- BIOPROCESSING OF SURGICAL PEDIATRIC BRAIN TUMOR SPECIMENS FOR GENOME-GUIDED PERSONALIZED DRUG TESTING OXFORD UNIV PRESS INC. 2023
THERAPY-INDUCED CHANGES BY BRAF AND MEK INHIBITORS IN BRAF V600E-MUTATED GLIOMA MODELS PROVIDE POTENTIAL NOVEL THERAPEUTIC OPPORTUNITIES
OXFORD UNIV PRESS INC. 2022: 34
View details for Web of Science ID 000888571000135
BIOPROCESSING OF SURGICAL PEDIATRIC BRAIN TUMOR SPECIMENS FOR GENOME-GUIDED PERSONALIZED DRUG TESTING
OXFORD UNIV PRESS INC. 2022: 232
View details for Web of Science ID 000888571001208
- Deep Immune Profiling of an Arginine-Enriched Nutritional Intervention in Patients Undergoing Surgery JOURNAL OF IMMUNOLOGY 2017; 199 (6): 2171–80
Deep Immune Profiling of an Arginine-Enriched Nutritional Intervention in Patients Undergoing Surgery.
Journal of immunology (Baltimore, Md. : 1950)
Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific, as they reduce infection rates in elective surgery, but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably, AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery, consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly, AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells, which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms.
View details for PubMedID 28794234
Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy.
Journal of immunology
Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4(+) and CD8(+) T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet(+)CD4(+) T cells, CD8(+) T cells, B cells, and CD56(lo)CD16(+) NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes.
View details for PubMedID 27793998
SYSTEMS-WIDE MODULATION OF PATIENTS IMMUNE RESPONSE TO SURGERY BY PRE-OPERATIVE IMMUNE ENHANCING NUTRIENTS
LIPPINCOTT WILLIAMS & WILKINS. 2016: 31–32
View details for Web of Science ID 000377692000058
COMPLEX MODULATION OF THE IMMUNE RESPONSE TO SURGERY BY IMMUNE ENHANCING NUTRIENTS
LIPPINCOTT WILLIAMS & WILKINS. 2016
View details for Web of Science ID 000403582200252
Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth.
Cytometry. Part A : the journal of the International Society for Analytical Cytology
2015; 87 (9): 817-829
Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1) ) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort. © 2015 International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.22720
View details for PubMedID 26190063
PRE-OPERATIVE EVOKED IMMUNE RESPONSES CORRELATE WITH RECOVERY FROM SURGICAL TRAUMA
LIPPINCOTT WILLIAMS & WILKINS. 2015: 65–66
View details for Web of Science ID 000354736000121
Pre-operative immune signatures correlate with recovery from surgical trauma
AMER ASSOC IMMUNOLOGISTS. 2015
View details for Web of Science ID 000379404500311
Cyclin-dependent kinase inhibitor 2B regulates efferocytosis and atherosclerosis
JOURNAL OF CLINICAL INVESTIGATION
2014; 124 (3): 1083-1097
Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.
View details for DOI 10.1172/JCI70391
View details for Web of Science ID 000332347700028
View details for PubMedID 24531546
RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2011; 301 (5): H1841-H1849
Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G(2)/M fraction consistent with a mitotic defect; 4',6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G(2)/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall.
View details for DOI 10.1152/ajpheart.00089.2011
View details for Web of Science ID 000296716900011
View details for PubMedID 21856925
Aspirin-Triggered Lipoxin and Resolvin E1 Modulate Vascular Smooth Muscle Phenotype and Correlate with Peripheral Atherosclerosis
AMERICAN JOURNAL OF PATHOLOGY
2010; 177 (4): 2116-2123
Atherosclerosis is a chronic inflammatory disease of the vessel wall. Recent evidence suggests that chronic vascular inflammation ensues as an imbalance between pro- and anti-inflammatory mediators. Recently identified lipid mediators (eg, lipoxins and resolvins) play active roles in promoting the resolution of inflammation. Alterations in vascular smooth muscle cell (VSMC) phenotype, which manifest as a loss of contractile protein expression and increased proliferation and migration, are prominent mechanistic features of both atherosclerosis and restenosis following various interventions (eg, angioplasty and bypass grafting). We sought to determine whether human atherosclerosis is associated with a "resolution deficit" and whether lipoxins and resolvins influence VSMC phenotype. Here we report that plasma levels of aspirin-triggered lipoxin are significantly lower in patients with symptomatic peripheral artery disease than in healthy volunteers. Both aspirin-triggered lipoxin and resolvin E1 block platelet-derived growth factor-stimulated migration of human saphenous vein SMCs and decrease phosphorylation of the platelet-derived growth factor receptor-?. Importantly, receptors for aspirin-triggered lipoxin and resolvin E1 (ALX and ChemR23, respectively) were identified in human VSMCs. Overall, these results demonstrate that stimulatory lipid mediators confer a protective phenotypic switch in VSMCs and elucidate new functions for these mediators in the regulation of SMC biology. These results also suggest that peripheral artery disease is associated with an inflammation-resolution deficit and highlight a potential therapeutic opportunity for the regulation of vascular injury responses.
