nanoparticles on U87 MG glioblastoma cells.A single emulsion technique using a TMZ saturated aqueous phase produced nanoparticles ≤200 nm in size allowing a maximal drug loading of 4.4% w/w of polymer. There was a bi-phasic drug release pattern, with 80% of TMZ released within the first 6 h. Nanoparticles accumulated in the cytoplasm after effective endocytosis. There was no significant difference in cytotoxic effect of TMZ encapsulated within PLGA nanoparticles and free TMZ.PLGA nanoparticles are not suitable as carriers of TMZ for glioblastoma drug delivery on account of the overall high IC50 values of glioblastoma cells to TMZ and poor loading and encapsulation efficiencies. Further biotechnological developments aimed at improving the loading of TMZ in PLGA nanoparticles or co-delivery of small molecule sensitizers to improve the response of human glioblastoma cells to TMZ are required for this approach to be considered and optimized for future clinical translation.
View details for DOI 10.1080/01616412.2015.1133025
View details for PubMedID 26905383
-
Imaging Histone Methylations in Living Animals
BIOLUMINESCENCE, 3 EDITION
2016; 1461: 203-215
View details for DOI 10.1007/978-1-4939-3813-1_17
View details for Web of Science ID 000685739700018
-
Theranostic Imaging of Cancer Gene Therapy
BIOLUMINESCENCE, 3 EDITION
2016; 1461: 241-254
View details for DOI 10.1007/978-1-4939-3813-1_20
View details for Web of Science ID 000685739700021
-
Pterostilbene Ameliorates Streptozotocin-Induced Diabetes through Enhancing Antioxidant Signaling Pathways Mediated by Nrf2
CHEMICAL RESEARCH IN TOXICOLOGY
2016; 29 (1): 47-57
Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) remains a master regulator of cytoprotective and antioxidant genes. In this study, we investigated the antidiabetic role of pterostilbene (PTS) in streptozotocin (STZ)-induced diabetic model through Nrf2-mediated antioxidant mechanisms. The ability of PTS to activate Nrf2 in MIN6 cells was assessed by dissociation of the Nrf2-Keap1 complex at different time points and by expression of ARE-driven downstream target genes of Nrf2. Immunoblot experiments examining Nrf2 activation and phosphorylation indicated that it conferred cytoprotection against STZ-induced cellular damage. In STZ-induced diabetic mice, PTS administration significantly decreased blood glucose levels through the improvement of insulin secretion. In addition, we also observed insulin-positive cells with recovered islet architecture in the pancreas of STZ-induced diabetic mice after treatment with PTS. The activation of Nrf2 and expression of its downstream target genes were observed upon PTS treatment, thereby reducing oxidative damage to pancreas. Furthermore, PTS treatment significantly reverted the abundance of key glucose metabolism enzymes, such as hexokinase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, and fructose-1,6-bisphosphatase, to near-normal levels in liver tissue of STZ-induced diabetic mice. These results clearly indicate that PTS maintains glucose homeostasis, suggesting the possibility that it is a future candidate for use in diabetes management.
View details for DOI 10.1021/acs.chemrestox.5b00378
View details for Web of Science ID 000368562400005
-
Imaging Histone Methylations in Living Animals.
Methods in molecular biology (Clifton, N.J.)
2016; 1461: 203-215
Abstract
Histone modifications (methylation, acetylation, phosphorylation, sumoylation, etc.,) are at the heart of cellular regulatory mechanisms, which control expression of genes in an orderly fashion and control the entire cellular regulatory networks. Histone lysine methylation has been identified as one of the several posttranslational histone modifications that plays crucial role in regulating gene expressions in facultative heterochromatic DNA regions while maintaining structural integrity in constitutive heterochromatic DNA regions. Since histone methylation is dysregulated in various cellular diseases, it has been considered a potential therapeutic target for drug development. Currently there is no simple method available to screen and preclinically evaluate drugs modulating this cellular process, we recently developed two different methods by adopting reporter gene technology to screen drugs and to preclinically evaluate them in living animals. Method detects and quantitatively monitors the level of histone methylations in intact cells, is of a prerequisite to screen small molecules that modulate histone lysine methylation. Here, we describe two independent optical imaging sensors developed to image histone methylations in cells and in living animals. Since we used standard PCR-based cloning strategies to construct different plasmid vectors shown in this chapter, we are not providing any details regarding the construction methods, instead, we focus on detailing various methods used for measuring histone methylation-assisted luciferase quantitation in cells and imaging in living animals.
View details for DOI 10.1007/978-1-4939-3813-1_17
View details for PubMedID 27424907
-
Theranostic Imaging of Cancer Gene Therapy.
Methods in molecular biology (Clifton, N.J.)
2016; 1461: 241-254
Abstract
Gene-directed enzyme prodrug therapy (GDEPT) is a promising therapeutic approach for treating cancers of various phenotypes. This strategy is independent of various other chemotherapeutic drugs used for treating cancers where the drugs are mainly designed to target endogenous cellular mechanisms, which are different in various cancer subtypes. In GDEPT an external enzyme, which is different from the cellular proteins, is expressed to convert the injected prodrug in to a toxic metabolite, that normally kill cancer cells express this protein. Theranostic imaging is an approach used to directly monitor the expression of these gene therapy enzymes while evaluating therapeutic effect. We recently developed a dual-GDEPT system where we combined mutant human herpes simplex thymidine kinase (HSV1sr39TK) and E. coli nitroreductase (NTR) enzyme, to improve therapeutic efficiency of cancer gene therapy by simultaneously injecting two prodrugs at a lower dose. In this approach we use two different prodrugs such as ganciclovir (GCV) and CB1954 to target two different cellular mechanisms to kill cancer cells. The developed dual GDEPT system was highly efficacious than that of either of the system used independently. In this chapter, we describe the complete protocol involved for in vitro and in vivo imaging of therapeutic cancer gene therapy evaluation.
View details for DOI 10.1007/978-1-4939-3813-1_20
View details for PubMedID 27424910
-
Nanoparticle-Delivered Antisense MicroRNA-21 Enhances the Effects of Temozolomide on Glioblastoma Cells
MOLECULAR PHARMACEUTICS
2015; 12 (12): 4509-4517
Abstract
Glioblastoma (GBM) generally exhibits high IC50 values for its standard drug treatment, temozolomide (TMZ). MicroRNA-21 (miR-21) is an oncomiR overexpressed in GBM, thus controlling important aspects of glioma biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 to glioblastoma cells might beneficially knock down endogenous miR-21 prior to TMZ treatment. PLGA nanoparticles encapsulating antisense miR-21 were effective in intracellular delivery and sustained silencing (p < 0.01) of miR-21 function in U87 MG, LN229, and T98G cells. Prior antisense miR-21 delivery significantly reduced the number of viable cells (p < 0.001), and increased (1.6-fold) cell cycle arrest at G2/M phase upon TMZ treatment in U87 MG cells. There was overexpression of the miR-21 target genes PTEN (by 67%) and caspase-3 (by 15%) upon cotreatment. This promising PLGA nanoparticle-based platform for antisense miR-21 delivery to GBM is an effective cotherapeutic strategy in cell culture, warranting the need for further studies prior to future clinical translation.
View details for DOI 10.1021/acs.molpharmaceut.5b00694
View details for PubMedID 26559642
-
3-D tumor models.
Materials today
2015; 18 (10): 539-553
Abstract
The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell-cell, cell-matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing.
View details for DOI 10.1016/j.mattod.2015.05.002
View details for PubMedID 28458612
-
Engineering cancer microenvironments for in vitro 3-D tumor models
MATERIALS TODAY
2015; 18 (10): 539-553
Abstract
The natural microenvironment of tumors is composed of extracellular matrix (ECM), blood vasculature, and supporting stromal cells. The physical characteristics of ECM as well as the cellular components play a vital role in controlling cancer cell proliferation, apoptosis, metabolism, and differentiation. To mimic the tumor microenvironment outside the human body for drug testing, two-dimensional (2-D) and murine tumor models are routinely used. Although these conventional approaches are employed in preclinical studies, they still present challenges. For example, murine tumor models are expensive and difficult to adopt for routine drug screening. On the other hand, 2-D in vitro models are simple to perform, but they do not recapitulate natural tumor microenvironment, because they do not capture important three-dimensional (3-D) cell-cell, cell-matrix signaling pathways, and multi-cellular heterogeneous components of the tumor microenvironment such as stromal and immune cells. The three-dimensional (3-D) in vitro tumor models aim to closely mimic cancer microenvironments and have emerged as an alternative to routinely used methods for drug screening. Herein, we review recent advances in 3-D tumor model generation and highlight directions for future applications in drug testing.
View details for DOI 10.1016/j.mattod.2015.05.002
View details for Web of Science ID 000366576000019
View details for PubMedCentralID PMC5407188
-
Monitoring the Antioxidant Mediated Chemosensitization and ARE-Signaling in Triple Negative Breast Cancer Therapy
PLOS ONE
2015; 10 (11)
Abstract
Chemotherapy often fails due to cellular detoxifying mechanisms, including phase-II enzymes. Activation of Nrf2-Keap1 pathway induces phase-II enzymes expression through ARE-signaling and prevents cancer development. Nrf2-overexpression in cancer cells results in chemo- and/or radioresistance. This necessitates understanding of Nrf2-regulation, and identification of Nrf2 activators/inhibitors sensitizing cancer cells to improve chemotherapy. N-terminal 435-amino acids of Nrf2 are crucial for Keap1 binding during ubiquitination. Identification of a minimum Nrf2-domain required for Keap1 binding without altering endogenous ARE-signaling would be a novel tool to study Nrf2-signaling. Current study developed firefly-luciferase reporter fusion with N-terminal Nrf2-domain of different lengths and examined its response to Nrf2-activators in cells. The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells. We used MDA-MB231 cells expressing this particular construct for studying antioxidant induced Nrf2-activation and chemosensitization in triple-negative breast cancer therapy. While antioxidant EGCG showed chemosensitization of MDA-MB231 cells to cisplatin by activating Nrf2-ARE signaling, PTS, another antioxidant showed chemoprotection. Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment. The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.
View details for DOI 10.1371/journal.pone.0141913
View details for Web of Science ID 000364298400100
View details for PubMedID 26536456
View details for PubMedCentralID PMC4633093
-
Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001.
Oncotarget
2015; 6 (25): 21547-21556
Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. RRx-001 is a new anticancer agent that generates reactive oxygen and nitrogen species, and leads to epigenetic alterations in cancer cells. Here we report the RRx-001 mediated nuclear translocation of Nrf2 and the activation of expression of its downstream enzymes HO-1 and NQO1 in tumor cells. Inhibition of intrinsic Nrf2 expression by Nrf2-specific siRNA increased cell sensitivity to RRx-001. Molecular imaging of tumor cells co-expressing pARE-Firefly luciferase and pCMV-Renilla luciferase-mRFP in vitro and in vivo in mice revealed that RRx-001 significantly increased ARE-FLUC signal in cells in a dose- and time-dependent manner, suggesting that RRx-001 is an effective activator of the Nrf2-ARE signaling pathway. The pre-treatment level of ARE-FLUC signal in cells, reflecting basal activity of Nrf2, negatively correlated with the tumor response to RRx-001. The results support the concept that RRx-001 activates Nrf2-ARE antioxidant signaling pathways in tumor cells. Hence measurement of Nrf2-mediated activation of downstream target genes through ARE signaling may constitute a useful molecular biomarker for the early prediction of response to RRx-001 treatment, and thereby guide therapeutic decision-making.
View details for PubMedID 26280276
-
Development of a High-Throughput Molecular Imaging-Based Orthotopic Hepatocellular Carcinoma Model.
Cureus
2015; 7 (6)
Abstract
We have developed a novel orthotopic rat hepatocellular (HCC) model and have assessed the ability to use bioluminescence imaging (BLI), positron emission tomography (PET), and ultrasound for early tumor detection and monitoring of disease progression. Briefly, rat HCC cells were stably transfected with click beetle red as a reporter gene for BLI. Tumor cells were injected under direct visualization into the left or middle lobe of the liver in 37 rats. In six animals, serial PET, BLI, and ultrasound imaging were performed at 10-time points in 28 days. The remainder of the animals underwent PET imaging at 14 days. Tumor implantation was successful in 34 of 37 animals (91.9%). In the six animals that underwent serial imaging, tumor formation was first detected with BLI on Day 4 with continued increase through Day 21, and hypermetabolic activity on PET was first noted on Days 14-15 with continued increase through Day 28. PET activity was seen on Day 14 in the 28 other animals that demonstrated tumor development. Anatomic tumor formation was detected with ultrasound at Days 10-12 with continued growth through Day 28. The first metastases were detected by PET after Day 24. We have successfully developed and validated a novel orthotopic HCC small animal model that permits longitudinal assessment of change in tumor size using molecular imaging techniques. BLI is the most sensitive imaging method for detection of early tumor formation and growth. This model permits high-throughput in vivo evaluation of image-guided therapies.
View details for DOI 10.7759/cureus.281
View details for PubMedID 26180705
View details for PubMedCentralID PMC4494575
-
Formulation of Anti-miR-21 and 4-Hydroxytamoxifen Co-loaded Biodegradable Polymer Nanoparticles and Their Antiproliferative Effect on Breast Cancer Cells.
Molecular pharmaceutics
2015; 12 (6): 2080-2092
Abstract
Breast cancer is the second leading cause of cancer-related death in women. The majority of breast tumors are estrogen receptor-positive (ER+) and hormone-dependent. Neoadjuvant anti-estrogen therapy has been widely employed to reduce tumor mass prior to surgery. Tamoxifen is a broadly used anti-estrogen for early and advanced ER+ breast cancers in women and the most common hormone treatment for male breast cancer. 4-Hydroxytamoxifen (4-OHT) is an active metabolite of tamoxifen that functions as an estrogen receptor antagonist and displays higher affinity for estrogen receptors than that of tamoxifen and its other metabolites. MicroRNA-21 (miR-21) is a small noncoding RNA of 23 nucleotides that regulates several apoptotic and tumor suppressor genes and contributes to chemoresistance in numerous cancers, including breast cancer. The present study investigated the therapeutic potential of 4-OHT and anti-miR-21 coadministration in an attempt to combat tamoxifen resistance, a common problem often encountered in anti-estrogen therapy. A biodegradable poly(d,l-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) copolymer was utilized as a carrier to codeliver 4-OHT and anti-miR-21 to ER+ breast cancer cells. 4-OHT and anti-miR-21 co-loaded PLGA-b-PEG nanoparticles (NPs) were developed using emulsion-diffusion evaporation (EDE) and water-in-oil-in-water (w/o/w) double emulsion methods. The EDE method was found to be best method for 4-OHT loading, and the w/o/w method proved to be more effective for coloading NPs with anti-miR-21 and 4-OHT. The optimal NPs, which were prepared using the double emulsion method, were evaluated for their antiproliferative and apoptotic effects against MCF7, ZR-75-1, and BT-474 human breast cancer cells as well as against 4T1 mouse mammary carcinoma cells. We demonstrated that PLGA-b-PEG NP encapsulation significantly extended 4-OHT's stability and biological activity compared to that of free 4-OHT. MTT assays indicated that treatment of MCF7 cells with 4-OHT-anti-miR-21 co-loaded NPs resulted in dose-dependent antiproliferative effects at 24 h, which was significantly higher than what was achieved with free 4-OHT at 48 and 72 h post-treatment. Cell proliferation analysis showed that 4-OHT and anti-miR-21 co-loaded NPs significantly inhibited MCF-7 cell growth compared to that of free 4-OHT (1.9-fold) and untreated cells (5.4-fold) at 1 μM concentration. The growth rate of MCF7 cells treated with control NPs or NPs loaded with anti-miR-21 showed no significant difference from that of untreated cells. These findings demonstrate the utility of the PLGA-b-PEG polymer NPs as an effective nanocarrier for co-delivery of anti-miR-21 and 4-OHT as well as the potential of this drug combination for use in the treatment of ER+ breast cancer.
