Lydia-Marie Joubert

All Publications

• Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism. bioRxiv : the preprint server for biology Findinier, J., Joubert, L. M., Schmid, M. F., Malkovskiy, A., Chiu, W., Burlacot, A., Grossman, A. R. 2024

  Abstract

  Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cell's apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cell's affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

  View details for DOI 10.1101/2024.03.25.586705

  View details for PubMedID 38585955

  View details for PubMedCentralID PMC10996633

• Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts. mBio Gerrick, E. R., DeSchepper, L. B., Mechler, C. M., Joubert, L. -., Dunker, F., Colston, T. J., Howitt, M. R. 2023: e0227323

  Abstract

  Environmental factors like climate change and captive breeding can impact the gut microbiota and host health. Therefore, conservation efforts for threatened species may benefit from understanding how these factors influence animal microbiomes. Parabasalid protists are members of the mammalian microbiota that can modulate the immune system and impact susceptibility to infections. However, little is known about parabasalids in reptiles. Here, we profile reptile-associated parabasalids in wild and captive reptiles and find that captivity has minimal impact on parabasalid prevalence or diversity. However, because reptiles are cold-blooded (ectothermic), their microbiotas experience wider temperature fluctuation than microbes in warm-blooded animals. To investigate whether extreme weather patterns affect parabasalid-host interactions, we analyzed the gene expression in reptile-associated parabasalids and found that temperature differences significantly alter genes associated with host health. These results expand our understanding of parabasalids in this vulnerable vertebrate group and highlight important factors to be taken into consideration for conservation efforts.

  View details for DOI 10.1128/mbio.02273-23

  View details for PubMedID 37962346

• Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts. bioRxiv : the preprint server for biology Gerrick, E. R., DeSchepper, L. B., Mechler, C. M., Joubert, L. M., Dunker, F., Colston, T. J., Howitt, M. R. 2023

  Abstract

  Parabasalid protists recently emerged as keystone members of the mammalian microbiota with important effects on their host's health. However, the prevalence and diversity of parabasalids in wild reptiles and the consequences of captivity and other environmental factors on these symbiotic protists are unknown. Reptiles are ectothermic, and their microbiomes are subject to temperature fluctuations, such as those driven by climate change. Thus, conservation efforts for threatened reptile species may benefit from understanding how shifts in temperature and captive breeding influence the microbiota, including parabasalids, to impact host fitness and disease susceptibility. Here, we surveyed intestinal parabasalids in a cohort of wild reptiles across three continents and compared these to captive animals. Reptiles harbor surprisingly few species of parabasalids compared to mammals, but these protists exhibited a flexible host-range, suggesting specific adaptations to reptilian social structures and microbiota transmission. Furthermore, reptile-associated parabasalids are adapted to wide temperature ranges, although colder temperatures significantly altered the protist transcriptomes, with increased expression of genes associated with detrimental interactions with the host. Our findings establish that parabasalids are widely distributed in the microbiota of wild and captive reptiles and highlight how these protists respond to temperature swings encountered in their ectothermic hosts.

  View details for DOI 10.1101/2023.05.25.542353

  View details for PubMedID 37292851

  View details for PubMedCentralID PMC10245904

• CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons. Nature communications Wu, G. H., Smith-Geater, C., Galaz-Montoya, J. G., Gu, Y., Gupte, S. R., Aviner, R., Mitchell, P. G., Hsu, J., Miramontes, R., Wang, K. Q., Geller, N. R., Hou, C., Danita, C., Joubert, L. M., Schmid, M. F., Yeung, S., Frydman, J., Mobley, W., Wu, C., Thompson, L. M., Chiu, W. 2023; 14 (1): 692

  Abstract

  Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

  View details for DOI 10.1038/s41467-023-36096-w

  View details for PubMedID 36754966

• Visualization of the distribution of covalently cross-linked hydrogels in CLARITY brain-polymer hybrids for different monomer concentrations. Scientific reports Malkovskiy, A. V., Tom, A., Joubert, L., Bao, Z. 2022; 12 (1): 13549

  Abstract

  CLARITY is a tissue preservation and optical clearing technique whereby a hydrogel is formed directly within the architectural confines of ex vivo brain tissue. In this work, the extent of polymer gel formation and crosslinking within tissue was assessed using Raman spectroscopy and rheology on CLARITY samples prepared with a range of acrylamide monomer (AAm) concentrations (1%, 4%, 8%, 12% w/v). Raman spectroscopy of individual neurons within hybrids revealed the chemical presence and distribution of polyacrylamide within the mouse hippocampus. Consistent with rheological measurements, lower %AAm concentration decreased shear elastic modulus G', providing a practical correlation with sample permeability and protein retention. Permeability of F(ab)'2 secondary fluorescent antibody changes from 9.3 to 1.4 m2s-1 going from 1 to 12%. Notably, protein retention increased linearly relative to standard PFA-fixed tissue from 96.6% when AAm concentration exceeded 1%, with 12% AAm samples retaining up to~99.3% native protein. This suggests that though 1% AAm offers high permeability, additional %AAm may be required to enhance protein. Our quantitative results on polymer distribution, stability, protein retention, and macromolecule permeability can be used to guide the design of future CLARITY-based tissue-clearing solutions, and establish protocols for characterization of novel tissue-polymer hybrid biomaterials using chemical spectroscopy and rheology.

