Faculty Bibliography

Epithelial cells form intercellular junctions to strengthen cell-cell adhesion and limit diffusion, allowing epithelia to function as dynamic tissues and barriers separating internal and external environments. Junctions form as epithelial cells differentiate; clusters of junction proteins first concentrate apically, then mature into continuous junctional belts that encircle and connect each cell. In mammals and Drosophila, atypical protein kinase C (aPKC) is required for junction maturation, although how it contributes to this process is poorly understood. A role for the Caenorhabditis elegans aPKC homologue PKC-3 in junction formation has not been described previously. Here, we show that PKC-3 is essential for junction maturation as epithelia first differentiate. Using a temperature-sensitive allele of pkc-3 that causes junction breaks in the spermatheca and leads to sterility, we identify intragenic and extragenic suppressors that render pkc-3 mutants fertile. Intragenic suppressors include an unanticipated stop-to-stop mutation in the pkc-3 gene, providing evidence for the importance of stop codon identity in gene activity. One extragenic pkc-3 suppressor is a loss-of-function allele of the lethal(2) giant larvae homologue lgl-1, which antagonizes aPKC within epithelia of Drosophila and mammals but was not known previously to function in C. elegans epithelia. Finally, two extragenic suppressors are loss-of-function alleles of sups-1, a previously uncharacterized gene. We show that SUPS-1 is an apical extracellular matrix protein expressed in epidermal cells, suggesting that it non-autonomously regulates junction formation in the spermatheca. These findings establish a foundation for dissecting the role of PKC-3 and interacting genes in epithelial junction maturation.

BACKGROUND:Pressure injury is seen across all healthcare settings and affects people of any age and health condition. It imposes a significant burden, with annual costs of up to $17.8 billion in the United States alone. Despite considerable resources it exhausts, the disease remains very prevalent, and the incidence is on the rise. This is in part due to aging population, growing number of nursing home residents, poorly understood biology, and dismal track record of clinical research in this field. METHODS:In our Review Article, we discuss the disease pathophysiology, clinical manifestation, evidence based recommendations for risk assessment, prevention and timely management, existing challenges, and directions to improve research on the field. This article encompasses dedicated sections on the full spectrum of the pressure related pathologies including "conventional pressure ulcers", "medical device related pressure injuries", "pressure injuries in mucosal membranes", "pressure injuries in pediatric population", "pressure injury at end of life", and the "role of pressure in pathogenesis of diabetic foot ulcers".

The purpose of this investigation was to determine the role of extracellular vesicles (EVs), released from articular chondrocytes in a physiological or pathological state, in cell-cell communication with other articular chondrocytes or chondrocytic precursor cells. Conditioned medium from interleukin-1beta (IL-1β)-treated human articular chondrocytes stimulated catabolic events and inhibited type II collagen expression in articular chondrocytes to a much greater degree than medium from IL-1β-treated chondrocytes after complete removal of EVs. Vehicle-treated and IL-1β-treated human articular chondrocytes released EVs of similar size; however the number of EVs released by IL-1β-treated chondrocytes was markedly higher than the number of EVs released from vehicle-treated cells. Furthermore, our findings demonstrate that similar to medium from IL-1β-treated chondrocytes containing EVs, EVs isolated from medium of IL-1β-treated chondrocytes stimulated catabolic events in articular chondrocytes, whereas EVs isolated from the medium of vehicle-treated chondrocytes inhibited catabolic events and increased mRNA levels of aggrecan and type II collagen in IL-1β-treated chondrocytes. Furthermore, medium containing EVs from vehicle-treated articular chondrocytes or EVs isolated from this medium stimulated chondrogenesis of C3H10T1/2 cells, whereas medium containing EVs from IL-1β-treated chondrocytes or EVs isolated from this medium inhibited chondrogenesis. Our findings suggest that EVs released by articular chondrocytes play a key role in the communication between joint cells and ultimately in joint homeostasis, maintenance, pathology, and repair. This article is protected by copyright. All rights reserved.

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.

NYU School of Medicine recently embarked on a re-design of its anatomy curriculum that decreased the use of cadavers with plastinated specimens. Plastinated models provide an authentic learning experience of the human body, but lack necessary labels outlining important structures. Due to the fragile nature of the specimens, we endeavored to solve the challenge of labeling by developing a digitized supplement and archive of plastinated and pathology specimens. An interdisciplinary team of faculty and multimedia designers at NYU School of Medicine designed and developed electronic resources related to the artistic models and plastinated specimens. Over the course of three months, 60 artistic and plastinated models of different sizes were captured from dozens of angles using a digital camera or an Artec Leo Scanner. The numerous image captures of the plastinated specimens were processed in Agisoft Metashape, a stand-alone software product, that performs photogrammetric processing of digital images and generates 3D spatial data. After Agisoft Metashape exported a complex 3D mesh with a high-resolution texture, anatomy faculty added labels to the digitized 3D anatomy specimens using the Sketchfab web platform. The labeled 3D anatomy models were then uploaded into the Living Anatomy site on NYU School of Medicine's learning management system for students to explore before, during, and after their anatomy lab sessions. Quizzes using these models also were created to help students identify the structures and link them to physiology and clinical scenarios. The digitized 3D models allow students to zoom in, rotate and explore the specimens in a more interactive way, thereby enhancing the process of just observing fragile plastination models. When asked, 84% of students reported that the 3D models of plastinated specimens contributed "very much so" to their learning of anatomical relationships. We will continue to find opportunities for the meaningful integration of these 3D models within the anatomy curriculum as well as into other pre-clerkship and clerkship modules. We will also assess the educational outcomes of the 3D models and, by doing so, will incorporate instructional design into the process.