View details for DOI 10.1021/mp500852s
View details for PubMedID 25880495
-
Development of a High-Throughput Molecular Imaging-Based Orthotopic Hepatocellular Carcinoma Model
CUREUS
2015; 7 (6)
View details for DOI 10.7759/cureus.281
View details for Web of Science ID 000453603500009
-
Ultrasound-guided delivery of microRNA loaded nanoparticles into cancer
JOURNAL OF CONTROLLED RELEASE
2015; 203: 99-108
Abstract
Ultrasound induced microbubble cavitation can cause enhanced permeability across natural barriers of tumors such as vessel walls or cellular membranes, allowing for enhanced therapeutic delivery into the target tissues. While enhanced delivery of small (<1nm) molecules has been shown at acoustic pressures below 1MPa both in vitro and in vivo, the delivery efficiency of larger (>100nm) therapeutic carriers into cancer remains unclear and may require a higher pressure for sufficient delivery. Enhanced delivery of larger therapeutic carriers such as FDA approved pegylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NP) has significant clinical value because these nanoparticles have been shown to protect encapsulated drugs from degradation in the blood circulation and allow for slow and prolonged release of encapsulated drugs at the target location. In this study, various acoustic parameters were investigated to facilitate the successful delivery of two nanocarriers, a fluorescent semiconducting polymer model drug nanoparticle as well as PLGA-PEG-NP into human colon cancer xenografts in mice. We first measured the cavitation dose produced by various acoustic parameters (pressure, pulse length, and pulse repetition frequency) and microbubble concentration in a tissue mimicking phantom. Next, in vivo studies were performed to evaluate the penetration depth of nanocarriers using various acoustic pressures, ranging between 1.7 and 6.9MPa. Finally, a therapeutic microRNA, miR-122, was loaded into PLGA-PEG-NP and the amount of delivered miR-122 was assessed using quantitative RT-PCR. Our results show that acoustic pressures had the strongest effect on cavitation. An increase of the pressure from 0.8 to 6.9MPa resulted in a nearly 50-fold increase in cavitation in phantom experiments. In vivo, as the pressures increased from 1.7 to 6.9MPa, the amount of nanoparticles deposited in cancer xenografts was increased from 4- to 14-fold, and the median penetration depth of extravasated nanoparticles was increased from 1.3-fold to 3-fold, compared to control conditions without ultrasound, as examined on 3D confocal microscopy. When delivering miR-122 loaded PLGA-PEG-NP using optimal acoustic settings with minimum tissue damage, miR-122 delivery into tumors with ultrasound and microbubbles was 7.9-fold higher compared to treatment without ultrasound. This study demonstrates that ultrasound induced microbubble cavitation can be a useful tool for delivery of therapeutic miR loaded nanocarriers into cancer in vivo.
View details for DOI 10.1016/j.jconrel.2015.02.018
View details for Web of Science ID 000351696600011
View details for PubMedID 25687306
-
Polymer Nanoparticles Mediated Codelivery of AntimiR-10b and AntimiR-21 for Achieving Triple Negative Breast Cancer Therapy
ACS NANO
2015; 9 (3): 2290-2302
Abstract
The current study shows the therapeutic outcome achieved in triple negative breast cancer (TNBC) by simultaneously antagonizing miR-21-induced antiapoptosis and miR-10b-induced metastasis, using antisense-miR-21-PS and antisense-miR-10b-PS delivered by polymer nanoparticles (NPs). We synthesized the antisense-miR-21 and antisense-miR-10b loaded PLGA-b-PEG polymer NPs and evaluated their cellular uptake, serum stability, release profile, and the subsequent synchronous blocking of endogenous miR-21 and miR-10b function in TNBC cells in culture, and tumor xenografts in living animals using molecular imaging. Results show that multitarget antagonization of endogenous miRNAs could be an efficient strategy for targeting metastasis and antiapoptosis in the treatment of metastatic cancer. Targeted delivery of antisense-miR-21 and antisense-miR-10b coloaded urokinase plasminogen activator receptor (uPAR) targeted polymer NPs treated mice showed substantial reduction in tumor growth at very low dose of 0.15 mg/kg, compared to the control NPs treated mice and 40% reduction in tumor growth compared to scramble peptide conjugated NPs treated mice, thus demonstrating a potential new therapeutic option for TNBC.
View details for DOI 10.1021/nn507465d
View details for PubMedID 25652012
-
Genetically encoded molecular biosensors to image histone methylation in living animals.
Analytical chemistry
2015; 87 (2): 892-899
Abstract
Post-translational addition of methyl groups to the amino terminal tails of histone proteins regulates cellular gene expression at various stages of development and the pathogenesis of cellular diseases, including cancer. Several enzymes that modulate these post-translational modifications of histones are promising targets for development of small molecule drugs. However, there is no promising real-time histone methylation detection tool currently available to screen and validate potential small molecule histone methylation modulators in small animal models. With this in mind, we developed genetically encoded molecular biosensors based on the split-enzyme complementation approach for in vitro and in vivo imaging of lysine 9 (H3-K9 sensor) and lysine 27 (H3-K27 sensor) methylation marks of histone 3. These methylation sensors were validated in vitro in HEK293T, HepG2, and HeLa cells. The efficiency of the histone methylation sensor was assessed by employing methyltransferase inhibitors (Bix01294 and UNC0638), demethylase inhibitor (JIB-04), and siRNA silencing at the endogenous histone K9-methyltransferase enzyme level. Furthermore, noninvasive bioluminescence imaging of histone methylation sensors confirmed the potential of these sensors in monitoring histone methylation status in response to histone methyltransferase inhibitors in living animals. Experimental results confirmed that the developed H3-K9 and H3-K27 sensors are specific and sensitive to image the drug-induced histone methylation changes in living animals. These novel histone methylation sensors can facilitate the in vitro screening and in vivo characterization of new histone methyltransferase inhibitors and accelerate the pace of introduction of epigenetic therapies into the clinic.
View details for DOI 10.1021/ac502629r
View details for PubMedID 25506787
View details for PubMedCentralID PMC4303335
-
Degron protease blockade sensor to image epigenetic histone protein methylation in cells and living animals.
ACS chemical biology
2015; 10 (1): 165-174
Abstract
Lysine methylation of histone H3 and H4 has been identified as a promising therapeutic target in treating various cellular diseases. The availability of an in vivo assay that enables rapid screening and preclinical evaluation of drugs that potentially target this cellular process will significantly expedite the pace of drug development. This study is the first to report the development of a real-time molecular imaging biosensor (a fusion protein, [FLuc2]-[Suv39h1]-[(G4S)3]-[H3-K9]-[cODC]) that can detect and monitor the methylation status of a specific histone lysine methylation mark (H3-K9) in live animals. The sensitivity of this sensor was assessed in various cell lines, in response to down-regulation of methyltransferase EHMT2 by specific siRNA, and in nude mice with lysine replacement mutants. In vivo imaging in response to a combination of methyltransferase inhibitors BIX01294 and Chaetocin in mice reveals the potential of this sensor for preclinical drug evaluation. This biosensor thus has demonstrated its utility in the detection of H3-K9 methylations in vivo and potential value in preclinical drug development.
View details for DOI 10.1021/cb5008037
View details for PubMedID 25489787
View details for PubMedCentralID PMC4301175
-
The emerging role of redox-sensitive Nrf2-Keap1 pathway in diabetes
PHARMACOLOGICAL RESEARCH
2015; 91: 104-114
Abstract
The pathogenic processes involving in the development of diabetes range from autoimmune destruction of pancreatic β-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The major contributing factor for excessive β-cell death includes oxidative stress-mediated mitochondrial damage, which creates an imbalance in redox homeostasis. Yet, β-cells have evolved adaptive mechanisms to endure a wide range of stress conditions to safeguard its potential functions. These include 'Nrf2/Keap1' pathway, a key cellular defense mechanism, to combat oxidative stress by regulating phase II detoxifying and antioxidant genes. During diabetes, redox imbalance provokes defective Nrf2-dependent signaling and compromise antioxidant capacity of the pancreas which turnout β-cells to become highly vulnerable against various insults. Hence, identification of small molecule activators of Nrf2/Keap1 pathway remains significant to enhance cellular defense to overcome the burden of oxidative stress related disturbances. This review summarizes the molecular mechanism behind Nrf2 activation and the impact of Nrf2 activators in diabetes and its complications.
View details for DOI 10.1016/j.phrs.2014.10.004
View details for Web of Science ID 000349273400012
-
Core - shell upconversion nanoparticle - semiconductor heterostructures for photodynamic therapy.
Scientific reports
2015; 5: 8252-?
Abstract
Core-shell nanoparticles (CSNPs) with diverse chemical compositions have been attracting greater attention in recent years. However, it has been a challenge to develop CSNPs with different crystal structures due to the lattice mismatch of the nanocrystals. Here we report a rational design of core-shell heterostructure consisting of NaYF4:Yb,Tm upconversion nanoparticle (UCN) as the core and ZnO semiconductor as the shell for potential application in photodynamic therapy (PDT). The core-shell architecture (confirmed by TEM and STEM) enables for improving the loading efficiency of photosensitizer (ZnO) as the semiconductor is directly coated on the UCN core. Importantly, UCN acts as a transducer to sensitize ZnO and trigger the generation of cytotoxic reactive oxygen species (ROS) to induce cancer cell death. We also present a firefly luciferase (FLuc) reporter gene based molecular biosensor (ARE-FLuc) to measure the antioxidant signaling response activated in cells during the release of ROS in response to the exposure of CSNPs under 980 nm NIR light. The breast cancer cells (MDA-MB-231 and 4T1) exposed to CSNPs showed significant release of ROS as measured by aminophenyl fluorescein (APF) and ARE-FLuc luciferase assays, and ~45% cancer cell death as measured by MTT assay, when illuminated with 980 nm NIR light.
View details for DOI 10.1038/srep08252
View details for PubMedID 25652742
View details for PubMedCentralID PMC4317689
-
The emerging role of redox-sensitive Nrf2-Keap1 pathway in diabetes.
Pharmacological research
2015; 91: 104-114
Abstract
The pathogenic processes involving in the development of diabetes range from autoimmune destruction of pancreatic β-cells with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The major contributing factor for excessive β-cell death includes oxidative stress-mediated mitochondrial damage, which creates an imbalance in redox homeostasis. Yet, β-cells have evolved adaptive mechanisms to endure a wide range of stress conditions to safeguard its potential functions. These include 'Nrf2/Keap1' pathway, a key cellular defense mechanism, to combat oxidative stress by regulating phase II detoxifying and antioxidant genes. During diabetes, redox imbalance provokes defective Nrf2-dependent signaling and compromise antioxidant capacity of the pancreas which turnout β-cells to become highly vulnerable against various insults. Hence, identification of small molecule activators of Nrf2/Keap1 pathway remains significant to enhance cellular defense to overcome the burden of oxidative stress related disturbances. This review summarizes the molecular mechanism behind Nrf2 activation and the impact of Nrf2 activators in diabetes and its complications.
View details for DOI 10.1016/j.phrs.2014.10.004
View details for PubMedID 25447793
-
Syntheses and Discovery of a Novel Class of Cinnamic Hydroxamates as Histone Deacetylase Inhibitors by Multimodality Molecular Imaging in Living Subjects
CANCER RESEARCH
2014; 74 (24): 7475-7486
Abstract
Histone deacetylases (HDAC) that regulate gene expression are being explored as cancer therapeutic targets. In this study, we focused on HDAC6 based on its ability to inhibit cancerous Hsp90 chaperone activities by disrupting Hsp90/p23 interactions. To identify novel HDAC6 inhibitors, we used a dual-luciferase reporter system in cell culture and living mice by bioluminescence imaging (BLI). On the basis of existing knowledge, a library of hydrazone compounds was generated for screening by coupling cinnamic hydroxamates with aldehydes and ketones. Potency and selectivity were determined by in vitro HDAC profiling assays, with further evaluation to inhibit Hsp90(α/β)/p23 interactions by BLI. In this manner, we identified compound 1A12 as a dose-dependent inhibitor of Hsp90(α/β)/p23 interactions, UKE-1 myeloid cell proliferation, p21(waf1) upregulation, and acetylated histone H3 levels. 1A12 was efficacious in tumor xenografts expressing Hsp90(α)/p23 reporters relative to carrier control-treated mice as determined by BLI. Small animal (18)F-FDG PET/CT imaging on the same cohort showed that 1A12 also inhibited glucose metabolism relative to control subjects. Ex vivo analyses of tumor lysates showed that 1A12 administration upregulated acetylated-H3 by approximately 3.5-fold. Taken together, our results describe the discovery and initial preclinical validation of a novel selective HDAC inhibitor.
View details for DOI 10.1158/0008-5472.CAN-14-0197
View details for Web of Science ID 000346363900031
View details for PubMedID 25320008
-
Noninvasive Reporter Gene Imaging of Human Oct4 (Pluripotency) Dynamics During the Differentiation of Embryonic Stem Cells in Living Subjects
MOLECULAR IMAGING AND BIOLOGY
2014; 16 (6): 865-876
Abstract
Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects.To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter.In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) (p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h (p = 0.001) and between 12 and 168 h (p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments (n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers.We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects.
View details for DOI 10.1007/s11307-014-0744-1
View details for Web of Science ID 000345281600014
-
Noninvasive reporter gene imaging of human Oct4 (pluripotency) dynamics during the differentiation of embryonic stem cells in living subjects.
Molecular imaging and biology
2014; 16 (6): 865-876
Abstract
Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects.To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter.In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) (p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h (p = 0.001) and between 12 and 168 h (p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments (n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers.We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects.
View details for DOI 10.1007/s11307-014-0744-1
View details for PubMedID 24845530
-
Dynamic Analysis of GH Receptor Conformational Changes by Split Luciferase Complementation
MOLECULAR ENDOCRINOLOGY
2014; 28 (11): 1807-1819
Abstract
The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharmacological manipulation, genetic alteration of JAK2 levels, and truncation of the GHR intracellular domain (ICD) tail suggested that GH acutely enhances proximity of the GHR homodimer partners independent of the presence of JAK2, phosphorylation of GHR-luciferase chimeras, or an intact ICD. However, subsequent reduction of complementation requires JAK2 kinase activity and the ICD tail. This conclusion is in contrast to existing models of the GHR activation process.