  View details for DOI 10.1038/s41598-022-17687-x

  View details for PubMedID 35941350

• Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt. The Journal of biological chemistry Yu, Z., Chen, J., Takagi, E., Wang, F., Saha, B., Liu, X., Joubert, L. M., Gleason, C. E., Jin, M., Li, C., Nowotny, C., Agard, D., Cheng, Y., Pearce, D. 2022: 102288

  Abstract

  Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously, however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

  View details for DOI 10.1016/j.jbc.2022.102288

  View details for PubMedID 35926713

• Correlative Light and Electron Microscopy (CLEM): Bringing Together the Best of Both Worlds to Study Neuronal Autophagy Imaging and Quantifying Neuronal Autophagy Kriel, J., Joubert, L. Springer. 2022

  View details for DOI DOI 10.1007/978-1-0716-1589-8_10

• Vision, challenges and opportunities for a Plant Cell Atlas. eLife Plant Cell Atlas Consortium, Jha, S. G., Borowsky, A. T., Cole, B. J., Fahlgren, N., Farmer, A., Huang, S. C., Karia, P., Libault, M., Provart, N. J., Rice, S. L., Saura-Sanchez, M., Agarwal, P., Ahkami, A. H., Anderton, C. R., Briggs, S. P., Brophy, J. A., Denolf, P., Di Costanzo, L. F., Exposito-Alonso, M., Giacomello, S., Gomez-Cano, F., Kaufmann, K., Ko, D. K., Kumar, S., Malkovskiy, A. V., Nakayama, N., Obata, T., Otegui, M. S., Palfalvi, G., Quezada-Rodriguez, E. H., Singh, R., Uhrig, R. G., Waese, J., Van Wijk, K., Wright, R. C., Ehrhardt, D. W., Birnbaum, K. D., Rhee, S. Y., Ahmed, J., Alaba, O., Ameen, G., Arora, V., Arteaga-Vazquez, M. A., Arun, A., Bailey-Serres, J., Bartley, L. E., Bassel, G. W., Bergmann, D. C., Bertolini, E., Bhati, K. K., Blanco-Tourinan, N., Briggs, S. P., Brumos, J., Buer, B., Burlaocot, A., Cervantes-Perez, S. A., Chen, S., Contreras-Moreira, B., Corpas, F. J., Cruz-Ramirez, A., Cuevas-Velazquez, C. L., Cuperus, J. T., David, L. I., de Folter, S., Denolf, P. H., Ding, P., Dwyer, W. P., Evans, M. M., George, N., Handakumbura, P. P., Harrison, M. J., Haswell, E. S., Herath, V., Jiao, Y., Jinkerson, R. E., John, U., Joshi, S., Joshi, A., Joubert, L., Katam, R., Kaur, H., Kazachkova, Y., Raju, S. K., Khan, M. A., Khangura, R., Kumar, A., Kumar, A., Kumar, P., Kumar, P., Lavania, D., Lew, T. T., Lewsey, M. G., Lin, C., Liu, D., Liu, L., Liu, T., Lokdarshi, A., My Luong, A., Macaulay, I. C., Mahmud, S., Mahonen, A. P., Malukani, K. K., Marand, A. P., Martin, C. A., McWhite, C. D., Mehta, D., Martin, M. M., Mortimer, J. C., Nikolov, L. A., Nobori, T., Nolan, T. M., Ogden, A. J., Otegui, M. S., Ott, M., Palma, J. M., Paul, P., Rehman, A. U., Romera-Branchat, M., Romero, L. C., Roth, R., Sah, S. K., Shahan, R., Solanki, S., Song, B., Sozzani, R., Stacey, G., Stepanova, A. N., Taylor, N. L., Tello-Ruiz, M. K., Tran, T. M., Tripathi, R. K., Vadde, B. V., Varga, T., Vidovic, M., Walley, J. W., Wang, Z., Weizbauer, R. A., Whelan, J., Wijeratne, A. J., Xiang, T., Xu, S., Yadegari, R., Yu, H., Yuan, H. Y., Zanini, F., Zhao, F., Zhu, J., Zhuang, X. 2021; 10

  Abstract

  With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.

  View details for DOI 10.7554/eLife.66877

  View details for PubMedID 34491200

• SEM and TEM for identification of capsular fibrosis and cellular behavior around breast implants - a descriptive analysis. BMC molecular and cell biology Kuehlmann, B., Zucal, I., Bonham, C. A., Joubert, L., Prantl, L. 2021; 22 (1): 25

  Abstract

  BACKGROUND: Capsular fibrosis (CF) is the most common long-term complication in implant-based breast augmentation. It is well accepted that the foreign body response (FBR) instigates the development of fibrotic disease. Our study aims to compare murine and human samples of CF and describe the cellular and extracellular matrix (ECM) composition using scanning and transmission electron microscopy (SEM and TEM).RESULTS: Miniature microtextured silicone breast implants were implanted in mice and subsequently harvested at days 15, 30, and 90 post-operation. Isolated human capsules with the most aggravated form of CF (Baker IV) were harvested post-operation. Both were analyzed with SEM and TEM to assess cellular infiltration and ECM structure. An architectural shift of collagen fiber arrangement from unidirectional to multidirectional was observed at day 90 when compared to days 15 and 30. Fibrosis was observed with an increase of histiocytic infiltration. Moreover, bacterial accumulation was seen around silicone fragments. These findings were common in both murine and human capsules.CONCLUSIONS: This murine model accurately recapitulates CF found in humans and can be utilized for future research on cellular invasion in capsular fibrosis. This descriptive study helps to gain a better understanding of cellular mechanisms involved in the FBR. Increases of ECM and cellularity were observed over time with SEM and TEM analysis.

  View details for DOI 10.1186/s12860-021-00364-8

  View details for PubMedID 33941075

• Combining the glioblastoma cell membrane-permeabilizing effect of tumor treating fields with chemotherapy Chang, E., Patel, C., Young, C. J., Flores, T., Joubert, L., Zeng, Y., Sinclair, R., Gambhir, S. AMER ASSOC CANCER RESEARCH. 2020

  View details for DOI 10.1158/1538-7445.AM2020-6258

  View details for Web of Science ID 000590059307155

• Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals. Science (New York, N.Y.) Liu, J. n., Kim, Y. S., Richardson, C. E., Tom, A. n., Ramakrishnan, C. n., Birey, F. n., Katsumata, T. n., Chen, S. n., Wang, C. n., Wang, X. n., Joubert, L. M., Jiang, Y. n., Wang, H. n., Fenno, L. E., Tok, J. B., Pașca, S. P., Shen, K. n., Bao, Z. n., Deisseroth, K. n. 2020; 367 (6484): 1372–76