Osteocutaneous reconstruction can be challenging because of concomitant injuries and limited donor sites. There is a paucity of data on limb salvage outcomes following combined soft-tissue reconstruction and bone transport or Masquelet procedures. The authors reviewed a consecutive series of open tibia fracture patients undergoing soft-tissue reconstruction with either distraction osteogenesis or Masquelet technique. Endpoints were perioperative flap complications and bone union. Fourteen patients with Gustilo type IIIB open tibia fractures were included. Half of the group received muscle flaps and the remaining half received fasciocutaneous flaps. Ten patients (71.4 percent) underwent distraction osteogenesis and the remaining patients underwent Masquelet technique. Average bone gap length was 65.7 ± 31.3 mm (range, 20 to 120 mm). In the bone transport group, the average external fixation duration was 245 days (range, 47 to 686 days). In the Masquelet group, the average duration of the first stage of this two-stage procedure (i.e., time from cement spacer placement to bone grafting) was 95 days (range, 42 to 181 days). Bone union rate, as determined by radiographic evidence, was 85.7 percent. There was one complete flap failure (7.1 percent). One patient underwent below-knee amputation after failing bone transport and developing chronic osteomyelitis and subsequent infected nonunion. Our case series demonstrates that nonosteocutaneous flap methods of limb reconstruction are a viable option in patients with segmental long bone defects, with a bone union rate of 85 percent and a limb salvage rate over 90 percent in patients with Gustilo type IIIB fractures. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Therapeutic, IV.

In OA chondrocytes, there is diminished mitochondrial production of ATP and diminished extracellular adenosine resulting in diminished adenosine A2A receptor (A2AR) stimulation and altered chondrocyte homeostasis which contributes to the pathogenesis of OA. We tested the hypothesis that A2AR stimulation maintains or enhances mitochondrial function in chondrocytes. The effect of A2AR signaling on mitochondrial health and function was determined in primary murine chondrocytes, a human chondrocytic cell line (T/C-28a2), primary human chondrocytes, and a murine model of OA by transmission electron microscopy analysis, mitochondrial stress testing, confocal live imaging for mitochondrial inner membrane polarity, and immunohistochemistry. In primary murine chondrocytes from A2AR^-/- null mice, which develop spontaneous OA by 16 weeks, there is mitochondrial swelling, dysfunction, and reduced mitochondrial content with increased reactive oxygen species (ROS) burden and diminished mitophagy, as compared to chondrocytes from WT animals. IL-1-stimulated T/C-28a2 cells treated with an A2AR agonist had reduced ROS burden with increased mitochondrial dynamic stability and function, findings which were recapitulated in primary human chondrocytes. In an obesity-induced OA mouse model, there was a marked increase in mitochondrial oxidized material which was markedly improved after intraarticular injections of liposomal A2AR agonist. These results are consistent with the hypothesis that A2AR ligation is mitoprotective in OA.

Neural stem cells (NSCs) are multipotent progenitors that are responsible for producing all of the neurons and macroglia in the nervous system. In adult mammals, NSCs reside predominantly in a mitotically dormant, quiescent state, but they can proliferate in response to environmental inputs such as feeding or exercise. It is hoped that quiescent NSCs could be activated therapeutically to contribute towards repair in humans. This will require an understanding of quiescent NSC heterogeneities and regulation during normal physiology and following brain injury. Non-mammalian vertebrates (zebrafish and salamanders) and invertebrates (Drosophila) offer insights into brain repair and quiescence regulation that are difficult to obtain using rodent models alone. We review conceptual progress from these various models, a first step towards harnessing quiescent NSCs for therapeutic purposes.

Radial glial progenitor cells (RGPs) are the major neural progenitor cells that generate neurons and glia in the developing mammalian cerebral cortex^1-4. In RGPs, the centrosome is positioned away from the nucleus at the apical surface of the ventricular zone of the cerebral cortex^5-8. However, the molecular basis and precise function of this distinctive subcellular organization of the centrosome are largely unknown. Here we show in mice that anchoring of the centrosome to the apical membrane controls the mechanical properties of cortical RGPs, and consequently their mitotic behaviour and the size and formation of the cortex. The mother centriole in RGPs develops distal appendages that anchor it to the apical membrane. Selective removal of centrosomal protein 83 (CEP83) eliminates these distal appendages and disrupts the anchorage of the centrosome to the apical membrane, resulting in the disorganization of microtubules and stretching and stiffening of the apical membrane. The elimination of CEP83 also activates the mechanically sensitive yes-associated protein (YAP) and promotes the excessive proliferation of RGPs, together with a subsequent overproduction of intermediate progenitor cells, which leads to the formation of an enlarged cortex with abnormal folding. Simultaneous elimination of YAP suppresses the cortical enlargement and folding that is induced by the removal of CEP83. Together, these results indicate a previously unknown role of the centrosome in regulating the mechanical features of neural progenitor cells and the size and configuration of the mammalian cerebral cortex.

Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD 'fusion' event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.