View details for DOI 10.1210/me.2014-1153
View details for Web of Science ID 000346837100006
View details for PubMedID 25188449
-
Molecular imaging of RRx-001-induced oxidative stress in Nrf2-luciferase expressing SCC VII tumors in mice
AMER ASSOC CANCER RESEARCH. 2014
View details for DOI 10.1158/1538-7445.AM2014-906
View details for Web of Science ID 000349906905366
-
Correlation of plasma biomarker levels with early-stage tumor viability in an orthotopic ovarian cancer mouse model
AMER ASSOC CANCER RESEARCH. 2014
View details for DOI 10.1158/1538-7445.AM2014-873
View details for Web of Science ID 000349906905331
-
Monitoring antioxidant induced chemosensitization in triple negative breast cancer cells by Nrf2-luciferase fusion protein
AMER ASSOC CANCER RESEARCH. 2014
View details for DOI 10.1158/1538-7445.AM2014-5120
View details for Web of Science ID 000349910204074
-
Proteomic Identification of Pterostilbene-Mediated Anticancer Activities in HepG2 Cells.
Chemical research in toxicology
2014; 27 (7): 1243-1252
Abstract
In the present study, we attempt to shed light on the underlying molecular mechanism of the anticancer activity of pterostilbene (PTS) in HepG2 cells through the proteomic approach. PTS was found to induce apoptosis by altering the expression of apoptotic genes and the G2/M phase of cell cycle arrest. Further, the 2-DE map showed the expression of 72 differentially regulated proteins in PTS-treated HepG2 cells, of which 8 spots with >2 fold up- or down-regulated level were identified by MALDI-TOF analysis, which has a regulatory role in apoptosis. These findings for the first time offer valuable insights into the mechanism of apoptotis by PTS in HepG2 cells.
View details for DOI 10.1021/tx5001392
View details for PubMedID 24936659
-
Quercetin ameliorates tunicamycin-induced endoplasmic reticulum stress in endothelial cells.
Cell proliferation
2014; 47 (3): 231-240
Abstract
Endothelial dysfunction highlights that it is a potential contributor in the pathogenesis of vascular complications arising from endoplasmic reticulum stress (ER stress) and has been emerging as a main causative factor in vascular failure. Here, we hypothesize that the natural flavonoid, quercetin plays an effective role in reducing ER stress in human umbilical vein endothelial cells.Human umbilical vein endothelial cells were pre-treated with different concentrations of quercetin (0-100 μm) before inducing ER stress using tunicamycin (TUN) (0.75 μg/ml); cytotoxicity was assessed by MTT assay. Expression levels of ER stress responsive genes, antioxidant enzymes and apoptotic markers were assessed by qRT-PCR, while roles of caspase-3 and PARP cleavage were measured by western blot analysis.Quercetin pre-treatment at 25 and 50 μm had a cytoprotective effect on cells against TUN-induced toxicity. Quercetin administration modulated expression level of ER stress genes coding for glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP), and antioxidant enzymes such as superoxide dismutase and catalase, along with free radical generation assessed by malondialdehyde assay. Induction of apoptosis was prevented with reduction in expression level of Bax, and concomitant increase in Bcl-2 levels, thus proving its potential against ER stress.The current study indicates that quercetin modulated stress responsive genes GRP78 and CHOP, helping endothelial cells prevent TUN-induced ER stress.
View details for DOI 10.1111/cpr.12102
View details for PubMedID 24666891
-
A titratable two-step transcriptional amplification strategy for targeted gene therapy based on ligand-induced intramolecular folding of a mutant human estrogen receptor.
Molecular imaging and biology
2014; 16 (2): 224-234
Abstract
The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ER(G521T)) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.
View details for DOI 10.1007/s11307-013-0673-4
View details for PubMedID 23955099
View details for PubMedCentralID PMC4154804
-
Therapeutic potential of pterostilbene against pancreatic beta-cell apoptosis mediated through Nrf2.
British journal of pharmacology
2014; 171 (7): 1747-1757
Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be a 'master regulator' of the antioxidant response as it regulates the expression of several genes including phase II metabolic and antioxidant enzymes and thus plays an important role in preventing oxidative stress-mediated disorders, including diabetes. In this study, for the first time, we investigated the protective properties of a naturally available antioxidant, pterostilbene (PTS), against pancreatic beta-cell apoptosis and the involvement of Nrf2 in its mechanism of action.Immunoblotting and quantitative reverse transcriptase (qRT)-PCR analysis were performed to identify PTS-mediated nuclear translocation of Nrf2 protein and the following activation of target gene expression, respectively, in INS-1E cells. In addition, an annexin-V binding assay was carried out to identify the apoptotic status of PTS-treated INS-1E cells, while confirming the anti-apoptotic potential of Nrf2 by qRT-PCR analysis of the expressions of both pro- and anti-apoptotic genes.PTS induced significant activation of Nrf2, in dose- and time-dependent manner, in streptozotocin-treated INS-1E rat pancreatic beta-cells. Furthermore, PTS increased the expression of target genes downstream of Nrf2, such as heme oxygenase 1 (HO1), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), that confer cellular protection. PTS also up-regulated the expression of anti-apoptotic gene, Bcl-2, with a concomitant reduction in pro-apoptotic Bax and caspase-3 expression.Collectively, our findings indicate the therapeutic potential of Nrf2 activation by PTS as a promising approach to safeguard pancreatic beta-cells against oxidative damage in diabetes.
View details for DOI 10.1111/bph.12577
View details for PubMedID 24417315
-
In vivo effects of the pure aryl hydrocarbon receptor antagonist GNF-351 after oral administration are limited to the gastrointestinal tract
BRITISH JOURNAL OF PHARMACOLOGY
2014; 171 (7): 1747-1757
Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be a 'master regulator' of the antioxidant response as it regulates the expression of several genes including phase II metabolic and antioxidant enzymes and thus plays an important role in preventing oxidative stress-mediated disorders, including diabetes. In this study, for the first time, we investigated the protective properties of a naturally available antioxidant, pterostilbene (PTS), against pancreatic beta-cell apoptosis and the involvement of Nrf2 in its mechanism of action.Immunoblotting and quantitative reverse transcriptase (qRT)-PCR analysis were performed to identify PTS-mediated nuclear translocation of Nrf2 protein and the following activation of target gene expression, respectively, in INS-1E cells. In addition, an annexin-V binding assay was carried out to identify the apoptotic status of PTS-treated INS-1E cells, while confirming the anti-apoptotic potential of Nrf2 by qRT-PCR analysis of the expressions of both pro- and anti-apoptotic genes.PTS induced significant activation of Nrf2, in dose- and time-dependent manner, in streptozotocin-treated INS-1E rat pancreatic beta-cells. Furthermore, PTS increased the expression of target genes downstream of Nrf2, such as heme oxygenase 1 (HO1), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), that confer cellular protection. PTS also up-regulated the expression of anti-apoptotic gene, Bcl-2, with a concomitant reduction in pro-apoptotic Bax and caspase-3 expression.Collectively, our findings indicate the therapeutic potential of Nrf2 activation by PTS as a promising approach to safeguard pancreatic beta-cells against oxidative damage in diabetes.
View details for DOI 10.1111/bph.12576
View details for Web of Science ID 000332903000014
View details for PubMedCentralID PMC3966753
-
Noninvasive Theranostic Imaging of HSV1-sr39TK-NTR/GCV-CB1954 Dual-Prodrug Therapy in Metastatic Lung Lesions of MDA-MB-231 Triple Negative Breast Cancer in Mice.
Theranostics
2014; 4 (5): 460-474
Abstract
Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 μg/ml GCV and 10 μM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following two consecutive doses of a combination of GCV (40 mg/kg) and CB1954 (40 mg/kg) administered at 5 day intervals. In contrast, the respective treatment condition in animals expressing HSV1-sr39TK or NTR separately, showed minimal or no effect on tumor reduction as measured by bioluminescence (tumor mass) and [(18)F]-FHBG microPET (TK expression) imaging. These highlight the strong therapeutic effect of the dual fusion prodrug therapy and its use in theranostic imaging of tumor monitoring in living animals by multimodality molecular imaging.
View details for DOI 10.7150/thno.8077
View details for PubMedID 24669276
View details for PubMedCentralID PMC3964441
-
Targeting SUMOylation cascade for diabetes management.
Current drug targets
2014; 15 (12): 1094-1106
Abstract
Post-translational modifications (PTMs) play important roles in regulating protein stability, trafficking, folding conformation, and functional activity. Small ubiquitin-like modifier (SUMO) protein mediates a distinct type of PTM called SUMOylation in which the SUMO protein is covalently ligated to the target protein and modifies its activities through a series of enzymatically-catalyzed reactions. SUMOylation regulates many cellular processes like transcription, the maintenance of the ion gradient across the cell membrane, stress response, autoimmunity, etc. Several target proteins of SUMOylation are involved in the biological pathways related to various human diseases, including cardiovascular diseases, diabetes, cancer, and neurodegenerative disorders. This review focuses on the SUMOylation process, regulatory roles of SUMOylation in diabetes, and prospects of developing novel anti-diabetic drugs targeting the SUMOylation process.
View details for PubMedID 25230117
-
Polymer nanoparticles for drug and small silencing RNA delivery to treat cancers of different phenotypes
WILEY INTERDISCIPLINARY REVIEWS-NANOMEDICINE AND NANOBIOTECHNOLOGY
2014; 6 (1): 40-60
Abstract
Advances in nanotechnology have provided powerful and efficient tools in the development of cancer diagnosis and therapy. There are numerous nanocarriers that are currently approved for clinical use in cancer therapy. In recent years, biodegradable polymer nanoparticles have attracted a considerable attention for their ability to function as a possible carrier for target-specific delivery of various drugs, genes, proteins, peptides, vaccines, and other biomolecules in humans without much toxicity. This review will specifically focus on the recent advances in polymer-based nanocarriers for various drugs and small silencing RNA's loading and delivery to treat different types of cancer.
View details for DOI 10.1002/wnan.1242
View details for Web of Science ID 000328354300003
View details for PubMedID 23996830
View details for PubMedCentralID PMC3865230
-
Polymer nanoparticles for drug and small silencing RNA delivery to treat cancers of different phenotypes.
Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology
2014; 6 (1): 40-60
Abstract
Advances in nanotechnology have provided powerful and efficient tools in the development of cancer diagnosis and therapy. There are numerous nanocarriers that are currently approved for clinical use in cancer therapy. In recent years, biodegradable polymer nanoparticles have attracted a considerable attention for their ability to function as a possible carrier for target-specific delivery of various drugs, genes, proteins, peptides, vaccines, and other biomolecules in humans without much toxicity. This review will specifically focus on the recent advances in polymer-based nanocarriers for various drugs and small silencing RNA's loading and delivery to treat different types of cancer.
View details for DOI 10.1002/wnan.1242
View details for PubMedID 23996830
View details for PubMedCentralID PMC3865230
-
Targeting SUMOylation Cascade for Diabetes Management
CURRENT DRUG TARGETS
2014; 15 (12): 1094-1106
Abstract
Post-translational modifications (PTMs) play important roles in regulating protein stability, trafficking, folding conformation, and functional activity. Small ubiquitin-like modifier (SUMO) protein mediates a distinct type of PTM called SUMOylation in which the SUMO protein is covalently ligated to the target protein and modifies its activities through a series of enzymatically-catalyzed reactions. SUMOylation regulates many cellular processes like transcription, the maintenance of the ion gradient across the cell membrane, stress response, autoimmunity, etc. Several target proteins of SUMOylation are involved in the biological pathways related to various human diseases, including cardiovascular diseases, diabetes, cancer, and neurodegenerative disorders. This review focuses on the SUMOylation process, regulatory roles of SUMOylation in diabetes, and prospects of developing novel anti-diabetic drugs targeting the SUMOylation process.
View details for Web of Science ID 000345225700002
-
MicroRNAs - a new generation molecular targets for treating cellular diseases.
Theranostics
2013; 3 (12): 927-9
Abstract
MicroRNAs (miRNAs) are a unique class of non-coding, small RNAs, similar to mRNAs, transcribed by cells, but for entirely different reasons. While mRNAs are transcribed to code for proteins, miRNAs are produced to regulate the production of proteins from mRNAs. miRNAs are central components that tightly and temporally regulating gene expression in cells. Dysregulation of miRNAs expressions in cellular pathogenesis, including cancer, has been reported, and it clearly supports the importance of miRNAs as therapeutic targets in almost every cellular disease. This special issue compiles manuscripts from different authors in order to highlight the importance of microRNAs in cancer and other cellular diseases and how to explore them as therapeutic targets to treat patients with these genetic and metabolic disorders. This issue also covers manuscripts that highlight the obstacles associated with the delivery of therapeutic microRNAs in cells and in vivo in living animals and how to improve and noninvasively monitor the delivery in living animals through molecular imaging, including potential clinical perspectives.
View details for DOI 10.7150/thno.8113
View details for PubMedID 24396503
View details for PubMedCentralID PMC3881094
-
Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia.
Human gene therapy methods
2013; 24 (5): 279-288
Abstract
Abstract Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in preclinical models of myocardial ischemia.
View details for DOI 10.1089/hgtb.2013.028
View details for PubMedID 23937265
-
Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia.
Human gene therapy methods
2013; 24 (5): 279-288
View details for DOI 10.1089/hgtb.2013.028
View details for PubMedID 23937265
-
H-PCV) Uptake Assay.
Bio-protocol
2013; 3 (17)
Abstract
Thymidine Kinase from human Herpes simplex virus type 1 (HSV1-TK) in combination with specific substrate prodrug nucleotide analogue ganciclovir (GCV) has been widely used as suicidal therapeutic gene for cancer gene therapy. HSV1, and its mutant (HSV1-sr39TK) with improved substrate specificity, were used as reporter genes for PET-imaging of various biological functions in small animals, by combining with radiolabeled substrates such as (18)F-FHBG and (124)I-FIAU. (3)H-Penciclovir (PCV) uptake assay is a method of choice used to determine the expression level of HSV1-TK in mammalian cells and tissues. HSV1-TK phosphorylate PCV and result in the formation of penciclovir monophosphate, and its subsequent phopsphorylation by cellular TK lead to the formation of penciclovir triphosphate, which is trapped selectively in cells expressing HSV-TK. (3)H-Penciclovir enables the detection of penciclovir uptake of mammalian cells and tissues by radioactive procedures such as scintillation counting. Here we describe the protocol to carry out (3)H-Penciclovir uptakes in mammalian cells.
View details for PubMedID 27446976
-
A c-Myc Activation Sensor-Based High-Throughput Drug Screening Identifies an Antineoplastic Effect of Nitazoxanide.
Molecular cancer therapeutics
2013; 12 (9): 1896-1905
Abstract
Deregulation of c-Myc plays a central role in the tumorigenesis of many human cancers. Yet, the development of drugs regulating c-Myc activity has been challenging. To facilitate the identification of c-Myc inhibitors, we developed a molecular imaging sensor based high throughput-screening (HTS) system. This system uses a cell-based assay to detect c-Myc activation in a HTS format, which is established from a pure clone of a stable breast cancer cell line that constitutively expresses a c-Myc activation sensor. Optimization of the assay performance in the HTS format resulted in uniform and robust signals at the baseline. Using this system, we performed a quantitative HTS against approximately 5,000 existing bioactive compounds from five different libraries. Thirty-nine potential hits were identified, including currently known c-Myc inhibitors. There are a few among the top potent hits that are not known for anti-c-Myc activity. One of these hits is nitazoxanide (NTZ), a thiazolide for treating human protozoal infections. Validation of NTZ in different cancer cell lines revealed a high potency for c-Myc inhibition with IC50 ranging between 10 - 500nM. Oral administration of NTZ in breast cancer xenograft mouse models significantly suppressed tumor growth by inhibition of c-Myc and induction of apoptosis. These findings suggest a potential of NTZ to be repurposed as a new anti-tumor agent for inhibition of c-Myc associated neoplasia. Our work also demonstrated the unique advantage of molecular imaging in accelerating discovery of drugs for c-Myc targeted cancer therapy.