  Abstract

  The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

  View details for DOI 10.1126/science.aay4866

  View details for PubMedID 32193327

• Designed Antimicrobial Peptides for Recurrent Vulvovaginal Candidiasis Treatment. Antimicrobial agents and chemotherapy Woodburn, K. W., Clemens, L. E., Jaynes, J., Joubert, L., Botha, A., Nazik, H., Stevens, D. A. 2019

  Abstract

  Background: Recurrent Vulvovaginal Candidiasis (RVVC) is a widespread chronic infection that has a substantial negative impact on work and quality of life. The development of antimicrobial resistance and biofilm formation are speculated to contribute to Candida pathogenicity and treatment ineffectiveness. Designed antimicrobial peptides (dAMPs) are chemically modified from endogenous antimicrobial peptides that provide the first line of defense against pathogens. The goal here is to identify a dAMP for the topical treatment of RVVC.Methods: The dAMP minimum inhibitory concentrations (MICs) were determined for 46 fluconazole-susceptible and fluconazole-resistant Candida spp. clinical isolates. The possibility of inducing dAMP drug resistance, and comparison of dAMP and fluconazole activity against preformed Candida biofilm and biofilm formation were evaluated. Assessment of mammalian cell viability was determined using bioluminescent human keratinocytes. The dAMP effect on fungus was probed via scanning electron microscopy and topically applied dAMP activity was evaluated in a rodent VVC infection model.Results: dAMPs demonstrated broad-spectrum antimicrobial activity against common causative clinical Candida isolates, reduced preformed biofilm and inhibited biofilm formation. An evaluated dAMP did not induce resistance after repeated exposure of C. tropicalis The dAMPs were selective for Candida cells with limited mammalian cytotoxicity with substantial activity in a rodent VVC model.Conclusions: dAMPs are described with potent antifungal and anti-biofilm activity, likely direct membrane action with selectivity for Candida cells, with limited resistance development. Combined with activity in a rodent VVC model, the data support clinical evaluation of dAMPs for topical treatment of VCC and recurrent VVC infections.

  View details for DOI 10.1128/AAC.02690-18

  View details for PubMedID 31451496

• Spatiotemporal Tracking of Brain-Tumor-Associated Myeloid Cells in Vivo through Optical Coherence Tomography with Plasmonic Labeling and Speckle Modulation ACS NANO SoRelle, E., Yecies, D., Liba, O., Bennett, F., Graef, C., Dutta, R., Mitra, S., Joubert, L., Cheshier, S., Grant, G. A., de la Zerda, A. 2019; 13 (7): 7985–95

  Abstract

  By their nature, tumors pose a set of profound challenges to the immune system with respect to cellular recognition and response coordination. Recent research indicates that leukocyte subpopulations, especially tumor-associated macrophages (TAMs), can exert substantial influence on the efficacy of various cancer immunotherapy treatment strategies. To better study and understand the roles of TAMs in determining immunotherapeutic outcomes, significant technical challenges associated with dynamically monitoring single cells of interest in relevant live animal models of solid tumors must be overcome. However, imaging techniques with the requisite combination of spatiotemporal resolution, cell-specific contrast, and sufficient signal-to-noise at increasing depths in tissue are exceedingly limited. Here we describe a method to enable high-resolution, wide-field, longitudinal imaging of TAMs based on speckle-modulating optical coherence tomography (SM-OCT) and spectral scattering from an optimized contrast agent. The approach's improvements to OCT detection sensitivity and noise reduction enabled high-resolution OCT-based observation of individual cells of a specific host lineage in live animals. We found that large gold nanorods (LGNRs) that exhibit a narrow-band, enhanced scattering cross-section can selectively label TAMs and activate microglia in an in vivo orthotopic murine model of glioblastoma multiforme. We demonstrated near real-time tracking of the migration of cells within these myeloid subpopulations. The intrinsic spatiotemporal resolution, imaging depth, and contrast sensitivity reported herein may facilitate detailed studies of the fundamental behaviors of TAMs and other leukocytes at the single-cell level in vivo, including intratumoral distribution heterogeneity and roles in modulating cancer proliferation.

  View details for DOI 10.1021/acsnano.9b02656

  View details for Web of Science ID 000477786400063

  View details for PubMedID 31259527

• Tumor treating fields increases membrane permeability in glioblastoma cells Chang, E., Patel, C., Pohling, C., Young, C., Song, J., Flores, T. A., Zeng, Y., Joubert, L., Arami, H., Natarajan, A., Sinclair, R., Gambhir, S. S. AMER ASSOC CANCER RESEARCH. 2019

  View details for DOI 10.1158/1538-7445.AM2019-250

  View details for Web of Science ID 000488129901424

• A Combination of Itraconazole and Amiodarone Is Highly Effective against Trypanosoma cruzi Infection of Human Stem Cell-Derived Cardiomyocytes. The American journal of tropical medicine and hygiene Sass, G., Madigan, R. T., Joubert, L., Bozzi, A., Sayed, N., Wu, J. C., Stevens, D. A. 2019

  Abstract

  Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other non-endemic countries, rising case numbers have also been observed. The currently used drugs are benznidazole (BNZ) and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole (ICZ), originally an antifungal, in combination with amiodarone (AMD), an antiarrhythmic, with the goal of interfering with T. cruzi infection. Human pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPSC-CMs). Vero cells or hiPSC-CMs were infected with T. cruzi trypomastigotes of the II or I strain in the presence of ICZ and/or AMD. After 48 hours, cells were Giemsa stained, and infection and multiplication were evalutated microscopically. Trypanosoma cruzi infection and multiplication were evalutated also by electron microscopy. BNZ was used as a reference compound. Cell viability in the presence of test substances was assessed. Itraconazole and AMD showed strain- and dose-dependent interference with T. cruzi infection and multiplication in Vero cells or hiPSC-CMs. Combinations of ICZ and AMD were more effective against T. cruzi than the single substances, or BNZ, without affecting host cell metabolism, and better preserving host cell integrity during infection. Our in vitro data in hiPSC-CMs suggest that a combination of ICZ and AMD might serve as a treatment option for CD in patients, but that different responses due to T. cruzi strain differences have to be taken into account.