View details for DOI 10.2353/ajpath.2010.091082
View details for Web of Science ID 000282496100052
View details for PubMedID 20709806
Cell cycle-dependent phosphorylation of human CDC5 regulates RNA processing
2008; 7 (12): 1795-1803
CDC5 proteins are components of the pre-mRNA splicing complex and essential for cell cycle progression in yeast, plants and mammals. Human CDC5 is phosphorylated in a mitogen-dependent manner, and its association with the spliceosome is ATP-dependent. Examination of the amino acid sequence suggests that CDC5L may be phosphorylated at up to 28 potential consensus recognition sequences for known kinases, however, the identity of actual phosphorylation sites, their role in regulating CDC5L activity, and the kinases responsible for their phosphorylation have not previously been determined. Using two-dimensional phosphopeptide mapping and nanoelectrospray mass spectrometry, we now show that CDC5L is phosphorylated on at least nine sites in vivo. We demonstrate that while CDC5L is capable of forming homodimers in vitro and in vivo, neither homodimerization nor nuclear localization is dependent on phosphorylation at these sites. Using an in vitro splicing assay, we show that phosphorylation of CDC5L at threonines 411 and 438 within recognition sequences for CDKs are required for CDC5L-mediated pre-mRNA splicing. We also demonstrate that a specific inhibitor of CDK2, CVT-313, inhibits CDC5L phosphorylation in both in vitro kinase assays and in vivo radiolabeling experiments in cycling cells. These studies represent the first demonstration of a regulatory role for phosphorylation of CDC5L, and suggest that targeting these sites or the implicated kinases may provide novel strategies for treating disorders of unguarded cellular proliferation, such as cancer.
View details for Web of Science ID 000257249300016
View details for PubMedID 18583928
Analysing protein-protein interactions of the Myxococcus xanthus Dif signalling pathway using the yeast two-hybrid system
2005; 151: 1535-1541
The dif operon is essential for fruiting body formation, fibril (exopolysaccharide) production and social motility of Myxococcus xanthus. The dif locus contains a gene cluster homologous to chemotaxis genes such as mcp (difA), cheW (difC), cheY (difD), cheA (difE) and cheC (difF), as well as an unknown ORF called difB. This study used yeast two-hybrid analysis to investigate possible interactions between Dif proteins, and determined that DifA, C, D and E interact in a similar fashion to chemotaxis proteins of Escherichia coli and Bacillus subtilis. It also showed that DifF interacted with DifD, and that the novel protein DifB did not interact with Dif proteins. Furthermore, DifA-F proteins were used to determine other possible protein-protein interactions in the M. xanthus genomic library. The authors not only confirmed the specific interactions among known Dif proteins, but also discovered two novel interactions between DifE and Nla19, and DifB and YidC, providing some new information about the Dif signalling pathway. Based on these findings, a model for the Dif signalling pathway is proposed.
View details for DOI 10.1099/mic.0.27743-0
View details for Web of Science ID 000229272100022
View details for PubMedID 15870463
Characterization of a Myxococcus xanthus mutant that is defective for adventurous motility and social motility
2004; 150: 4085-4093
Myxococcus xanthus is a gliding bacterium that possesses two motility systems, the adventurous (A-motility) and social (S-motility) systems. A-motility is used for individual cell gliding, while S-motility is used for gliding in multicellular groups. Video microscopy studies showed that nla24 cells are non-motile on agar surfaces, suggesting that the nla24 gene product is absolutely required for both A-motility and S-motility under these assay conditions. S-motility requires functional type IV pili, wild-type LPS O-antigen, and an extracellular matrix of exopolysaccharide (EPS) and protein called fibrils. The results of expression studies and tethering assays indicate that the nla24 mutant has functional type IV pili. The nla24 mutant also produces wild-type LPS. However, several lines of evidence suggest that the nla24 mutant is defective for production of the EPS portion of the fibril matrix. The nla24 mutant is also defective for transcription of two genes (aglU and cglB) known to be required for A-motility, which is consistent with the idea that nla24 cells are defective for A-motility. Based on these findings, it is proposed that the putative transcriptional activator Nla24 regulates a subset of genes that are important for A-motility and S-motility in M. xanthus.
View details for DOI 10.1099/mic.0.27381-0
View details for Web of Science ID 000226043000021
View details for PubMedID 15583161
Mapping of Myxococcus xanthus social motility dsp mutations to the dif genes
JOURNAL OF BACTERIOLOGY
2002; 184 (5): 1462-1465
Myxococcus xanthus dsp and dif mutants have similar phenotypes in that they are deficient in social motility and fruiting body development. We compared the two loci by genetic mapping, complementation with a cosmid clone, DNA sequencing, and gene disruption and found that 16 of the 18 dsp alleles map to the dif genes. Another dsp allele contains a mutation in the sglK gene. About 36.6 kb around the dsp-dif locus was sequenced and annotated, and 50% of the genes are novel.