View details for DOI 10.1158/1535-7163.MCT-12-1243
View details for PubMedID 23825064
-
Reporter protein complementation imaging assay to screen and study nrf2 activators in cells and living animals.
Analytical chemistry
2013; 85 (15): 7542-7549
Abstract
NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform. Further, PTS increased the expression of Nrf2 downstream target genes, which was confirmed using luciferase reporter driven by ARE-NQO1 and ARE-GST1 promoters. Daily administration of PTS disturbed Nrf2/Keap1 interaction and reduced complemented luciferase signals in HEK293TNKS mouse tumor xenografts. This study reveals the potentials of Nrf2 activators as chemosensitizing agents' for therapeutic intervention in cancer treatment. Hence, the validated assay can be used to evaluate the identified activators preclinically in small animal models by noninvasive molecular imaging approach.
View details for DOI 10.1021/ac401569j
View details for PubMedID 23826874
-
Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.
Gene therapy
2013; 20 (5): 529-537
Abstract
Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.
View details for DOI 10.1038/gt.2012.66
View details for PubMedID 22914496
-
The Impact of Oxidative Stress on Islet Transplantation and Monitoring the Graft Survival by Non-Invasive Imaging
CURRENT MEDICINAL CHEMISTRY
2013; 20 (9): 1127-1146
Abstract
Islet transplantation is an attractive strategy to treat severe diabetic conditions in patients suffering from autoimmune derived diabetes, and it has currently been considered a forefront research arena in diabetes. Major aim of islet transplantation is to achieve successful insulin independent disease free survival. The key challenges in transplanted islets are the generation of reactive oxygen species (ROS) and associated oxidative stress, pro-inflammatory cytokine - (TNFα) mediated apoptotic induction, attack by immune cells, and achieving revascularization with minimal hypoxic microenvironment. Free radicals and their derivatives are constantly produced in living systems, but at relatively low level, and in a balanced state. Oxidative stress, which occurs as a result of an imbalance between the intracellular free radicals production and the cellular antioxidant defense mechanisms in the transplanted islets, can lead to cell death. The balance between oxidants and antioxidants in a cell can be easily disturbed by increase in ROS production or reduction in the level of cellular antioxidant defensive substances, which can cause many metabolic complications, including pancreatic β-cell damage. Antioxidants function as blockers of radical processes by eliminating harmful ROS produced during normal cellular metabolism. A complex antioxidant defense mechanism has been developed by nature in cells to protect the cellular homeostasis. This system mainly includes antioxidant enzymes, vitamins and minerals. As transplanted islet survival is crucial for achieving successful therapy, most of these antioxidants can be used as a supplement to scavenge the local ROS thereby improving the survival of transplanted islets. Currently, very few techniques have been routinely used to qualitatively and quantitatively assess the survival and function of islet grafts, especially to confirm the success of treatment, which includes metabolic parameters such as blood glucose, insulin and C-peptide levels. These biochemical measurements provide markers at only the late stages of islet rejection. Use of molecular imaging techniques has the potential for real-time non-invasive monitoring of the functional status and viability of transplanted islet grafts in living animals. This review mainly focuses on the current status of islet transplantations, potential preventive strategies used to reduce oxidative stress-mediated toxicity in islet grafts, and use of molecular imaging as a tool to quantitatively evaluate the functional status of the transplanted islets in living animals.
View details for Web of Science ID 000316615700003
View details for PubMedID 23317098
-
Real time dynamic imaging and current targeted therapies in the war on cancer: a new paradigm.
Theranostics
2013; 3 (6): 437-447
Abstract
In biology, as every science student is made to learn, ontology recapitulates phylogeny. In medicine, however, oncology recapitulates polemology, the science of warfare: The medical establishment is transitioning from highly toxic poisons that kill rapidly dividing normal and malignant cells with little specificity to tailored therapies that target the tumors with the lethality of the therapeutic warhead. From the advent of the information age with the incorporation of high-tech intelligence, reconnaissance, and surveillance has resulted in "data fusion" where a wide range of information collected in near real-time can be used to redesign most of the treatment strategies currently used in the clinic. The medical community has begun to transition from the 'black box' of tumor therapy based solely on the clinical response to the 'glass box' of dynamic imaging designed to bring transparency to the clinical battlefield during treatment, thereby informing the therapeutic decision to 'retreat or repeat'. The tumor microenvironment is dynamic, constantly changing in response to therapeutic intervention, and therefore the therapeutic assessment must map to this variable and ever-changing landscape with dynamic and non-static imaging capabilities. The path to personalized medicine will require incorporation and integration of dynamic imaging at the bedside into clinical practice for real-time, interactive assessment of response to targeted therapies. The application of advanced real time imaging techniques along with current molecularly targeted anticancer therapies which alter cellular homeostasis and microenvironment can enhance therapeutic interventions in cancer patients and further improve the current status in clinical management of patients with advanced cancers.
View details for DOI 10.7150/thno.5658
View details for PubMedID 23781290
View details for PubMedCentralID PMC3677414
-
Therapeutic evaluation of microRNAs by molecular imaging.
Theranostics
2013; 3 (12): 964-985
Abstract
MicroRNAs (miRNAs) function as regulatory molecules of gene expression with multifaceted activities that exhibit direct or indirect oncogenic properties, which promote cell proliferation, differentiation, and the development of different types of cancers. Because of their extensive functional involvement in many cellular processes, under both normal and pathological conditions such as various cancers, this class of molecules holds particular interest for cancer research. MiRNAs possess the ability to act as tumor suppressors or oncogenes by regulating the expression of different apoptotic proteins, kinases, oncogenes, and other molecular mechanisms that can cause the onset of tumor development. In contrast to current cancer medicines, miRNA-based therapies function by subtle repression of gene expression on a large number of oncogenic factors, and therefore are anticipated to be highly efficacious. Given their unique mechanism of action, miRNAs are likely to yield a new class of targeted therapeutics for a variety of cancers. More than thousand miRNAs have been identified to date, and their molecular mechanisms and functions are well studied. Furthermore, they are established as compelling therapeutic targets in a variety of cellular complications. However, the notion of using them as therapeutic tool was proposed only recently, given that modern imaging methods are just beginning to be deployed for miRNA research. In this review, we present a summary of various molecular imaging methods, which are instrumental in revealing the therapeutic potential of miRNAs, especially in various cancers. Imaging methods have recently been developed for monitoring the expression levels of miRNAs and their target genes by fluorescence-, bioluminescence- and chemiluminescence-based imaging techniques. Mature miRNAs bind to the untranslated regions (UTRs) of the target mRNAs and regulate target genes expressions. This concept has been used for the development of fluorescent reporter-based imaging strategies to monitor the functional status of endogenous miRNAs, or the respective miRNAs transiently co-expressed in cells. Bioluminescence-based imaging strategies have been used to investigate various stages of miRNA processing and its involvement in different cellular processes. Similarly, chemiluminsecence methods were developed for in vitro miRNA imaging such as monitoring their therapeutic roles in various cancer cell lines.
View details for DOI 10.7150/thno.4928
View details for PubMedID 24396507
- Noninvasive imaging of hypoxia-inducible factor-1? gene therapy for myocardial ischemia Hum Gene Ther Methods. 2013 Aug 12. [Epub ahead of print] 2013
-
Therapeutic Evaluation of microRNAs by Molecular Imaging
THERANOSTICS
2013; 3 (12): 953-974
Abstract
MicroRNAs (miRNAs) function as regulatory molecules of gene expression with multifaceted activities that exhibit direct or indirect oncogenic properties, which promote cell proliferation, differentiation, and the development of different types of cancers. Because of their extensive functional involvement in many cellular processes, under both normal and pathological conditions such as various cancers, this class of molecules holds particular interest for cancer research. MiRNAs possess the ability to act as tumor suppressors or oncogenes by regulating the expression of different apoptotic proteins, kinases, oncogenes, and other molecular mechanisms that can cause the onset of tumor development. In contrast to current cancer medicines, miRNA-based therapies function by subtle repression of gene expression on a large number of oncogenic factors, and therefore are anticipated to be highly efficacious. Given their unique mechanism of action, miRNAs are likely to yield a new class of targeted therapeutics for a variety of cancers. More than thousand miRNAs have been identified to date, and their molecular mechanisms and functions are well studied. Furthermore, they are established as compelling therapeutic targets in a variety of cellular complications. However, the notion of using them as therapeutic tool was proposed only recently, given that modern imaging methods are just beginning to be deployed for miRNA research. In this review, we present a summary of various molecular imaging methods, which are instrumental in revealing the therapeutic potential of miRNAs, especially in various cancers. Imaging methods have recently been developed for monitoring the expression levels of miRNAs and their target genes by fluorescence-, bioluminescence- and chemiluminescence-based imaging techniques. Mature miRNAs bind to the untranslated regions (UTRs) of the target mRNAs and regulate target genes expressions. This concept has been used for the development of fluorescent reporter-based imaging strategies to monitor the functional status of endogenous miRNAs, or the respective miRNAs transiently co-expressed in cells. Bioluminescence-based imaging strategies have been used to investigate various stages of miRNA processing and its involvement in different cellular processes. Similarly, chemiluminsecence methods were developed for in vitro miRNA imaging such as monitoring their therapeutic roles in various cancer cell lines.
View details for DOI 10.7150/thno.4928
View details for Web of Science ID 000328162400004
View details for PubMedCentralID PMC3881098
-
A transgenic tri-modality reporter mouse.
PloS one
2013; 8 (8)
Abstract
Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent (tdTomato), and positron emission tomography (PET) (ttk) modalities. Transgenic colonies with different levels of tri-fusion reporter gene expression showed a linear correlation between all three-reporter proteins (R(2)=0.89 for TdTomato vs Fluc, R(2)=0.94 for Fluc vs TTK, R(2)=0.89 for TdTomato vs TTK) in vitro from tissue lysates and in vivo by optical and PET imaging. Mesenchymal stem cells (MSCs) isolated from this transgenics showed high level of reporter gene expression, which linearly correlated with the cell numbers (R(2)=0.99 for bioluminescence imaging (BLI)). Both BLI (R(2)=0.93) and micro-PET (R(2)=0.94) imaging of the subcutaneous implants of Tri-Modality Reporter Mouse derived MSCs in nude mice showed linear correlation with the cell numbers and across different imaging modalities (R(2)=0.97). Serial imaging of MSCs transplanted to mice with acute myocardial infarction (MI) by intramyocardial injection exhibited significantly higher signals in MI heart at days 2, 3, 4, and 7 (p<0.01). MSCs transplanted to the ischemic hindlimb of nude mice showed significantly higher BLI and PET signals in the first 2 weeks that dropped by 4(th) week due to poor cell survival. However, laser Doppler perfusion imaging revealed that blood circulation in the ischemic limb was significantly improved in the MSCs transplantation group compared with the control group. In summary, this mouse can be used as a source of donor cells and organs in various research areas such as stem cell research, tissue engineering research, and organ transplantation.
View details for DOI 10.1371/journal.pone.0073580
View details for PubMedID 23951359
View details for PubMedCentralID PMC3739740
-
FEASIBILITY OF AN INTRAMOLECULAR COMPLEMENTATION STRATEGY FOR SPLIT-REPORTER GENE IMAGING OF DRUGGABLE PROTEIN MISFOLDING IN BRAIN CANCER
17th Annual Scientific Meeting and Education Day of the Society-for-Neuro-Oncology (SNO)
OXFORD UNIV PRESS INC. 2012: 11–11
View details for Web of Science ID 000310971300048
-
Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging in living subjects
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (37): E2476-E2485
Abstract
Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/β)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90β/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/β)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.
View details for DOI 10.1073/pnas.1205459109
View details for PubMedID 22895790
-
Cationic versus Neutral Microbubbles for Ultrasound-mediated Gene Delivery in Cancer
RADIOLOGY
2012; 264 (3): 721-732
Abstract
To test whether plasmid-binding cationic microbubbles (MBs) enhance ultrasound-mediated gene delivery efficiency relative to control neutral MBs in cell culture and in vivo tumors in mice.Animal studies were approved by the institutional animal care committee. Cationic and neutral MBs were characterized in terms of size, charge, circulation time, and DNA binding. Click beetle luciferase (CBLuc) reporter plasmids were mixed with cationic or neutral MBs. The ability of cationic MBs to protect bound plasmids from nuclease degradation was tested by means of a deoxyribonuclease (DNase) protection assay. Relative efficiencies of ultrasound-mediated transfection (ultrasound parameters: 1 MHz, 1 W/cm(2), 20% duty cycle, 1 minute) of CBLuc to endothelial cells by using cationic, neutral, or no MBs were compared in cell culture. Ultrasound-mediated gene delivery to mouse hind limb tumors was performed in vivo (n = 24) with insonation (1 MHz, 2 W/cm(2), 50% duty cycle, 5 minutes) after intravenous administration of CBLuc with cationic, neutral, or no MBs. Tumor luciferase activity was assessed by means of serial in vivo bioluminescence imaging and ex vivo analysis. Results were compared by using analysis of variance.Cationic MBs (+15.8 mV; DNA binding capacity, 0.03 pg per MB) partially protected bound DNA from DNase degradation. Mean CBLuc expression of treated endothelial cells in culture was 20-fold higher with cationic than with neutral MBs (219.0 relative light units [RLUs]/µg protein ± 92.5 [standard deviation] vs 10.9 RLUs/µg protein ± 2.7, P = .001) and was significantly higher (P < .001) than that in the no MB and no ultrasound control groups. Serial in vivo bioluminescence of mouse tumors was significantly higher with cationic than with neutral MBs ([5.9 ± 2.2] to [9.3 ± 5.2] vs [2.4 ± 0.8] to [2.9 ± 1.1] × 10(4) photons/sec/cm(2)/steradian, P < .0001) and versus no MB and no ultrasound controls (P < .0001). Results of ex vivo analysis confirmed these results (ρ = 0.88, P < .0001).Plasmid-binding cationic MBs enhance ultrasound-mediated gene delivery efficiency relative to neutral MBs in both cell culture and mouse hind limb tumors.