  View details for DOI 10.4269/ajtmh.19-0023

  View details for PubMedID 31219005

• Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy NATURE NANOTECHNOLOGY Prigozhin, M. B., Maurer, P. C., Courtis, A. M., Liu, N., Wisser, M. D., Siefe, C., Tian, B., Chan, E., Song, G., Fischer, S., Aloni, S., Ogletree, D., Barnard, E. S., Joubert, L., Rao, J., Alivisatos, A., Macfarlane, R. M., Cohen, B. E., Cui, Y., Dionne, J. A., Chu, S. 2019; 14 (5): 420-+

  View details for DOI 10.1038/s41565-019-0395-0

  View details for Web of Science ID 000467053100016

• Integration of electron microscopy and solid-state NMR analysis for new views and compositional parameters of Aspergillus fumigatus biofilms. Medical mycology Reichhardt, C., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2019; 57 (Supplement_2): S239–S244

  Abstract

  The general ability and tendency of bacteria and fungi to assemble into bacterial communities, termed biofilms, poses unique challenges to the treatment of human infections. Fungal biofilms, in particular, are associated with enhanced virulence in vivo and decreased sensitivity to antifungals. Much attention has been given to the complex cell wall structures in fungal organisms, yet beyond the cell surface, Aspergillus fumigatus and other fungi assemble a self-secreted extracellular matrix that is the hallmark of the biofilm lifestyle, protecting and changing the environment of resident members. Elucidation of the chemical and molecular detail of the extracellular matrix is crucial to understanding how its structure contributes to persistence and antifungal resistance in the host. We present a summary of integrated analyses of A. fumigatus biofilm architecture, including hyphae and the extracellular matrix, by scanning electron microscopy and A. fumigatus matrix composition by new top-down solid-state NMR approaches coupled with biochemical analysis. This combined methodology will be invaluable in formulating quantitative and chemical comparisons of A. fumigatus isolates that differ in virulence and are more or less resistant to antifungals. Ultimately, knowledge of the chemical and molecular requirements for matrix formation and function will drive the identification and development of new strategies to interfere with biofilm formation and virulence.

  View details for PubMedID 30816969

• Integration of electron microscopy and solid-state NMR analysis for new views and compositional parameters of Aspergillus fumigatus biofilms MEDICAL MYCOLOGY Reichhardt, C., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2019; 57: S239–S244

  View details for DOI 10.1093/mmy/myy140

  View details for Web of Science ID 000463726800020

• Bright sub-20-nm cathodoluminescent nanoprobes for electron microscopy. Nature nanotechnology Prigozhin, M. B., Maurer, P. C., Courtis, A. M., Liu, N., Wisser, M. D., Siefe, C., Tian, B., Chan, E., Song, G., Fischer, S., Aloni, S., Ogletree, D. F., Barnard, E. S., Joubert, L., Rao, J., Alivisatos, A. P., Macfarlane, R. M., Cohen, B. E., Cui, Y., Dionne, J. A., Chu, S. 2019

  Abstract

  Electron microscopy has been instrumental in our understanding of complex biological systems. Although electron microscopy reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An electron-microscopy-based bioimaging technology capable of localizing individual proteins and resolving protein-protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. Here, we synthesize small lanthanide-doped nanoparticles and measure the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results suggest that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nanolabels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colours, so the imaging of biomolecules in a multicolour electron microscopy modality may be possible.

  View details for PubMedID 30833691

• The hippocampal extracellular matrix regulates pain and memory after injury MOLECULAR PSYCHIATRY Tajerian, M., Hung, V., Huy Nguyen, Lee, G., Joubert, L., Malkovskiy, A., Zou, B., Xie, S., Huang, T., Clark, J. 2018; 23 (12): 2302–13

  View details for DOI 10.1038/s41380-018-0209-z

  View details for Web of Science ID 000453243900006

• Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers. Science translational medicine Tsai, J. M., Sinha, R., Seita, J., Fernhoff, N., Christ, S., Koopmans, T., Krampitz, G. W., McKenna, K. M., Xing, L., Sandholzer, M., Sales, J. H., Shoham, M., McCracken, M., Joubert, L., Gordon, S. R., Poux, N., Wernig, G., Norton, J. A., Weissman, I. L., Rinkevich, Y. 2018; 10 (469)

  Abstract

  Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPN+MSLN+ mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1alpha) that preceded adhesion development. Inhibition of HIF1alpha with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.

  View details for PubMedID 30487249

• Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers SCIENCE TRANSLATIONAL MEDICINE Tsai, J. M., Sinha, R., Seita, J., Fernhoff, N., Christ, S., Koopmans, T., Krampitz, G. W., McKenna, K. M., Xing, L., Sandholzer, M., Sales, J., Shoham, M., McCracken, M., Joubert, L., Gordon, S. R., Poux, N., Wernig, G., Norton, J. A., Weissman, I. L., Rinkevich, Y. 2018; 10 (469)

  View details for DOI 10.1126/scitranslmed.aan6735

  View details for Web of Science ID 000451465600001

• The hippocampal extracellular matrix regulates pain and memory after injury. Molecular psychiatry Tajerian, M., Hung, V., Nguyen, H., Lee, G., Joubert, L., Malkovskiy, A. V., Zou, B., Xie, S., Huang, T., Clark, J. D. 2018

  Abstract

  Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.