View details for DOI 10.1128/JB.184.5.1462-1465.2002
View details for Web of Science ID 000173932600031
View details for PubMedID 11844780
Phenotypic analyses of frz and dif double mutants of Myxococcus xanthus
FEMS MICROBIOLOGY LETTERS
2000; 192 (2): 211-215
Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.
View details for Web of Science ID 000165211000010
View details for PubMedID 11064197
Function of gingival fibroblasts and periodontal ligament cells in the presence of methyl mercaptan
1999; 30 (5): 343-349
This study examined the effects of methyl mercaptan, a product of the bacterial putrefaction of protein in periodontal pockets, on the function of cells in culture.Human gingival fibroblasts and periodontal ligament cells were exposed to a constant, continuous flow of methyl mercaptan in vitro. Control and test cultures were then examined for changes in intracellular pH, an event often associated with alterations in cellular function. Intracellular pH was determined by single-cell image analysis of cells loaded with a fluorescent, pH-sensitive dye. Periodontal ligament cells were also tested for changes in synthesis of total protein and fibronectin.Test cells exhibited a consistent decrease in intracellular pH following exposure to methyl mercaptan. Measurements of total protein production showed that test periodontal ligament cell cultures produced approximately 30% less protein than control cultures (P < 0.05). Western-blot analysis of fibronectin in medium demonstrated that abnormal monomeric fibronectins were a major protein in test, but not in control, cell cultures.Exposure to methyl mercaptan induced alterations in intracellular events that paralleled changes in extracellular matrix proteins. The observed changes in extracellular matrix proteins support the hypothesis that methyl mercaptan contributes to the progression of periodontal disease.
View details for Web of Science ID 000080325100011
View details for PubMedID 10635290
The response of periodontal ligament cells to fibronectin
JOURNAL OF PERIODONTOLOGY
1998; 69 (9): 1008-1019
Fibronectin (fn) is an extracellular matrix (ECM) molecule important in cell adhesion and migration and in wound healing. It is also likely important in periodontal ligament (PDL) cell-ECM interactions, and thus in regenerating periodontal tissues. In this study we characterized PDL cells and their interactions with FN, testing different PDL cell isolates taken from healthy and diseased conditions. PDL cells were characterized by their morphology, integrin profile, motility, and bone nodule formation. Cells were then assayed for adhesion, proliferation, and chemotaxis in response to FN or FN fragments. Cell isolates were morphologically heterogeneous and fibroblastic, had a normal-appearing actin cytoskeleton and a wide range of migration potentials, and formed bone-like nodules in vitro. They expressed alpha5, beta1, alpha v, and alpha4 integrin subunits, known receptors for FN, and in fact they bound FN preferentially at 5 and 10 microg/ml. Intact FN induced greater PDL cell proliferation and chemotaxis than did FN fragments (120-kDa cell-binding, 60-kDa heparin-binding, and 45-kDa collagen-binding). PDL cells harvested from diseased and healthy conditions were no different on the basis of these assays. These data demonstrate that PDL cells are a mixed population of fibroblastic cells, capable of forming a mineralized matrix. They also suggest that maximal proliferation and chemotaxis require specific FN domains that are present on the intact molecule but not its fragments.
View details for Web of Science ID 000076302600008
View details for PubMedID 9776029
Exposure of periodontal ligament cells to methyl mercaptan reduces intracellular pH and inhibits cell migration
JOURNAL OF DENTAL RESEARCH
1996; 75 (12): 1994-2002
Volatile sulfur compounds such as hydrogen sulfide and methyl mercaptan have been associated with adult periodontitis as well as with healing surgical wounds. To examine the effects of these compounds on the periodontium, we assayed periodontal ligament (PDL) cells for changes in intracellular pH, total protein, and cell migration following chronic exposure to CH3SH. Intracellular pH was quantitated by fluorescence measurements of cells loaded with BCECF, a pH-sensitive dye. Data show that 48-hour exposure to mercaptan lowered resting intracellular pH but did not consistently alter activity of the Na/H exchanger. This effect was seen in PDL cells from three different patients. Lowered pH was accompanied by decreases in both total protein and mature alpha 1 and alpha 2 chains of type I collagen. Since reductions in intracellular pH and total protein have been associated with inhibition of cell motility, migration was quantitated by sequential computer imaging, which measured the increase in size of plated cell circles at different times of migration. Incubation of PDL cells in pH 7.4 and 6.6 buffers reversibly altered intracellular pH. Migration was reversibly inhibited in pH 6.8 buffer. Exposure to CH3SH reduced intracellular pH in pH 7.4 buffer and in three independent assays inhibited enlargement of cell circles in pH 7.4 medium. These effects were therefore not related to alterations of extracellular pH, which remained at 7.4. The results support the hypothesis that gases such as methyl mercaptan may play a role in both surgical wound healing and periodontal disease by adversely affecting cell function and suggest that alterations in intracellular pH may be part of the mechanism for these changes.
View details for Web of Science ID A1996WG21700012
View details for PubMedID 9033455