View details for DOI 10.1148/radiol.12112368
View details for Web of Science ID 000308645500013
View details for PubMedID 22723497
View details for PubMedCentralID PMC3426857
-
Oxidative stress-mediated cytotoxicity and apoptosis induction by TiO2 nanofibers in HeLa cells
EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS
2012; 81 (2): 324-333
Abstract
Titanium dioxide nanoparticles are increasingly being used in pharmaceutical and cosmetic products. The high aspect ratio of fibrous nanomaterials, such as carbon nanotubes and TiO(2) nanofibers (TiO(2)NFs), similar to the one used in this study makes them an attractive structural material and has attracted a lot of attention due to their possible negative health effects as suggested by their morphological similarities with asbestos. In the present study, therefore, toxicity of TiO(2)NFs was evaluated in human cervical adenocarcinoma HeLa cells. The TEM and XRD analyses showed that TiO(2)NFs used in this study are pure with uniform diameter of around 200 nm, and their length to width aspect ratio ranged between 5 and 15. Exposure of HeLa cells to TiO(2)NFs induced significant cytotoxicity even at doses as low as 2 μg/ml. The intracellular uptake of TiO(2)NFs in cells was shown by Alizarin Red S (ARS) labeled nanofibers. The mechanism of toxicity is mainly due to the induction of cellular oxidative stress, as revealed by elevated ROS levels, reduced antioxidant levels, and increased lipid peroxidation leading to apoptosis. The cell cycle analysis indicated G(2)/M cell cycle arrest in the cells exposed to TiO(2)NF. TiO(2)NFs treatment to HeLa cells resulted in increased expression of proapoptotic proteins Bax with an increase in cytosolic Cytochrome-C and inhibition of anti-apoptotic protein Bcl-2. Our results revealed the potential mechanism of cellular effects of TiO(2)NFs.
View details for DOI 10.1016/j.ejpb.2012.02.013
View details for Web of Science ID 000305591700011
View details for PubMedID 22446064
-
Intratumoral versus Intravenous Gene Therapy Using a Transcriptionally Targeted Viral Vector in an Orthotopic Hepatocellular Carcinoma Rat Model
JOURNAL OF VASCULAR AND INTERVENTIONAL RADIOLOGY
2012; 23 (5): 704-711
Abstract
To evaluate the feasibility of intratumoral delivery of adenoviral vector carrying a bidirectional two-step transcriptional amplification (TSTA) system to amplify transcriptional strength of cancer-specific Survivin promoter in a hepatocellular carcinoma model.MCA-RH7777 cells were implanted in rat liver, and tumor formation was confirmed with [(18)F]fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). The adenoviral vector studied had Survivin promoter driving a therapeutic gene (tumor necrosis factor-α-related apoptosis-inducing ligand [TRAIL]) and a reporter gene (firefly luciferase [FL]; Ad-pSurvivin-TSTA-TRAIL-FL). Tumor-bearing rats were administered Ad-pSurvivin-TSTA-TRAIL-FL intravenously (n = 7) or intratumorally (n = 8). For control groups, adenovirus FL under cytomegalovirus (CMV) promoter (Ad-pCMV-FL) was administered intravenously (n = 3) or intratumorally (n = 3). One day after delivery, bioluminescence imaging was performed to evaluate transduction. At 4 and 7 days after delivery, 18F-FDG-PET was performed to evaluate therapeutic efficacy.With intravenous delivery, Ad-pSurvivin-TSTA-TRAIL-FL showed no measurable liver tumor FL signal on day 1 after delivery, but showed better therapeutic efficacy than Ad-pCMV-FL on day 7 (PET tumor/liver ratio, 3.5 ± 0.58 vs 6.0 ± 0.71; P = .02). With intratumoral delivery, Ad-pSurvivin-TSTA-TRAIL-FL showed positive FL signal from all tumors and better therapeutic efficacy than Ad-pCMV-FL on day 7 (2.4 ± 0.50 vs 5.4 ± 0.78; P = .01). In addition, intratumoral delivery of Ad-pSurvivin-TSTA-TRAIL-FL demonstrated significant decrease in tumoral viability compared with intravenous delivery (2.4 ± 0.50 vs 3.5 ± 0.58; P = .03).Intratumoral delivery of a transcriptionally targeted therapeutic vector for amplifying tumor-specific effect demonstrated better transduction efficiency and therapeutic efficacy for liver cancer than systemic delivery, and may lead to improved therapeutic outcome for future clinical practice.
View details for DOI 10.1016/j.jvir.2012.01.053
View details for Web of Science ID 000303557000020
View details for PubMedID 22387029
View details for PubMedCentralID PMC4132166
-
Noninvasive optical imaging of nitroreductase gene-directed enzyme prodrug therapy system in living animals
GENE THERAPY
2012; 19 (3): 295-302
Abstract
Gene-directed enzyme prodrug therapy (GDEPT) is a promising and emerging strategy that attempts to limit the systemic toxicity inherent to cancer chemotherapy by means of tumor-targeted delivery and expression of an exogenous gene whose product converts nontoxic prodrug(s) into activated cytotoxic agent(s). The bacterial nitroreductase (NTR) enzyme, coupled with its substrate prodrug 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), is a promising GDEPT strategy that has reached clinical trials. However, no strategy exists to visually monitor and quantitatively evaluate the therapeutic efficacy of NTR/CB1954 prodrug therapy in cells and imaging in living animals. As the success of any GDEPT is dependent upon the efficiency of transgene expression in vivo, we developed a safe, sensitive and reproducible noninvasive imaging method to monitor NTR transgene expression that would allow quantitative assessment of both therapeutic efficacy and diagnostic outcome of NTR/CB1954 prodrug therapy in the future. Here, we investigate the use of a novel fluorescent imaging dye CytoCy5S (a Cy5-labeled quenched substrate of NTR enzyme) on various cancer cell lines in vitro and in NTR-transfected tumor-bearing animals in vivo. CytoCy5S-labeled cells become fluorescent at 'red-shifted' wavelengths (638 nm) when reduced by cellular NTR enzyme and remains trapped within the cells for extended periods of time. The conversion and entrapment was dynamically recorded using a time-lapsed microscopy. Systemic and intratumoral delivery of CytoCy5S to NTR-expressing tumors in animals indicated steady and reproducible signals even 16 h after delivery (P<0.001). This is the first study to address visual monitoring and quantitative evaluation of NTR activity in small animals using CytoCy5S, and establishes the capability of NTR to function as an imageable reporter gene.
View details for DOI 10.1038/gt.2011.101
View details for Web of Science ID 000301355800008
View details for PubMedID 21753794
- Therapeutic Evaluation of microRNAs by Molecular Imaging. Theranostics 2012: (in press)
-
Introduction to Cancer Biology
MOLECULAR IMAGING PROBES FOR CANCER RESEARCH
2012: 3–27
View details for Web of Science ID 000320761300002
-
Biodegradable Polymer Nanocarriers for Therapeutic Antisense microRNA Delivery in Living Animals
Conference on Colloidal Nanocrystals for Biomedical Applications VII
SPIE-INT SOC OPTICAL ENGINEERING. 2012
View details for DOI 10.1117/12.914830
View details for Web of Science ID 000302562300004
-
Ultrasound-Mediated Gene Delivery with Cationic Versus Neutral Microbubbles: Effect of DNA and Microbubble Dose on In Vivo Transfection Efficiency
THERANOSTICS
2012; 2 (11): 1078-1091
Abstract
To assess the effect of varying microbubble (MB) and DNA doses on the overall and comparative efficiencies of ultrasound (US)-mediated gene delivery (UMGD) to murine hindlimb skeletal muscle using cationic versus neutral MBs.Cationic and control neutral MBs were characterized for size, charge, plasmid DNA binding, and ability to protect DNA against endonuclease degradation. UMGD of a codon optimized firefly luciferase (Fluc) reporter plasmid to endothelial cells (1 MHz, 1 W/cm², 20% duty cycle, 1 min) was performed in cell culture using cationic, neutral, or no MBs. In vivo UMGD to mouse hindlimb muscle was performed by insonation (1 MHz, 2 W/cm², 50% duty cycle, 5 min) after intravenous administration of Fluc combined with cationic, neutral, or no MBs. Gene delivery efficiency was assessed by serial in vivo bioluminescence imaging. Efficiency of in vivo UMGD with cationic versus neutral MBs was systematically evaluated by varying plasmid DNA dose (10, 17.5, 25, 37.5, and 50 µg) while maintaining a constant MB dose of 1x10(8) MBs and by changing MB dose (1x10(7), 5x10(7), 1x10(8), or 5x10(8) MBs) while keeping a constant DNA dose of 50 µg.Cationic and size-matched control neutral MBs differed significantly in zeta potential with cationic MBs being able to bind plasmid DNA (binding capacity of 0.03 pg/MB) and partially protect DNA from nuclease degradation while neutral MBs could not. Cationic MBs enhanced UMGD compared to neutral MBs as well as no MB and no US controls both in cell culture (P < 0.001) and in vivo (P < 0.05). Regardless of MB type, in vivo UMGD efficiency increased dose-dependently with DNA dose and showed overall maximum transfection with 50 µg DNA. However, there was an inverse correlation (ρ = -0.90; P = 0.02) between DNA dose and the degree of enhanced UMGD efficiency observed with using cationic MBs instead of neutral MBs. The delivery efficiency advantage associated with cationic MBs was most prominent at the lowest investigated DNA dose (7.5-fold increase with cationic versus neutral MBs at a DNA dose of 10 µg; P = 0.02) compared to only a 1.4-fold increase at a DNA dose of 50 µg (P < 0.01). With increasing MB dose, overall in vivo UMGD efficiency increased dose-dependently with a maximum reached at a dose of 1x10(8) MBs with no further significant increase with 5x10(8) MBs (P = 0.97). However, compared to neutral MBs, cationic MBs enhanced UMGD efficiency the most at low MB doses. Relative enhancement of UMGD efficiency using cationic over neutral MBs decreased from a factor of 27 for 1x10(7) MBs (P = 0.02) to a factor of 1.4 for 1x10(8) MBs (P < 0.01) and no significant difference for 5x10(8) MBs.Cationic MBs enhance UMGD to mouse skeletal muscle relative to neutral MBs but this is dependent on MB and DNA dose. The enhancement effect of cationic MBs on UMGD efficiency is more evident when lower doses of MBs or DNA are used, whereas the advantage of cationic MBs over neutral MBs is substantially reduced in the presence of excess MBs or DNA.
View details for DOI 10.7150/thno.4240
View details for Web of Science ID 000312955800005
View details for PubMedID 23227124
View details for PubMedCentralID PMC3516840
- Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals. Proceedings of SPIE 2012; 8232 (1) (Jan 21)
-
Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer
MOLECULAR IMAGING AND BIOLOGY
2011; 13 (6): 1124-1132
Abstract
We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1α (HIF-1α-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively.The plasmid pUbi-hrl-pUbi-HIF-1α-VP2, which expresses both hrl and HIF-1α-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1α-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI).Strong correlations existed between the expression of hRL and each of HIF-1α-VP2, VEGF, and PlGF (r(2) > 0.83, r(2) > 0.82, and r(2) > 0.97, respectively). In vivo, both transplanted cells and HIF-1α-VP2 transgene expression were successfully imaged using BLI.An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.
View details for DOI 10.1007/s11307-011-0471-9
View details for Web of Science ID 000296794400009
View details for PubMedID 21267661
View details for PubMedCentralID PMC4657136
-
In Vitro and in Vivo Molecular Imaging of Estrogen Receptor alpha and beta Homo- and Heterodimerization: Exploration of New Modes of Receptor Regulation
MOLECULAR ENDOCRINOLOGY
2011; 25 (12): 2029-2040
Abstract
Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses.
View details for DOI 10.1210/me.2011-1145
View details for PubMedID 22052998
-
Potent, tumor-specific gene expression in an orthotopic hepatoma rat model using a Survivin-targeted, amplifiable adenoviral vector
GENE THERAPY
2011; 18 (6): 606-612
Abstract
Ideal cancer gene therapies should have high tumor specificity and efficacy, and allow systemic administration to target metastases. We recently developed a bi-directional, two-step transcriptional amplification (TSTA) system driven by the tumor-specific Survivin promoter (pSurv) to amplify the correlated expression of both the reporter gene firefly luciferase (FL) and therapeutic gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we compare the specificity and potency of an adenovirus carrying this system (Ad-pSurv-TSTA-TRAIL-FL) to a nonspecific vector (Ad-pCMV-FL) in an orthotopic hepatocellular carcinoma (HCC) rat model after systemic administration. At 24 h after injection of Ad-pCMV-FL, bioluminescence imaging revealed a trend (P=0.30) towards greater FL expression in liver versus tumor. In striking contrast, Ad-pSurv-TSTA-TRAIL-FL showed increased FL activity within the tumor compared with the liver (P<0.01), a strong trend towards reduced liver expression compared with Ad-pCMV-FL (P=0.07), and importantly, similar FL levels within tumor compared with Ad-pCMV-FL (P=0.32). Hence, this vector shows potent, tumor-specific transgene expression even after extensive liver transduction and may be of significant value in avoiding hepatotoxicity in HCC patients. Future studies will explore the benefits of tumor-specific TRAIL expression in this model, the potential to target metastases and the extension of this vector for the treatment of other Survivin-positive tumors is warranted.
View details for DOI 10.1038/gt.2011.5
View details for Web of Science ID 000291438900010
View details for PubMedID 21307888
View details for PubMedCentralID PMC4154811
-
Exploring Gaussia luciferase as a novel secretion system for eukaryotic protein delivery for bacteria-mediated cancer treatment
SOC NUCLEAR MEDICINE INC. 2011
View details for Web of Science ID 000443798902153
-
The Fate and Toxicity of Raman-Active Silica-Gold Nanoparticles in Mice
SCIENCE TRANSLATIONAL MEDICINE
2011; 3 (79)
Abstract
Raman spectroscopy is an optical imaging method that is based on the Raman effect, the inelastic scattering of a photon when energy is absorbed from light by a surface. Although Raman spectroscopy is widely used for chemical and molecular analysis, its clinical application has been hindered by the inherently weak nature of the Raman effect. Raman-silica-gold-nanoparticles (R-Si-Au-NPs) overcome this limitation by producing larger Raman signals through surface-enhanced Raman scattering. Because we are developing these particles for use as targeted molecular imaging agents, we examined the acute toxicity and biodistribution of core polyethylene glycol (PEG)-ylated R-Si-Au-NPs after different routes of administration in mice. After intravenous administration, PEG-R-Si-Au-NPs were removed from the circulation by macrophages in the liver and spleen (that is, the reticuloendothelial system). At 24 hours, PEG-R-Si-Au-NPs elicited a mild inflammatory response and an increase in oxidative stress in the liver, which subsided by 2 weeks after administration. No evidence of significant toxicity was observed by measuring clinical, histological, biochemical, or cardiovascular parameters for 2 weeks. Because we are designing targeted PEG-R-Si-Au-NPs (for example, PEG-R-Si-Au-NPs labeled with an affibody that binds specifically to the epidermal growth factor receptor) to detect colorectal cancer after administration into the bowel lumen, we tested the toxicity of the core nanoparticle after administration per rectum. We observed no significant bowel or systemic toxicity, and no PEG-R-Si-Au-NPs were detected systemically. Although additional studies are required to investigate the long-term effects of PEG-R-Si-Au-NPs and their toxicity when carrying the targeting moiety, the results presented here support the idea that PEG-R-Si-Au-NPs can be safely used in living subjects, especially when administered rectally.