  View details for PubMedID 30254235

• Single upconversion nanoparticle imaging at sub-10 W cm(-2) irradiance NATURE PHOTONICS Liu, Q., Zhang, Y., Peng, C., Yang, T., Joubert, L., Chu, S. 2018; 12 (9): 548–53

  View details for DOI 10.1038/s41566-018-0217-1

  View details for Web of Science ID 000443022200014

• Single upconversion nanoparticle imaging at sub-10 W cm-2 irradiance. Nature photonics Liu, Q., Zhang, Y., Peng, C. S., Yang, T., Joubert, L. M., Chu, S. 2018; 12 (9): 548-553

  Abstract

  Lanthanide-doped upconversion nanoparticles (UCNPs) are promising single-molecule probes given their non-blinking, photobleach-resistant luminescence upon infrared excitation. However, the weak luminescence of sub-50 nm UCNPs limits their single-particle detection to above 10 kWcm-2 that is impractical for live cell imaging. Here, we systematically characterize single-particle luminescence for UCNPs with various formulations over a 106 variation in incident power, down to 8 Wcm-2. A core-shell-shell (CSS) structure (NaYF4@NaYb1-xF4:Erx@NaYF4) is shown to be significantly brighter than the commonly used NaY0.78F4:Yb0.2Er0.02. At 8 Wcm-2, the 8% Er3+ CSS particles exhibit a 150-fold enhancement given their high sensitizer Yb3+ content and the presence of an inert shell to prevent energy migration to defects. Moreover, we reveal power-dependent luminescence enhancement from the inert shell, which explains the discrepancy in enhancement factors reported by ensemble and previous single-particle measurements. These brighter probes open the possibility of cellular and single-molecule tracking at low irradiance.

  View details for DOI 10.1038/s41566-018-0217-1

  View details for PubMedID 31258619

  View details for PubMedCentralID PMC6599589

• Tumor Treating Fields Increases Membrane Permeability in Glioblastoma Cells Cell Death Discovery Chang, E., Patel, C. B., Pohling, C., Young, C., Song, J., Flores, T., Zeng, Y., Joubert, L. M., Arami, H., Natarajan, A., Sinclair, R., Gambhir, S. S. 2018; 4

  View details for DOI 10.1038/s41420-018-0130-x

• Tumor treating fields increases membrane permeability in glioblastoma cells. Cell death discovery Chang, E. n., Patel, C. B., Pohling, C. n., Young, C. n., Song, J. n., Flores, T. A., Zeng, Y. n., Joubert, L. M., Arami, H. n., Natarajan, A. n., Sinclair, R. n., Gambhir, S. S. 2018; 4: 113

  Abstract

  Glioblastoma is the most common yet most lethal of primary brain cancers with a one-year post-diagnosis survival rate of 65% and a five-year survival rate of barely 5%. Recently the U.S. Food and Drug Administration approved a novel fourth approach (in addition to surgery, radiation therapy, and chemotherapy) to treating glioblastoma; namely, tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but its mechanisms of action are not fully understood. Current theories involve TTFields disrupting mitosis due to interference with proper mitotic spindle assembly. We show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. Increased membrane permeability through the imposition of TTFields was shown by several approaches. For example, increased permeability was indicated through increased bioluminescence with TTFields exposure or with the increased binding and ingress of membrane-associating reagents such as Dextran-FITC or ethidium D or with the demonstration by scanning electron microscopy of augmented number and sizes of holes on the cellular membrane. Further investigations showed that increases in bioluminescence and membrane hole production with TTFields exposure disappeared by 24 h after cessation of alternating electric fields thus demonstrating that this phenomenom is reversible. Preliminary investigations showed that TTFields did not induce membrane holes in normal human fibroblasts thus suggesting that the phenomenom was specific to cancer cells. With TTFields, we present evidence showing augmented membrane accessibility by compounds such as 5-aminolevulinic acid, a reagent used intraoperatively to delineate tumor from normal tissue in glioblastoma patients. In addition, this mechanism helps to explain previous reports of additive and synergistic effects between TTFields and other chemotherapies. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies for these diseases.

  View details for PubMedID 30534421

• Formation of Polymeric Nanocubes by Self-Assembly and Crystallization of Dithiolane-Containing Triblock Copolymers ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Margulis, K., Zhang, X., Joubert, L., Bruening, K., Tassone, C. J., Zare, R. N., Waymouth, R. M. 2017; 56 (51): 16357–62

  View details for DOI 10.1002/anie.201709564

  View details for Web of Science ID 000419399800041

• Formation of Polymeric Nanocubes by Self-Assembly and Crystallization of Dithiolane-Containing Triblock Copolymers. Angewandte Chemie (International ed. in English) Margulis, K., Zhang, X., Joubert, L., Bruening, K., Tassone, C. J., Zare, R. N., Waymouth, R. M. 2017

  Abstract

  Template-free fabrication of non-spherical polymeric nanoparticles is desirable for various applications, but has had limited success owing to thermodynamic favorability of sphere formation. Herein we present a simple way to prepare cubic nanoparticles of block copolymers by self-assembly from aqueous solutions at room temperature. Nanocubes with edges of 40-200 nm are formed spontaneously on different surfaces upon water evaporation from micellar solutions of triblock copolymers containing a central poly(ethylene oxide) block and terminal trimethylene carbonate/dithiolane blocks. These polymers self-assemble into 28±5 nm micelles in water. Upon drying, micelle aggregation and a kinetically controlled crystallization of central blocks evidently induce solid cubic particle formation. An approach for preserving the structures of these cubes in water by thiol- or photo-induced crosslinking was developed. The ability to solubilize a model hydrophobic drug, curcumin, was also explored.

  View details for PubMedID 29080292

• Atomic structure of sensitive battery materials and Interfaces revealed by cryo-electron microscopy SCIENCE Li, Y., Li, Y., Pei, A., Yan, K., Sun, Y., Wu, C., Joubert, L., Chin, R., Koh, A., Yu, Y., Perrino, J., Butz, B., Chu, S., Cui, Y. 2017; 358 (6362): 506–10

  Abstract

  Whereas standard transmission electron microscopy studies are unable to preserve the native state of chemically reactive and beam-sensitive battery materials after operation, such materials remain pristine at cryogenic conditions. It is then possible to atomically resolve individual lithium metal atoms and their interface with the solid electrolyte interphase (SEI). We observe that dendrites in carbonate-based electrolytes grow along the <111> (preferred), <110>, or <211> directions as faceted, single-crystalline nanowires. These growth directions can change at kinks with no observable crystallographic defect. Furthermore, we reveal distinct SEI nanostructures formed in different electrolytes.