View details for DOI 10.1126/scitranslmed.3001963
View details for PubMedID 21508310
-
Imaging Cellular Receptors in Breast Cancers: An Overview
CURRENT PHARMACEUTICAL BIOTECHNOLOGY
2011; 12 (4): 508-527
Abstract
Breast cancer, a leading cause of cancer death in women, is strongly correlated with the up- and down-regulation of hormone and growth factor receptors. Therefore, improving our understanding of such receptor status in different stages of breast cancer will help in the development of novel diagnostic and therapeutic solutions. In particular, molecular imaging technology in association with advanced molecular and cell biology techniques could reveal in detail dynamic molecular events in cells, allowing the study of crucial molecular pathological changes occurring in cancer and other diseases. Molecular imaging techniques such as PET, SPECT, MRI, and the combinatorial techniques have made tremendous strides in elucidating the role of cellular receptors, helping to monitor the course of breast cancer development and the therapeutic efficacy of novel drugs. Optical imaging of cellular receptors is emerging as a powerful tool given the advancement of fluorescent and bioluminescent proteins. Estrogen receptor, progesterone receptor, and HER2/neu have been adopted clinically to detect different types of breast cancer and to test novel treatment strategies; however, other cellular receptors may also be involved in breast cancer subtyping and could emerge as treatment prospects. This review will focus on the recent developments of imaging various cellular receptors pertaining to the growth and development of breast cancer.
View details for Web of Science ID 000289770000006
View details for PubMedID 21342102
-
Non-Invasive Imaging of Ferucarbotran Labeled INS-1E Cells and Rodent Islets in Vitro and in Transplanted Diabetic Rats
CURRENT PHARMACEUTICAL BIOTECHNOLOGY
2011; 12 (4): 488-496
Abstract
Transplantation of pancreatic islets is a promising strategy for restoring insulin secretion in diabetes mellitus. To monitor transplanted islets, a method to evaluate the distribution in a non-invasive manner in vivo is needed. INS-1E, a stable differentiated insulin secreting cell line, and rodent islets were used to monitor cell transplantation by MRI. For labeling INS-1E cells in vitro, increasing concentrations of Resovist in culture medium were tested. For MR imaging in a clinical 3T scanner, we placed a layer of labeled INS-1E cells between two layers of 4% gelatin. Viability assay was performed. Cell function was evaluated by static incubation assay to assess insulin secretion. For in vivo imaging, iron labeled rodent islets were transplanted into the liver of streptozotocin induced diabetic rats and visualized by MRI. Blood sugar values were controlled and liver tissue was removed for histological analysis. SPIO labeled INS-1E cells did not show altered viability or reduced glucose stimulated insulin secretion in vitro. Double staining of labeled and unlabeled INS-1E cells showed no difference in the staining pattern. Labeling of rodent islets with SPIOs does not reduce their secretory activity or alter their viability. We visualized SPIO-labeled INS-1E cells and rat islets in vitro using a clinical 3T scanner. Diabetic rats transplanted with SPIO-labeled islets became normoglycemic. MR imaging successfully verified the distribution of labeled transplanted cells in vivo. Labeling INS-1E cells and rat islets with SPIOs does not alter their viability, while enabling MR imaging of labeled cells in vitro and within the living organism.
View details for Web of Science ID 000289770000004
View details for PubMedID 21342106
-
Molecular Imaging in Current Pharmaceuticals
CURRENT PHARMACEUTICAL BIOTECHNOLOGY
2011; 12 (4): 458-458
View details for Web of Science ID 000289770000001
View details for PubMedID 21342109
-
Oxidative Stress Mediates the Effects of Raman-Active Gold Nanoparticles in Human Cells
SMALL
2011; 7 (1): 126-136
Abstract
Polyethylene glycol (PEG)ylated Raman-active gold nanoparticles (PEG-R-AuNPs) consist of an interchangeable Raman organic molecule layer held onto a gold nanocore by a silica shell. PEG-R-AuNPs have been shown preclinically to increase the sensitivity and specificity of Raman spectroscopy, with picomolar sensitivity and multiplexing capabilities. Although clinical trials are being designed to use functionalized PEG-R-AuNPs in various applications (e.g., to target dysplastic bowel lesions during colonoscopy), the effects of these nanoparticles on human cells remain unknown. The occurrence and mechanisms underlying any potential cytotoxicity induced by these nanoparticles (0-1000 PEG-R-AuNPs/cell) are investigated in immortalized human HeLa and HepG2 cell lines at several time points (0-48 h) after exposure. Using fluorometric assays, cell viability (MTT), reactive oxygen species (ROS) generation (dichlorofluorescein diacetate), protein oxidation (protein carbonyl content), and total cellular antioxidant concentrations the concentrations (metmyoblobin-induced oxidation of ABTS) are assessed. Analysis of lipid oxidation using an enzyme immunoassay (8-isoprostane concentrations), gene expression of antioxidant enzymes using quantitative reverse transcription polymerase chain reactions, and the intracellular location of PEG-R-AuNPs using transmission electron microscopy is also undertaken. PEG-R-AuNPs cause no cytotoxicity in either HeLa or HepG2 cells in the acute setting as ROS generation is balanced by antioxidant enzyme upregulation. Following prolonged exposures (48 h) at relatively high concentrations (1000 PEG-R-AuNPs/cell), nanoparticles are found within vesicles inside cells. Under these conditions, a minimal amount of cytotoxicity is seen in both cell lines owing to increases in cellular oxidative stress, most likely due to ROS overwhelming the antioxidant defenses. Evidence of oxidative stress-induced damage includes increased lipid and protein oxidation. Although further in vivo toxicity studies are necessary, these initial encouraging results show that PEG-R-AuNPs cause minimal toxicity in human cells in the acute setting, which bodes well for potential future applications of these nanoparticles in living subjects.
View details for DOI 10.1002/smll.201001466
View details for PubMedID 21104804
-
Noninvasive molecular imaging of c-Myc activation in living mice
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (36): 15892-15897
Abstract
The cytoplasmic Myc protein (c-Myc) regulates various human genes and is dysregulated in many human cancers. Phosphorylation mediates the protein activation of c-Myc and is essential for the function of this transcription factor in normal cell behavior and tumor growth. To date, however, the targeting of Myc as a therapeutic approach for cancer treatment has been achieved primarily at the nonprotein level. We have developed a molecular imaging sensor for noninvasive imaging of c-Myc activity in living subjects using a split Firefly luciferase (FL) complementation strategy to detect and quantify the phosphorylation-mediated interaction between glycogen synthase kinase 3beta (GSK3beta) and c-Myc. This sensor system consists of two fusion proteins, GSK 35-433-CFL and NFL-c-Myc, in which specific fragments of GSK3beta and c-Myc are fused with C-terminal and N-terminal fragments of the split FL, respectively. The sensor detects phosphorylation-specific GSK3beta-c-Myc interaction, the imaging signal of which correlates with the steady-state and temporal regulation of c-Myc phosphorylation in cell culture. The sensor also detects inhibition of c-Myc activity via differential pathways, allowing noninvasive monitoring of c-Myc-targeted drug efficacy in intact cells and living mice. Notably, this drug inhibition is detected before changes in tumor size are apparent in mouse xenograft and liver tumor models. This reporter system not only provides an innovative way to investigate the role of functional c-Myc in normal and cancer-related biological processes, but also facilitates c-Myc-targeted drug development by providing a rapid quantitative approach to assessing cancer response to therapy in living subjects.
View details for DOI 10.1073/pnas.1007443107
View details for PubMedID 20713710
-
A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography
NATURE MEDICINE
2010; 16 (8): 921-U123
Abstract
Improved techniques to noninvasively image protein-protein interactions (PPIs) are essential. We molecularly engineered a positron emission tomography (PET)-based split reporter (herpes simplex virus type 1 thymidine kinase), cleaved between Thr265 and Ala266, and used this in a protein-fragment complementation assay (PCA) to quantify PPIs in mammalian cells and to microPET image them in living mice. An introduced point mutation (V119C) markedly enhanced thymidine kinase complementation in PCAs, on the basis of rapamycin modulation of FKBP12-rapamycin-binding domain (FRB) and FKBP12 (FK506 binding protein), the interaction of hypoxia-inducible factor-1alpha with the von Hippel-Lindau tumor suppressor, and in an estrogen receptor intramolecular protein folding assay. Applications of this unique split thymidine kinase are potentially far reaching, including, for example, considerably more accurate monitoring of immune and stem cell therapies, allowing for fully quantitative and tomographic PET localization of PPIs in preclinical small- and large-animal models of disease.
View details for DOI 10.1038/nm.2185
View details for Web of Science ID 000280649200033
View details for PubMedID 20639890
View details for PubMedCentralID PMC2917476
-
Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy
GENE THERAPY
2010; 17 (7): 827-838
Abstract
Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.
View details for DOI 10.1038/gt.2010.30
View details for Web of Science ID 000279614600002
View details for PubMedID 20237511
View details for PubMedCentralID PMC2900530
-
Antioxidants Improve Early Survival of Cardiomyoblasts After Transplantation to the Myocardium
MOLECULAR IMAGING AND BIOLOGY
2010; 12 (3): 325-334
Abstract
We tested the hypothesis that modulation of the microenvironment (using antioxidants) will increase stem cell survival in hypoxia and after transplantation to the myocardium.Rat cardiomyoblasts were stably transfected with a reporter gene (firefly luciferase) for bioluminescence imaging (BLI). First, we examined the role of oxidative stress in cells under hypoxic conditions. Subsequently, stem cells were transplanted to the myocardium of rats using high-resolution ultrasound, and their survival was monitored daily using BLI.Under hypoxia, oxidative stress was increased together with decreased cell survival compared to control cells, both of which were preserved by antioxidants. In living subjects, oxidative stress blockade increased early cell survival after transplantation to the myocardium, compared to untreated cells/animals.Modulation of the local microenvironment (with antioxidants) improves stem cell survival. Increased understanding of the interaction between stem cells and their microenvironment will be critical to advance the field of regenerative medicine.
View details for DOI 10.1007/s11307-009-0274-4
View details for Web of Science ID 000277375300011
View details for PubMedID 20013064
View details for PubMedCentralID PMC2865580
-
A Novel Estrogen Receptor Intramolecular Folding-based Titratable Transgene Expression System
MOLECULAR THERAPY
2009; 17 (10): 1703-1711
Abstract
The use of regulated gene expression systems is important for successful gene therapy applications. In this study, ligand-induced structural change in the estrogen receptor (ER) was used to develop a novel ER intramolecular folding-based transcriptional activation system. The system was studied using ER-variants of different lengths, flanked on either side by the GAL4-DNA-binding domain and the VP16-transactivation domain (GAL4(DBD)-ER-VP16). The ER ligands of different types showed efficient ligand-regulated transactivation. We also characterized a bidirectional transactivation system based on the ER and demonstrated its utility in titrating both reporter and therapeutic gene expression. The ligand-regulated transactivation system developed by using a mutant form of the ER (G521T, lacking affinity for the endogenous ligand 17beta-estradiol, whereas maintaining affinity for other ligands) showed efficient activation by the ligand raloxifene in living mice without significant interference from the circulating endogenous ligand. The ligand-regulated transactivation system was used to test the therapeutic efficiency of the tumor suppressor protein p53 in HepG2 (p53(+/+)) and SKBr3 (p53(-/-)/mutant-p53(+/+)) cells in culture and tumor xenografts in living mice. The multifunctional capabilities of this system should be useful for gene therapy applications, to study ER biology, to evaluate gene regulation, ER ligand screening, and ER ligand biocharacterization in cells and living animals.
View details for DOI 10.1038/mt.2009.171
View details for PubMedID 19654568
-
Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals
RADIOLOGY
2009; 252 (1): 117-127
Abstract
To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed.In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs.Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.
View details for DOI 10.1148/radiol.2513081616
View details for Web of Science ID 000268362900015
View details for PubMedID 19366903
View details for PubMedCentralID PMC2702468
-
Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation
MOLECULAR IMAGING AND BIOLOGY
2009; 11 (3): 178-187
Abstract
In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively.Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis.BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days).Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.
View details for DOI 10.1007/s11307-008-0182-z
View details for PubMedID 19034584
-
Molecular Imaging of Phosphorylation Events for Drug Development
MOLECULAR IMAGING AND BIOLOGY
2009; 11 (3): 144-158
Abstract
Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging.An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA).The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity.This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events.
View details for DOI 10.1007/s11307-008-0187-7
View details for Web of Science ID 000265686900002
View details for PubMedID 19048345
View details for PubMedCentralID PMC4154800
-
Uptake kinetics and biodistribution of C-14-D-luciferin-a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging
EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING
2008; 35 (12): 2275-2285
Abstract
Firefly luciferase catalyzes the oxidative decarboxylation of D: -luciferin to oxyluciferin in the presence of cofactors, producing bioluminescence. This reaction is used in optical bioluminescence-based molecular imaging approaches to detect the expression of the firefly luciferase reporter gene. Biokinetics and distribution of the substrate most likely have a significant impact on levels of light signal and therefore need to be investigated.Benzene ring (14)C(U)-labeled D-luciferin was utilized. Cell uptake and efflux assays, murine biodistribution, autoradiography and CCD-camera based optical bioluminescence imaging were carried out to examine the in vitro and in vivo characteristics of the tracer in cell culture and in living mice respectively.Radiolabeled and unlabeled D-luciferin revealed comparable levels of light emission when incubated with equivalent amounts of the firefly luciferase enzyme. Cell uptake assays in pCMV-luciferase-transfected cells showed slow trapping of the tracer and relatively low uptake values (up to 22.9-fold higher in firefly luciferase gene-transfected vs. nontransfected cells, p = 0.0002). Biodistribution studies in living mice after tail-vein injection of (14)C-D-luciferin demonstrated inhomogeneous tracer distribution with early predominant high radioactivity levels in kidneys (10.6% injected dose [ID]/g) and liver (11.9% ID/g), followed at later time points by the bladder (up to 81.3% ID/g) and small intestine (6.5% ID/g), reflecting the elimination routes of the tracer. Kinetics and uptake levels profoundly differed when using alternate injection routes (intravenous versus intraperitoneal). No clear trapping of (14)C-D-luciferin in firefly luciferase-expressing tissues could be observed in vivo.The data obtained with (14)C-D-luciferin provide insights into the dynamics of D: -luciferin cell uptake, intracellular accumulation, and efflux. Results of the biodistribution and autoradiographic studies should be useful for optimizing and adapting optical imaging protocols to specific experimental settings when utilizing the firefly luciferase and D: -luciferin system.
View details for DOI 10.1007/s00259-008-0870-6
View details for Web of Science ID 000261654000015
View details for PubMedID 18661130
-
Noninvasive Imaging of Therapeutic Gene Expression Using a Bidirectional Transcriptional Amplification Strategy
MOLECULAR THERAPY
2008; 16 (11): 1848-1856
Abstract
Promoters that limit transgene expression to tumors play a vital role in cancer gene therapy. Although tumor specific, the human Survivin promoter (pSurv) elicits low levels of transcription. A bidirectional two-step transcriptional amplification (TSTA) system was designed to enhance expression of the therapeutic gene (TG) tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL or TR) and the reporter gene firefly luciferase (FL) from pSurv. An adenoviral vector carrying the enhanced targeting apparatus (Ad-pSurv-TR-G8-FL) was tested for efficiency and specificity of gene expression in cells and in living animals. Compared to the one-step systems (Ad-pSurv-FL or Ad-pSurv-TR), the bidirectional TSTA system showed tenfold higher expression of both the therapeutic and the reporter gene and their expression correlated in cells (R(2) = 0.99) and in animals (R(2) = 0.67). Noninvasive quantitative monitoring of magnitude and time variation of TRAIL gene expression was feasible by bioluminescence imaging of the transcriptionally linked FL gene in xenograft tumors following intratumoral adenoviral injection. Moreover, the TSTA adenovirus maintained promoter specificity in nontarget tissues following tail vein administration. These studies demonstrate the potential of the bidirectional TSTA system to achieve high levels of gene expression from a weak promoter, while preserving specificity and the ability to image expression of the TG noninvasively.