  View details for PubMedID 29074771

• Revealing the Cell-Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy ACS NANO Santoro, F., Zhao, W., Joubert, L., Duan, L., Schnitker, J., van de Burgt, Y., Lou, H., Liu, B., Salleo, A., Cui, L., Cui, Y., Cui, B. 2017; 11 (8): 8320–28

  Abstract

  The interface between cells and nonbiological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influence cellular responses; for example, titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compared to a smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-material interface at the relevant nanometer length scale. Here, we present a method for in situ examination of the cell-to-material interface at any desired location, based on focused ion beam milling and scanning electron microscopy imaging to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary by more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future.

  View details for DOI 10.1021/acsnano.7b03494

  View details for Web of Science ID 000408520900084

  View details for PubMedID 28682058

  View details for PubMedCentralID PMC5806611

• Intratendinous Injection of Hydrogel for Reseeding Decellularized Human Flexor Tendons. Plastic and reconstructive surgery Long, C., Galvez, M. G., Legrand, A., Joubert, L., Wang, Z., Chattopadhyay, A., Chang, J., Fox, P. M. 2017; 139 (6): 1305e-1314e

  Abstract

  Decellularized cadaveric tendons are a potential source for reconstruction. Reseeding to enhance healing is ideal; however, cells placed on the tendon surface result in inadequate delivery. The authors used an injection technique to evaluate intratendinous cell delivery.Decellularized tendons were reseeded with adipose-derived stem cells in culture, and injected with fetal bovine serum or hydrogel. PKH26-stained cells in cross-section were quantified. To evaluate cell viability, the authors delivered luciferase-labeled cells and performed bioluminescent imaging. To evaluate synthetic ability, the authors performed immunohistochemistry of procollagen. Adipose-derived stem cells' ability to attract tenocytes was assessed using transwell inserts. Cell-to-cell interaction was assessed by co-culturing, measuring proliferation and collagen production, and quantifying synergy. Finally, tensile strength was tested.Both fetal bovine serum (p < 0.001) and hydrogel (p < 0.001) injection led to more cells inside the tendon compared with culturing. Hydrogel injection initially demonstrated greater bioluminescence than culturing (p < 0.005) and fetal bovine serum injection (p < 0.05). Injection groups demonstrated intratendinous procollagen staining correlating with the cells' location. Co-culture led to greater tenocyte migration (p < 0.05). Interaction index of proliferation and collagen production assays were greater than 1 for all co-culture ratios, demonstrating synergistic proliferation and collagen production compared with controls (p < 0.05). There were no differences in tensile strength.Hydrogel injection demonstrated the greatest intratendinous seeding efficiency and consistency, without compromising tensile strength. Intratendinous cells demonstrated synthetic capabilities and can potentially attract tenocytes inside the tendon, where synergy would promote intrinsic tendon healing.Therapeutic, V.

  View details for DOI 10.1097/PRS.0000000000003359

  View details for PubMedID 28538572

• Visualization of Aspergillus fumigatus biofilms with Scanning Electron Microscopy and Variable Pressure-Scanning Electron Microscopy: A comparison of processing techniques JOURNAL OF MICROBIOLOGICAL METHODS Joubert, L., Ferreira, J. A., Stevens, D. A., Nazik, H., Cegelski, L. 2017; 132: 46-55

  Abstract

  Aspergillus fumigatus biofilms consist of a three-dimensional network of cellular hyphae and extracellular matrix. They are involved in infections of immune-compromised individuals, particularly those with cystic fibrosis. These structures are associated with persistence of infection, resistance to host immunity, and antimicrobial resistance. Thorough understanding of structure and function is imperative in the design of therapeutic drugs. Optimization of processing parameters, including aldehyde fixation, heavy metal contrasting, drying techniques and Ionic Liquid treatment, was undertaken for an ultrastructural approach to understand cellular and extracellular biofilm components. Conventional and Variable Pressure Scanning Electron Microscopy were applied to analyze the structure of biofilms attached to plastic and formed at an air-liquid interface.

  View details for DOI 10.1016/j.mimet.2016.11.002

  View details for Web of Science ID 000393017100009

  View details for PubMedID 27836634

• Pseudomonas phage inhibition of Candida albicans. Microbiology (Reading, England) Nazik, H. n., Joubert, L. M., Secor, P. R., Sweere, J. M., Bollyky, P. L., Sass, G. n., Cegelski, L. n., Stevens, D. A. 2017

  Abstract

  Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

  View details for PubMedID 28982395

• Engulfed cadherin fingers are polarized junctional structures between collectively migrating endothelial cells NATURE CELL BIOLOGY Hayer, A., Shao, L., Chung, M., Joubert, L., Yang, H. W., Tsai, F., Bisaria, A., Betzig, E., Meyer, T. 2016; 18 (12): 1311-?

  Abstract

  The development and maintenance of tissues requires collective cell movement, during which neighbouring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, 'cadherin fingers', which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature-sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighbouring cell, to promote follower behaviour. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.

  View details for DOI 10.1038/ncb3438

  View details for Web of Science ID 000389134600009

  View details for PubMedID 27842057

• Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration MICROBIOLOGY-SGM Penner, J. C., Ferreira, J. A., Secor, P. R., Sweere, J. M., Birukova, M. K., Joubert, L., Haagensen, J. A., Garcia, O., Malkovskiy, A. V., Kaber, G., Nazik, H., Manasherob, R., Spormann, A. M., Clemons, K. V., Stevens, D. A., Bollyky, P. L. 2016; 162 (9): 1583-1594

  Abstract

  Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

  View details for DOI 10.1099/mic.0.000344

  View details for Web of Science ID 000385273100008

  View details for PubMedID 27473221

• Isolation and trans-differentiation of mesenchymal stromal cells into smooth muscle cells: Utility and applicability for cell-sheet engineering. Cytotherapy Shudo, Y., Cohen, J. E., Goldstone, A. B., MacArthur, J. W., Patel, J., Edwards, B. B., Hopkins, M. S., Steele, A. N., Joubert, L., Miyagawa, S., Sawa, Y., Woo, Y. J. 2016; 18 (4): 510-517