View details for DOI 10.1038/mt.2008.180
View details for Web of Science ID 000260472600015
View details for PubMedID 18766175
View details for PubMedCentralID PMC3195556
-
Dual-targeted contrast agent for US assessment of tumor angiogenesis in vivo
RADIOLOGY
2008; 248 (3): 936-944
Abstract
To develop and validate a dual-targeted ultrasonographic (US) imaging agent with microbubbles (MBs) that attaches to both vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and alpha(v)beta(3) integrin and to compare the US imaging signal obtained from dual-targeted MBs (MB(D)) with that from single-targeted MBs (MB(S)) in a murine model of tumor angiogenesis.Animal protocols were approved by the institutional Administrative Panel on Laboratory Animal Care. Single- and dual-targeted US imaging agents were prepared by attaching anti-VEGFR2, anti-alpha(v)beta(3) integrin, or both antibodies to the shell of perfluorocarbon-filled MBs. Binding specificities of targeted MBs compared with isotype-matched immunoglobulin G-labeled control MBs (MB(C)) and nontargeted nonlabeled MBs (MB(N)) were tested with VEGFR2-positive and alpha(v)beta(3) integrin-positive cells (mouse SVR cells) and control cells (mouse 4T1 cells). In vivo imaging signals of contrast material-enhanced US by using anti-VEGFR2-targeted MBs (MB(V)), anti-alpha(v)beta(3) integrin-targeted MBs (MB(I)), MB(D), and MB(C) were quantified in 49 mice bearing SK-OV-3 tumors (human ovarian cancer). Tumor tissue was stained for VEGFR2, alpha(v)beta(3) integrin, and CD31.Attachment of MB(D) to SVR cells (mean, 0.74 MBs per cell +/- 0.05 [standard deviation]) was significantly higher than attachment to 4T1 cells (mean, 0.04 +/- 0.03), and attachment to SVR cells was higher for MB(D) than for MB(V) (mean, 0.58 +/- 0.09), MB(I) (mean, 0.42 +/- 0.21), MB(C) (mean, 0.11 +/- 0.13), and MB(N) (mean, 0.01 +/- 0.01) (P < .05). Imaging signal in the murine tumor angiogenesis model was significantly higher (P < .001) for MB(D) (mean, 16.7 +/- 7.2) than for MB(V) (mean, 11.3 +/- 5.7), MB(I) (mean, 7.8 +/- 5.3), MB(C) (mean, 2.8 +/- 0.9), and MB(N) (mean, 1.1 +/- 0.4). Immunofluorescence confirmed expression of VEGFR2 and alpha(v)beta(3) integrin on tumor vasculature.Dual-targeted contrast-enhanced US directed at both VEGFR2 and alpha(v)beta(3) integrin improves in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft tumor model in mice.http://radiology.rsnajnls.org/cgi/content/full/248/3/936/DC1.
View details for DOI 10.1148/radiol.2483072231
View details for Web of Science ID 000258541500031
View details for PubMedID 18710985
View details for PubMedCentralID PMC2798094
-
The death domain-containing kinase RIP1 regulates p27(Kip1) levels through the PI3K-Akt-forkhead pathway
EMBO REPORTS
2008; 9 (8): 766-773
Abstract
Elucidating the cross-talk between inflammatory and cell proliferation pathways might provide important insights into the pathogenesis of inflammation-induced cancer. Here, we show that the receptor-interacting protein 1 (RIP1)-an essential mediator of inflammation-induced nuclear factor-kappaB (NF-kappaB) activation-regulates p27(Kip1) levels and cell-cycle progression. RIP1 regulates p27(Kip1) levels by an NF-kappaB-independent signal that involves activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-forkhead pathway. Mouse embryonic fibroblasts (MEFs) from RIP1-knockout mice express high levels of p27(Kip1). Reconstitution of MEFs with RIP1 downregulates p27(Kip1) levels in a PI3K-dependent manner. RIP1 regulates p27(Kip1) at the messenger RNA level by regulating the p27(Kip1) promoter through the forkhead transcription factors. RIP1 expression blocks accumulation of cells in G(1) in response to serum starvation and favours cell-cycle progression. Finally, we show that overexpression of p27(Kip1) blocks the effects of RIP1 on the cell cycle. Thus, our study provides a new insight into how components of inflammatory and immune signalling pathways regulate cell-cycle progression.
View details for DOI 10.1038/embor.2008.102
View details for Web of Science ID 000258146000015
View details for PubMedID 18566599
View details for PubMedCentralID PMC2515213
-
A human estrogen receptor (ER)alpha mutation with differential responsiveness to nonsteroidal ligands: Novel approaches for studying mechanism of ER action
MOLECULAR ENDOCRINOLOGY
2008; 22 (7): 1552-1564
Abstract
Estrogens, acting through the estrogen receptors (ERs), play crucial roles in regulating the function of reproductive and other systems under physiological and pathological conditions. ER activity in regulating target genes is modulated by the binding of both steroidal and synthetic nonsteroidal ligands, with ligand binding inducing ERs to adopt various conformations that control their interactions with transcriptional coregulators. Previously, we developed an intramolecular folding sensor with a mutant form of ERalpha (ER(G521T)) that proved to be essentially unresponsive to the endogenous ligand 17beta-estradiol, yet responded very well to certain synthetic ligands. In this study, we have characterized this G521T-ER mutation in terms of the potency and efficacy of receptor response toward several steroidal and nonsteroidal ligands in two different ways: directly, by ligand effects on mutant ER conformation (by the split-luciferase complementation system), and indirectly, by ligand effects on mutant ER transactivation. Full-length G521T-ER shows no affinity for estradiol and does not activate an estrogen-responsive reporter gene. The synthetic pyrazole agonist ligand propyl-pyrazole-triol is approximately 100-fold more potent than estradiol in inducing intramolecular folding and reporter gene transactivation with the mutant ER, whereas both ligands have high potency on wild-type ER. This estradiol-unresponsive mutant ER can also specifically highlight the agonistic property of the selective ER modulator, 4-hydroxytamoxifen, by reporter gene transactivation, even in the presence of estradiol, and it can exert a dominant-negative effect on estrogen-stimulated wild-type ER. This system provides a model for ER-mutants that show differential ligand responsiveness to gene activation to gain insight into the phenomenon of hormone resistance observed in endocrine therapies of ER-positive breast cancers.
View details for DOI 10.1210/me.2007-0570
View details for PubMedID 18451095
-
Molecular Imaging of Hypoxia-Inducible Factor 1 alpha and von Hippel-Lindau Interaction in Mice
MOLECULAR IMAGING
2008; 7 (3): 139-146
Abstract
Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.
View details for DOI 10.2310/7290.2008.00017
View details for Web of Science ID 000260954700004
View details for PubMedID 19123984
-
Monitoring of the biological response to murine Hindlimb ischemia with Cu-64-labeled vascular endothelial growth factor-121 positron emission tomography
CIRCULATION
2008; 117 (7): 915-922
Abstract
Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training.64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem gamma-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P < 0.001) from a control level of 0.61+/-0.17% ID/g (percentage of injected dose per gram) to 1.62+/-0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59+/-0.14% ID/g at day 29), and correlated with gamma-counting (R2 = 0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P < or = 0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2 = 0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice.64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.
View details for DOI 10.1161/CIRCULATIONAHA.107.733220
View details for PubMedID 18250264
-
US imaging of tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice
RADIOLOGY
2008; 246 (2): 508-518
Abstract
To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance.In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types.US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.
View details for DOI 10.1148/radio1.2462070536
View details for Web of Science ID 000252796300021
View details for PubMedID 18180339
-
RIP1 links inflammatory and growth factor signaling pathways by regulating expression of the EGFR
CELL DEATH AND DIFFERENTIATION
2008; 15 (2): 344-353
Abstract
There is considerable interest in understanding how inflammatory responses influence cell proliferation and cancer. In this study, we show that the receptor-interacting protein (RIP1), a critical mediator of inflammation and stress-induced NF-kappaB activation, regulates the expression of the epidermal growth factor receptor (EGFR). Mouse embryo fibroblasts (MEFs) derived from RIP1 knockout mice express very high levels of the EGFR. Reconstitution of RIP1(-/-) MEFs with RIP1 results in a lowering of EGFR levels. RIP1 influences EGFR at the mRNA level by regulating the EGFR promoter. Expression of RIP1 inhibits the EGFR promoter. RIP1 downregulates EGFR expression by interfering with the function of Sp1, which is a key activator of EGFR transcription. RIP1 suppresses Sp1 activity and overexpression of Sp1 reverses RIP1-mediated repression of the EGFR promoter. RIP1 is present both in the cytoplasm and in the nucleus. RIP1 coimmunoprecipitates with Sp1 in vivo and binds directly to Sp1 in vitro. A RIP1 mutant lacking the death domain fails to suppress Sp1 activity and the EGFR promoter, suggesting a critical role for the RIP1 death domain in EGFR regulation. Thus, our study identifies a new link between inflammatory and growth factor signaling pathways mediated by RIP1 and provides insight into the mechanism used by RIP1 to regulate EGFR levels.
View details for DOI 10.1038/sj.cdd.4402268
View details for Web of Science ID 000252387900014
View details for PubMedID 18007664
-
Molecular imaging of the efficacy of heat shock protein 90 inhibitors in living subjects
CANCER RESEARCH
2008; 68 (1): 216-226
Abstract
Heat shock protein 90 alpha (Hsp90 alpha)/p23 and Hsp90 beta/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90 alpha/p23 and Hsp90 beta/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purine-scaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90 alpha/p23 and Hsp90 beta/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90 alpha/p23 and Hsp90 beta/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoform-selective Hsp90 inhibitors.
View details for DOI 10.1158/0008-5472.CAN-07-2268
View details for Web of Science ID 000252072100029
View details for PubMedID 18172314
View details for PubMedCentralID PMC4146344
-
Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions
ANALYTICAL CHEMISTRY
2007; 79 (6): 2346-2353
Abstract
Split reporter-based bioluminescence imaging is a useful strategy for studying protein-protein as well as other intracellular interactions. We have used a combinatorial strategy to identify a novel split site for firefly luciferase with improved characteristics over previously published split sites. A combination of fragments with greater absolute signal with near-zero background signals was achieved by screening 115 different combinations. The identified fragments were further characterized by using five different interacting protein partners and an intramolecular folding strategy. Cell culture studies and imaging in living mice was performed to validate the new split sites. In addition, the signal generated by the newly identified combination of fragments (Nfluc 398/ Cfluc 394) was compared with different split luciferase fragments currently in use for studying protein-protein interactions and was shown to be markedly superior with a lower self-complementation signal and equal or higher postinteraction absolute signal. This study also identified many different combinations of nonoverlapping and overlapping firefly luciferase fragments that can be used for studying different cellular events such as subcellular localization of proteins, cell-cell fusion, and evaluating cell delivery vehicles, in addition to protein-protein interactions, both in cells and small living animals.
View details for DOI 10.1021/ac062053q
View details for Web of Science ID 000244867100020
View details for PubMedID 17295448
View details for PubMedCentralID PMC3198827
-
Reporter gene imaging of protein-protein interactions in living subjects
CURRENT OPINION IN BIOTECHNOLOGY
2007; 18 (1): 31-37
Abstract
In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.
View details for DOI 10.1016/j.copbio.2007.01.007
View details for Web of Science ID 000244593000006
View details for PubMedID 17254764
-
Noninvasive monitoring of ligand-dependent VEGF receptor-2 dimerization with split firefly luciferase
49th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO)
ELSEVIER SCIENCE INC. 2007: S96–S97
View details for Web of Science ID 000249950200173
- Antibacterial activity of subterranean termites used in South Indian folk medicine. Indian Journal of Traditional Knowledge 2007; 6(4) (Oct): 559-562
-
An intramolecular folding sensor for imaging estrogen receptor-ligand interactions
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (43): 15883-15888
Abstract
Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla/firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.
View details for DOI 10.1073/pnas.0607385103
View details for Web of Science ID 000241568500029
View details for PubMedID 17043219
View details for PubMedCentralID PMC1635097
-
Visualization of telomerase reverse transcriptase (hTERT) promoter activity using a trimodality fusion reporter construct
JOURNAL OF NUCLEAR MEDICINE
2006; 47 (2): 270-277
Abstract
Our goal was to noninvasively measure chemotherapy-induced changes in the expression of critical tumor growth genes. To achieve this goal, we used radionuclide and optical methods to measure changes in human telomerase reverse transcriptase (hTERT) gene expression in tumor cells before and after 5-fluorouracil treatment.A fusion reporter construct, containing humanized Renilla luciferase (hrl, for bioluminescent imaging), monomeric red fluorescence protein 1 (mrfp1, for fluorescent imaging), and a truncated thymidine kinase (ttk, for imaging of radiolabeled acycloguanosines), was placed under the control of hTERT promoter fragments. These constructs were introduced into tumor cell lines with and without hTERT expression. Transfected cells were treated with 5-fluorouracil, a chemotherapeutic that decreases hTERT gene expression, and treatment-induced changes in hTERT promoter activity were imaged.When the fusion construct is introduced into cell lines that express hTERT, all 3 reporter systems are highly expressed and hTERT promoter activity can be visualized. Cell lines lacking hTERT transcription show no significant reporter expression. Decreases in hTERT gene expression caused by 5-fluorouracil treatment could be visualized in living 293T cells by both fluorescent microscopy and bioluminescent imaging.hTERT promoter activity can be monitored by 1 radionuclide and 2 optical reporter systems using a single reporter construct. This in vitro study provides evidence that our multimodality reporter construct can be used to study the expression of a critical tumor growth gene in living subjects.
View details for Web of Science ID 000235283500027
View details for PubMedID 16455633
-
Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.
FASEB journal
2006; 20 (1): 106-108
Abstract
Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.
View details for PubMedID 16246867
- Effect of the subterranean termite used in the South Indian folk medicine. Indian Journal of Traditional Knowledge 2006; 5(3) (Jul): 376-9
- Split luciferase complementation assay for studying interaction of proteins x and y in cells. CSH Protoc 2006; Nov1;(6) (10.1101/): pdb.prot4596
- Split luciferase complementation assay for studying interaction of proteins x and y in living mice. CSH Protoc 2006; Nov1(6) (10.1101/): pdb.prot4596
-
Split luciferase complementation assay for studying interaction of proteins x and y in cells.
CSH protocols
2006; 2006 (6)
Abstract
This protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the "split protein" strategy, a single reporter protein/enzyme (in this case, firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X and Y. Physical interactions between the two proteins, X and Y, reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assays. It is important to perform the assay initially in cell culture before proceeding with animal imaging, not only to conserve animals, but also to establish the sensitivity of the assay.