  Abstract

  Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) have shown potential to differentiate into various cell types, including smooth muscle cells (SMCs). The extracellular matrix (ECM) represents an appealing and readily available source of SMCs for use in tissue engineering. In this study, we hypothesized that the ECM could be used to induce MSC differentiation to SMCs for engineered cell-sheet construction.Primary MSCs were isolated from the BM of Wistar rats, transferred and cultured on dishes coated with 3 different types of ECM: collagen type IV (Col IV), fibronectin (FN), and laminin (LM). Primary MSCs were also included as a control. The proportions of SMC (a smooth muscle actin [aSMA] and SM22a) and MSC markers were examined with flow cytometry and Western blotting, and cell proliferation rates were also quantified.Both FN and LM groups were able to induce differentiation of MSCs toward smooth muscle-like cell types, as evidenced by an increase in the proportion of SMC markers (aSMA; Col IV 42.3 ± 6.9%, FN 65.1 ± 6.5%, LM 59.3 ± 7.0%, Control 39.9 ± 3.1%; P = 0.02, SM22; Col IV 56.0 ± 7.7%, FN 74.2 ± 6.7%, LM 60.4 ± 8.7%, Control 44.9 ± 3.6%) and a decrease in that of MSC markers (CD105: Col IV 64.0 ± 5.2%, FN 57.6 ± 4.0%, LM 60.3 ± 7.0%, Control 85.3 ± 4.2%; P = 0.03). The LM group showed a decrease in overall cell proliferation, whereas FN and Col IV groups remained similar to control MSCs (Col IV, 9.0 ± 2.3%; FN, 9.8 ± 2.5%; LM, 4.3 ± 1.3%; Control, 9.8 ± 2.8%).Our findings indicate that ECM selection can guide differentiation of MSCs into the SMC lineage. Fibronectin preserved cellular proliferative capacity while yielding the highest proportion of differentiated SMCs, suggesting that FN-coated materials may be facilitate smooth muscle tissue engineering.

  View details for DOI 10.1016/j.jcyt.2016.01.012

  View details for PubMedID 26971679

• Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy. Eukaryotic cell Reichhardt, C., Ferreira, J. A., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2015; 14 (11): 1064-1072

  Abstract

  Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

  View details for DOI 10.1128/EC.00050-15

  View details for PubMedID 26163318

  View details for PubMedCentralID PMC4621319

• Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy. Eukaryotic cell Reichhardt, C., Ferreira, J. A., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2015; 14 (11): 1064-1072

  Abstract

  Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

  View details for DOI 10.1128/EC.00050-15

  View details for PubMedID 26163318

  View details for PubMedCentralID PMC4621319

• Inner ear hair cell-like cells from human embryonic stem cells. Stem cells and development Ronaghi, M., Nasr, M., Ealy, M., Durruthy-Durruthy, R., Waldhaus, J., Diaz, G. H., Joubert, L., Oshima, K., Heller, S. 2014; 23 (11): 1275-1284

  Abstract

  In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.

  View details for DOI 10.1089/scd.2014.0033

  View details for PubMedID 24512547

  View details for PubMedCentralID PMC4028088

• Design and Characterization of an Injectable Tendon Hydrogel: A Novel Scaffold for Guided Tissue Regeneration in the Musculoskeletal System TISSUE ENGINEERING PART A Farnebo, S., Woon, C. Y., Schmitt, T., Joubert, L., Kim, M., Hung Pham, H., Chang, J. 2014; 20 (9-10): 1550-1561

  Abstract

  A biocompatible hydrogel consisting of extracellular matrix (ECM) from human tendons is described as a potential scaffold for guided tissue regeneration and tissue engineering purposes. Lyophilized decellularized tendons were milled and enzymatically digested to form an ECM solution. The ECM solution properties are assessed by proteome analysis with mass spectrometry, and the material's rheological properties are determined as a function of frequency, temperature, and time. In vivo application of the gel in a rat model is assessed for remodeling and host cell repopulation. Histology for macrophage invasion, fibroblast repopulation, and nanoscale properties of the gel is assessed. Gel interaction with multipotent adipoderived stem cells (ASCs) is also addressed in vitro to assess possible cytotoxicity and its ability to act as a delivery vehicle for cells. Proteome analysis of the ECM-solution and gel mass spectroscopy identified the most abundant 150 proteins, of which two isoforms of collagen I represented more than 55% of the sample. Rheology showed that storage (G') and loss (G″) of the ECM solution were stable at room temperature but displayed sigmoidal increases after ∼15 min at 37°C, matching macroscopic observations of its thermo responsiveness. G' and G″ of the gel at 1 rad/s were 213.1±19.9 and 27.1±2.4 Pa, respectively. Electron microscopy revealed fiber alignment and good structural porosity in the gel, as well as invasion of cells in vivo. Histology also showed early CD68(+) macrophage invasion throughout the gel, followed by increasing numbers of fibroblast cells. ASCs mixed with the gel in vitro proliferated, indicating good biocompatibility. This ECM solution can be delivered percutaneously into a zone of tendon injury. After injection, the thermoresponsive behavior of the ECM solution allows it to polymerize and form a porous gel at body temperature. A supportive nanostructure of collagen fibers is established that conforms to the three-dimensional space of the defect. This hydrogel holds the distinctive composition specific for tendon ECM, where tissue-specific cues facilitate host cell infiltration and remodeling. The results presented indicate that injectable ECM materials from tendon may offer a promising alternative in the treatment of tendinopathies and acute tendon injuries.

  View details for DOI 10.1089/ten.tea.2013.0207

  View details for Web of Science ID 000335661400020

  View details for PubMedID 24341855

• Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles. Biomaterials Jiang, X., Malkovskiy, A. V., Tian, W., Sung, Y. K., Sun, W., Hsu, J. L., Manickam, S., Wagh, D., Joubert, L., Semenza, G. L., Rajadas, J., Nicolls, M. R. 2014; 35 (2): 803-813

  Abstract

  Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation.

  View details for DOI 10.1016/j.biomaterials.2013.09.092

  View details for PubMedID 24161166

• External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane NATURE CELL BIOLOGY Galic, M., Jeong, S., Tsai, F., Joubert, L., Wu, Y. I., Hahn, K. M., Cui, Y., Meyer, T. 2012; 14 (8): 874-U212

  Abstract

  Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.