View details for DOI 10.1101/pdb.prot4596
View details for PubMedID 22485983
-
Split luciferase complementation assay for studying interaction of proteins x and y in living mice.
CSH protocols
2006; 2006 (6)
Abstract
This protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), as described below, or with tumor models grown from tumor cells stably expressing the complete reporter system. For optical imaging, the number of cells implanted can be relatively low (~1-5 × 10(6)), and imaging can begin even before the tumors are palpable. When using a regulated reporter gene, it may be necessary to perform a dose-dependent pilot experiment with the inducer or repressor before performing the primary experiments. Animal models other than mice can be used, including rats and, in theory, transgenic animals in which the reporter constructs have been stably integrated into the genome. Animals larger than the rat would be difficult to image due to poor penetration of light. For these larger-animal models, the use of other imaging technologies such as microPET should be considered.
View details for DOI 10.1101/pdb.prot4595
View details for PubMedID 22485982
-
Molecular imaging of HIF-1 alpha and pVHL interaction in living subjects using firefly luciferase complementation - Improvement over the Renilla luciferase system
48th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO)
ELSEVIER SCIENCE INC. 2006: S48–S49
View details for Web of Science ID 000241221600083
-
Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging
FASEB JOURNAL
2005; 19 (12): 106-?
Abstract
Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.
View details for DOI 10.1096/fj.05-4551fje
View details for Web of Science ID 000232991100011
View details for PubMedCentralID PMC3625424
-
Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals
CANCER RESEARCH
2005; 65 (16): 7413-7420
Abstract
Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.
View details for DOI 10.1158/0008-5472.CAN-05-0588
View details for Web of Science ID 000231188600049
View details for PubMedID 16103094
-
Imaging protein-protein interactions in living subjects
TRAC-TRENDS IN ANALYTICAL CHEMISTRY
2005; 24 (5): 446-458
View details for Web of Science ID 000234203000028
-
Firefly luciferase enzyme fragment complementation for imaging in cells and living animals
ANALYTICAL CHEMISTRY
2005; 77 (5): 1295-1302
Abstract
We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering TAT-Cfluc (265-550) to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.
View details for DOI 10.1021/ac0484777
View details for Web of Science ID 000227409800021
View details for PubMedID 15732910
-
Molecular imaging of HIF-1 alpha and pVHL interaction in vivo: The study of structure/function relationship guiding HIF-1 alpha and pVHL
47th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology
ELSEVIER SCIENCE INC. 2005: S133–S134
View details for Web of Science ID 000232083300224
- Imaging protein-protein interactions in living subjects. TRAC: Trends in Analytical Chemistry 2005; 24(5): 446-458
-
Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression
HUMAN GENE THERAPY
2004; 15 (7): 681-690
Abstract
Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.
View details for Web of Science ID 000222786900006
View details for PubMedID 15242528
View details for PubMedCentralID PMC4153396
-
Molecular imaging of homodimeric protein-protein interactions in living subjects.
FASEB journal
2004; 18 (10): 1105-1107
Abstract
Homodimeric protein interactions are potent regulators of cellular functions, but are particularly challenging to study in vivo. We used a split synthetic renilla luciferase (hRLUC) complementation-based bioluminescence assay to study homodimerization of herpes simplex virus type 1 thymidine kinase (TK) in mammalian cells and in living mice. We quantified and imaged homodimerization of TK chimeras containing N-terminal (N-hRLUC) or C-terminal (C-hRLUC) fragments of hRLUC in the upstream and downstream positions, respectively (tail-to-head homodimer). This was monitored using luminometry (68-fold increase, and was significantly [P<0.01] above background light emission) and by CCD camera imaging of living mice implanted with ex vivo transfected 293T cells (2.7-fold increase, and is significantly [P<0.01] above background light emission). We also made a mutant-TK to generate N-hRLUC mutant TK and mutant TK-C-hRLUC by changing a single amino acid at position 318 from arginine to cysteine, a key site that has previously been reported to be essential for TK homo-dimerization, to support the specificity of the hRLUC complementation signal from TK homodimerization. Ex vivo substrate (8-3H Penciclovir) accumulation assays in 293T cells expressing the TK protein chimeras showed active TK enzyme. We also devised an experimental strategy by constructing variant TK chimeras (possessing extra N-hRLUC or C-hRLUC 'spacers') to monitor incremental lack of association of the tail-to-head TK homodimer. Application of this potentially generalizable assay to screen for molecules that promote or disrupt ubiquitous homodimeric protein-protein interactions could serve not only as an invaluable tool to understand biological networks but could also be applied to drug discovery and validation in living subjects.
View details for PubMedID 15132989
-
Molecular imaging of homodimeric protein-protein interactions in living subjects
FASEB JOURNAL
2004; 18 (7): 1105-?
View details for DOI 10.1096/fj.03-1128fje
View details for Web of Science ID 000221883200025
-
MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene
GENE THERAPY
2004; 11 (7): 609-618
Abstract
Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.
View details for DOI 10.1038/sj.gt.3302194
View details for Web of Science ID 000220281300006
View details for PubMedID 14724687
-
Molecular imaging of drug-modulated protein-protein interactions in living subjects
CANCER RESEARCH
2004; 64 (6): 2113-2119
Abstract
Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor alpha, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.
View details for PubMedID 15026351
- Impact of physico- chemical parameters on the microbial population and its nature in a major retting zone of Kerala, S. India. International Journal of Environmental Studies, UK 2004; 61(5): 595-597
- Traditional therapeutic uses of animals among tribal population of Tamil Nadu. Indian Journal of Traditional Knowledge 2004; 3(2) (Apr): 198-205
-
Loss of expression, and mutations of Smad 2 and Smad 4 in human cervical cancer
ONCOGENE
2003; 22 (31): 4889-4897
Abstract
Mutations in Smads, intermediates of transforming growth factor-beta signaling, are known to contribute to the loss of sensitivity to transforming growth factor-beta, a common feature of many neoplastic cells. However, not much information is available on Smad alterations in cervical cancer and so we probed, for the first time, for alterations in Smad 2 and Smad 4 genes using human cervical cancer cell lines and human cervical tissue samples. Using PCR/reverse transcription-PCR, single-stranded conformation polymorphism analysis and DNA sequencing, we observed a deletion of 'G' in the L3 loop (crucial in Smad-receptor interaction) in C-33A cells, and an insertion of 'A' in codon 122 (loss of MH2 domain) from a cervical tumor sample, both of which caused frame shift and pretermination in Smad 2. In addition, a G/A transition at 31 bp upstream-nontranslated regions of exon 8 of Smad 4 was found in Bu 25TK cells. Smad 2 expression was less in some of the cervical tumor samples than that of nonmalignant samples and six cancer samples showed C-terminal deletions that abolish Smad 2 phosphorylation sites. The loss of expression of Smad 4 found in some cervical tumor samples was due to transcription loss rather than deletion of the gene. Our results highlight an important role for Smad 2 and Smad 4 in human cervical tumors.
View details for DOI 10.1038/sj.onc.1206806
View details for Web of Science ID 000184344600013
View details for PubMedID 12894231
-
Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation
ANALYTICAL CHEMISTRY
2003; 75 (7): 1584-1589
Abstract
In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.
View details for DOI 10.1021/ac020731c
View details for Web of Science ID 000181993600013
View details for PubMedID 12705589
-
Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (24): 15608-15613
Abstract
In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.
View details for DOI 10.1073/pnas.242594299
View details for Web of Science ID 000179530000065
View details for PubMedID 12438689
View details for PubMedCentralID PMC137764
-
Noninvasive quantitative imaging of protein-protein interactions in living subjects
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (5): 3105-3110
Abstract
We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.
View details for DOI 10.1073/pnas.052710999
View details for Web of Science ID 000174284600092
View details for PubMedID 11854471
View details for PubMedCentralID PMC122480
-
Simple radioactive assay for the estimation of DNA breaks
JOURNAL OF APPLIED TOXICOLOGY
2002; 22 (1): 19-23
Abstract
The intactness of DNA is an important part of the normal cellular structure. Any change to the DNA in the form of breaks leads to a change in the integrity, which in turn leads to abnormality in the cellular activity. Many discrepancies have been reported among the various methods of detecting DNA damage. Here, a simple, sensitive and reproducible method has been developed for the detection of DNA breaks by radioactive labelling of 5' broken ends. The method was evaluated by studying chemically induced DNA damage by using both organochloride (2,4-dichlorophenoxyacetic acid and lindane) and organophosphorus (sevin and phosphamidon) compounds at different concentrations. Phosphamidon, one of the organophosphorus compounds studied, showed complete degradation of the DNA after treatment. Radioactive analysis of phosphamidon showed higher counts at the lowest concentration (20 microg) of the chemical when compared with the control (2752 scintillation counts per minute, scm). Studies on the chemically induced DNA breaks by radiolabelling revealed that the cumulative effect of the organophosphorus and organochloride compounds showed maximum counts in all the samples (the highest being 2904 scm) when compared with the organophosphorus and organochloride compounds studied separately (the highest being 1881 and 2260 scm, respectively). Radiolabelling studies on the blood samples of 23 pesticide workers by the newly developed assay showed a significant positive correlation (0.893) between the number of years of exposure and the scintillation counts. A maximum of 11,702 scm (for 18 years of exposure) and a minimum of 1682 scm (for 4 years of exposure) were recorded compared with 1253 scm for the negative control. This method can be used effectively for estimation of the DNA breaks, irrespective of its nature.
View details for DOI 10.1002/jat.807
View details for Web of Science ID 000173603300003
View details for PubMedID 11807925
- Distribution of HCH residues in the Kuttanad wetland ecosystem of Kerala. Inter. J. Ecology and Environmental Science 2001; 7(1): 105-109
- Studies on the water quality of Kuttanad wetland ecosystem of Kerala. Pollution Research 2001; 20(1) (62): 138-148
-
Duplex RT-PCR for simultaneous detection of hepatitis A and hepatitis E virus isolated from drinking water samples
JOURNAL OF ENVIRONMENTAL MONITORING
2000; 2 (6): 587-590
Abstract
A duplex reverse transcription polymerase chain reaction (RT-PCR) protocol for simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in water samples has been developed and demonstrated. Both HAV and HEV were concentrated from drinking water samples through a one-step concentration protocol. Different cDNA could be produced in the RT step carried out with a random primer in a single reaction tube. Two different sets of primers specific for HAV-cDNA and HEV-cDNA were used for PCR amplification. Amplified DNA products representing HAV and HEV were identified by gel electrophoresis at 247 and 327 bp (base pair) sequences, respectively. Specific sets of primers amplified a single type of virus and no cross-reactivity of the primers was noticed in duplex RT-PCR. The protocol was used for direct isolation and detection of HAV and HEV from 23 water samples in urban areas of Chennai city. Out of these, nine water samples were positive for HAV, and three for HEV. All three samples positive for HEV were also positive for HAV. The test provides a rapid and economical means of water quality surveillance to specifically detect HAV and HEV.
View details for Web of Science ID 000165993200013
View details for PubMedID 11296746
- Pollution and infectious diseases. Science, India 2000; 3: 39-42
- Drinking water quality status of Kottarakara area, Kollam District, Kerala. Indian J. Environ. & Ecoplanning 2000; 3(1): 143-145
- Spatio temporal distribution and ecology of benthos in the Kuttanad wetland ecosystem. Pollution Research 1999; 18(3): 235-243
- A study on the distribution and ecology of phytoplankton in the Kuttanad wetland ecosystem, Kerala. Pollution Research 1999; 18(3): 261-269
- Distribution of heavy metals in Kuttanad wetland ecosystem of Kerala, India. Inter. J. Ecology and Environmental Science 1999; 25: 91-95
-
Membrane-impregnated probe for simultaneous PCR amplification and detection
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
1998; 14 (6): 933-934
View details for Web of Science ID 000078562600024
- Analysis of human immune response to potential hepatitis C viral epitopes. Acta Virologica 1998; 42: 141-145
- Membrane impregnated probe for simultaneous PCR amplification and detection. World Journal of Microbiology and Biotechnology 1998; 14: 933-934
-
A SIMPLE DEVICE FOR THE CONCENTRATION AND DETECTION OF ENTEROVIRUS, HEPATITIS-E VIRUS AND ROTAVIRUS FROM WATER SAMPLES BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION
JOURNAL OF VIROLOGICAL METHODS
1995; 55 (3): 401-415
Abstract
A simultaneous concentration of enteroviruses, hepatitis E virus, and rotavirus from drinking water samples through a filtration column filled with granular activated carbon (GAC) was achieved. Urea-arginine phosphate buffer (UAPB) as an eluent at pH 9.0 was used for effective desorption and elution of viruses from GAC. Further concentration of viruses with magnesium chloride enabled nucleic acid extraction, cDNA synthesis, amplification with a specific set of primers for enterovirus, hepatitis E virus and rotavirus. Polymerase chain reaction (PCR) products were then confirmed by Southern transfer and hybridization with the relevant probes. The efficacy of the protocol was established with 100 1 of water samples seeded with poliovirus-1, providing 74% recovery in granular activated carbon based UAPB-RT-PCR. The GAC-based method for concentration of viruses from water samples was preferred, despite its somewhat lower efficacy compared to 80% in membrane filter based UAPB-RT-PCR protocol, due to the specific requirements of short-time and savings in cost of analyses. The protocol was used for the detection of waterborne viruses from 24 drinking water sources in urban areas of New Delhi. Direct isolation of viruses from water samples revealed that the 4 samples were positive for enteroviruses, two for hepatitis E virus, and 10 samples for rotavirus. One sample was positive for both hepatitis E virus and rotavirus, and another for all the 3 types of viruses.
View details for Web of Science ID A1995TH30800012
View details for PubMedID 8609205
- Concentration and detection of rota virus in water samples using polymerase chain reaction during a gasteroenteries epidemic outbreak in Madras city. International Journal of Environmental Studies 1994; 46: 323-327
-
DETECTION OF HEPATITIS-E VIRUS IN RAW AND TREATED WASTE-WATER WITH THE POLYMERASE CHAIN-REACTION
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
1993; 59 (8): 2558-2562
Abstract
The main objective of this study was to determine the applicability of the polymerase chain reaction (PCR) to detection of hepatitis E virus (HEV) in sewage treatment plants and establishment of the prevalence of hepatitis viral diseases in a population. Epidemics of HEV infection because of inadequate public sanitation have been reported in several developing countries. A procedure for concentration of HEV in sewage samples through adsorption to membrane filters, elution with urea-arginine phosphate buffer, and subsequent reconcentration with magnesium chloride enabled us to concentrate HEV to volumes in the microliter range. HEV-specific cDNA was prepared by reverse transcription of the total RNA extracted from samples. Specific DNA amplification by PCR in combination with slot blot hybridization was used to demonstrate the presence of HEV in sewage samples from the inlets and outlets of three sewage treatment plants. The assay was specific for HEV, and a 240-bp amplified product was visualized by ethidium bromide fluorescence. Sewage samples adjusted to pH 5.0 for adsorption of viruses to membrane filters were PCR positive, while samples adjusted to pH 3.5 were PCR negative.
View details for Web of Science ID A1993LP83500032
View details for PubMedID 8368844
View details for PubMedCentralID PMC182320
- Rapid detection of waterborne viruses using the polymerase chain reaction and a gene probe. Intervirology 1991; 34: 184-191