  View details for DOI 10.1038/ncb2533

  View details for Web of Science ID 000307115900017

  View details for PubMedID 22750946

  View details for PubMedCentralID PMC3519285

• Airtight container for the transfer of atmosphere-sensitive materials into vacuum-operated characterization instruments REVIEW OF SCIENTIFIC INSTRUMENTS Gaume, R. M., Joubert, L. 2011; 82 (12): 123705

  Abstract

  This paper describes the design and operation of a simple airtight container devised to facilitate the transfer of atmosphere-sensitive samples from a glovebox to the vacuum chamber of an analytical instrument such as a scanning electron microscope. The use of this device for characterizing the microstructure of highly hygroscopic strontium iodide ceramics by scanning electron microscopy is illustrated as an application example.

  View details for DOI 10.1063/1.3669784

  View details for Web of Science ID 000298643100033

  View details for PubMedID 22225222

• Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis CELLULAR MICROBIOLOGY Peng, K., Broz, P., Jones, J., Joubert, L., Monack, D. 2011; 13 (10): 1586-1600

  Abstract

  Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1β, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g. FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signalling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered.

  View details for DOI 10.1111/j.1462-5822.2011.01643.x

  View details for Web of Science ID 000294924500012

  View details for PubMedID 21883803

  View details for PubMedCentralID PMC3173570

• Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds. Journal of biomedical materials research. Part A Neofytou, E. A., Chang, E., Patlola, B., Joubert, L., Rajadas, J., Gambhir, S. S., Cheng, Z., Robbins, R. C., Beygui, R. E. 2011; 98 (3): 383-393

  Abstract

  Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.

  View details for DOI 10.1002/jbm.a.33113

  View details for PubMedID 21630430

• Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Neofytou, E. A., Chang, E., Patloia, B., Joubert, L., Rajadas, J., Gambhir, S. S., Cheng, Z., Robbins, R. C., Beygui, R. E. 2011; 98A (3): 383-393

  Abstract

  Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.

  View details for DOI 10.1002/jbm.a.33113

  View details for Web of Science ID 000293699800007

• ChePep Controls Helicobacter pylori Infection of the Gastric Glands and Chemotaxis in the Epsilonproteobacteria MBIO Howitt, M. R., Lee, J. Y., Lertsethtakarn, P., Vogelmann, R., Joubert, L., Ottemann, K. M., Amieva, M. R. 2011; 2 (4)

  Abstract

  Microbes use directed motility to colonize harsh and dynamic environments. We discovered that Helicobacter pylori strains establish bacterial colonies deep in the gastric glands and identified a novel protein, ChePep, necessary to colonize this niche. ChePep is preferentially localized to the flagellar pole. Although mutants lacking ChePep have normal flagellar ultrastructure and are motile, they have a slight defect in swarming ability. By tracking the movement of single bacteria, we found that ΔChePep mutants cannot control the rotation of their flagella and swim with abnormally frequent reversals. These mutants even sustain bursts of movement backwards with the flagella pulling the bacteria. Genetic analysis of the chemotaxis signaling pathway shows that ChePep regulates flagellar rotation through the chemotaxis system. By examining H. pylori within a microscopic pH gradient, we determined that ChePep is critical for regulating chemotactic behavior. The chePep gene is unique to the Epsilonproteobacteria but is found throughout this diverse group. We expressed ChePep from other members of the Epsilonproteobacteria, including the zoonotic pathogen Campylobacter jejuni and the deep sea hydrothermal vent inhabitant Caminibacter mediatlanticus, in H. pylori and found that ChePep is functionally conserved across this class. ChePep represents a new family of chemotaxis regulators unique to the Epsilonproteobacteria and illustrates the different strategies that microbes have evolved to control motility.Helicobacter pylori strains infect half of all humans worldwide and contribute to the development of peptic ulcers and gastric cancer. H. pylori cannot survive within the acidic lumen of the stomach and uses flagella to actively swim to and colonize the protective mucus and epithelium. The chemotaxis system allows H. pylori to navigate by regulating the rotation of its flagella. We identified a new protein, ChePep, which controls chemotaxis in H. pylori. ChePep mutants fail to colonize the gastric glands of mice and are completely outcompeted by normal H. pylori. Genes encoding ChePep are found only in the class Epsilonproteobacteria, which includes the human pathogen Campylobacter jejuni and environmental microbes like the deep-sea hydrothermal vent colonizer Caminibacter mediatlanticus, and we show that ChePep function is conserved in this class. Our study identifies a new colonization factor in H. pylori and also provides insight into the control and evolution of bacterial chemotaxis.

  View details for DOI 10.1128/mBio.00098-11

  View details for PubMedID 21791582

• Contributions of Francisella tularensis subsp novicida Chitinases and Sec Secretion System to Biofilm Formation on Chitin APPLIED AND ENVIRONMENTAL MICROBIOLOGY Margolis, J. J., El-Etr, S., Joubert, L., Moore, E., Robison, R., Rasley, A., Spormann, A. M., Monack, D. M. 2010; 76 (2): 596-608

  Abstract

  Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studying F. tularensis pathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology of Francisella species. F. tularensis has been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show that F. tularensis subsp. novicida forms biofilms during the colonization of chitin surfaces. The ability of F. tularensis to persist using chitin as a sole carbon source is dependent on chitinases, since mutants lacking chiA or chiB are attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1 and secB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-type F. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation by F. tularensis promotes persistence on chitin surfaces. Further study of the interaction of F. tularensis with the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.

  View details for DOI 10.1128/AEM.02037-09

  View details for Web of Science ID 000273354200027

  View details for PubMedID 19948864

  View details for PubMedCentralID PMC2805214

• Visualization of Hydrogels with Variable-Pressure SEM Joubert, L. CAMBRIDGE UNIV PRESS. 2009: 1308–9

  View details for DOI 10.1017/S1431927609096263

  View details for Web of Science ID 000208119100645