Faculty Bibliography

INTRODUCTION: Osteoarthritis (OA) is a degenerative disease of the entire joint.¹ In healthy joints the articulating surfaces are encased in a smooth layer of hyaline cartilage and are further protected by the surrounding synovial fluid. High molecular weight hyaluronan (HMWHA) is present at relatively high levels in the synovial fluid and cartilage matrix.'²' HMWHA provides viscoelastic protection and lubrication of the cartilage surfaces, and has also been shown to have important anti-inflammatory properties.'³-⁵' During inflammation hyaluronan (HA) can be degraded to lower molecular weight HA (LMWHA) by the increased levels of free radicals and endogenous hyaluronidases present in the inflamed joint.'⁶' Interestingly, LMWHA, which can signal through a number of different receptors (TLR-2 and 4, CD44 and RHAMM), was suggested to act as potent inflammatory mediator in the joint.'⁷' Our collaborators identified a 15-mer peptide that binds to LMWHA and reduced inflammation and fibrogenesis in excisional skin wounds.^<8' In this study we hypothesized that LMWHA generated in the OA joint is a key mediator for stimulating catabolic and inflammatory events in joint cells and that interfering with LMWHA signaling using the novel peptide inhibitor will attenuate joint inflammation. Methods: Human articular chondrocytes were isolated from articular cartilage samples obtained from patients (donor age range 48 - 67) undergoing total knee replacement surgery at NYU Hospital for Joint Diseases. Knee cartilage was harvested from regions with no macroscopically evident degeneration. The collection of tissue from patients undergoing knee replacement surgery was approved by the Institutional Regulatory Board at NYU School of Medicine. Human chondrocytes were isolated from these cartilage samples and cultured as described previously.'⁹' Cultured chondrocyte were switched to serum-free medium for 24 h and then treated with inflammatory stimuli (interleukin-lbeta (IL-lp), LMWHA; average molecular weight of lOkDa). In addition, serum-starved cells were treated with the 15-mer peptide at various concentrations. A synovial fibroblast cell line (SW982) was also used and was cultured as described previously.'¹⁰' The analysis of HA concentrations in the culture medium was done as described by us previously.'¹¹' Western blotting and real time PCR analysis was performed as described by us.^<12) Results: In the present study we used human articular chondrocytes and a synovial fibroblast cell line (SW982) to determine the involvement of LMWHA promoting inflammation in the joint cavity. We show that LMWHA stimulated catabolic markers (Cox-2, IL-6, iNOS, MMP-13) and inhibited the expression of articular cartilage markers (aggrecan, type II collagen) in human articular chondrocytes and SW982 synovial cells. LMWHA treatment stimulated the NF-KB and the MAP kinase (ERK, JNK, p38) signaling pathways in human chondrocytes similar to IL-lp treatment. Whereas the stimulation of these signaling pathways occurred within the first hour of treatment with IL-lp, the stimulation of these pathways by LMWHA occurred at later time points (6h and 24h). The novel peptide inhibitor markedly reduced the activation of these signaling pathways in LMWHA-treated chondrocytes. In addition, it markedly decreased the expression of catabolic markers and increased the expression of articular cartilage markers in LMWHA-treated chondrocytes. The peptide also inhibited the expression of catabolic markers in IL-1 p-treated human articular chondrocytes and SW982 cells and increased articular cartilage marker expression in IL-1 p-treaded chondrocytes. Treatment of human articular chondrocytes with IL-lp resulted in a marked increase of HA released into the culture medium over time. This increase in HA release correlated with an increase in catabolic markers and a reduction in anabolic markers over time, similar to the effects of the LMWHA treatment. Discussion: Our findings demonstrate that LMWHA stimulates inflammatory and catabolic events in joint cells via activation of NF-Kb and ERK, JNK and p38 signaling pathways. In addition, we demonstrate that a novel peptide that binds to LMWHA and interferes with LMWHA signaling attenuates inflammatory and catabolic events in joint cells mediated by LMWHA and IL-lp. More specifically, we have shown that the novel peptide inhibitor dramatically reduced IL-6 and Cox-2 levels in articular chondrocytes and synovial fibroblasts. Both of these catabolic markers have been previously shown to be associated with increased pain levels in patients with OA.'¹³' These findings suggest that LMWHA plays a key role in mediating inflammation in joint cells during OA pathology, and that a novel peptide inhibitor of LMWHA signaling may act as a novel compound to specifically reduce inflammation and pain in the joint and ultimately slow down cartilage degradation during OA. SIGNIFICANCE: This study identified LMWHA as a key mediator of inflammatory and catabolic events in joint cells. In addition, we determined that interfering with LMWHA signaling using a novel peptide that binds LMWHA may provide a novel therapeutic strategy to reduce inflammation in the OA joint and ultimately slow down cartilage degradation during OA

Electron crystallography of two-dimensional crystalline arrays is a powerful alternative for the structure determination of membrane proteins. The advantages offered by this technique include a native membrane environment and the ability to closely correlate function and dynamics with crystalline preparations and structural data. Herein, we provide a detailed protocol for the reconstitution and two-dimensional crystallization of the sarcoplasmic reticulum calcium pump (also known as Ca(2+)-ATPase or SERCA) and its regulatory subunits phospholamban and sarcolipin.

Vitiligo, an acquired depigmentation disorder, manifests as white macules on the skin and can cause significant psychological stress and stigmatization. Recent advances have shed light on key components that drive disease onset and progression as well as therapeutic approaches. Vitiligo can be triggered by stress to the melanin pigment-producing cells of the skin, the melanocytes. The triggers, which range from sunburn to mechanical trauma and chemical exposures, ultimately cause an autoimmune response that targets melanocytes, driving progressive skin depigmentation. The most significant progress in our understanding of disease etiology has been made on three fronts: (1) identifying cellular responses to stress, including antioxidant pathways and the unfolded protein response (UPR), as key players in disease onset, (2) characterizing immune responses that target melanocytes and drive disease progression, and (3) identifying major susceptibility genes. The current model for vitiligo pathogenesis postulates that oxidative stress causes cellular disruptions, including interruption of protein maturation in the endoplasmic reticulum (ER), leading to the activation of the UPR and expression of UPR-regulated chemokines such as interleukin 6 (IL-6) and IL-8. These chemokines recruit immune components to the skin, causing melanocytes to be targeted for destruction. Oxidative stress can further increase melanocyte targeting by promoting antigen presentation. Two key components of the autoimmune response that promote disease progression are the interferon (IFN)-γ/CXCL10 axis and IL-17-mediated responses. Several genome-wide association studies support a role for these pathways, with the antioxidant gene NRF2, UPR gene XBP1, and numerous immune-related genes including class I and class II major histocompatibility genes associated with a risk for developing vitiligo. Novel approaches to promote repigmentation in vitiligo are being investigated and may yield effective, long-lasting therapies.

Germ granules are the hallmark of all germ cells. These membrane-less, electron-dense structures were first observed over 100 years ago. Today, their role in regulating and processing transcripts critical for the establishment, maintenance, and protection of germ cells is well established, and pathways outlining the biochemical mechanisms and physical properties associated with their biogenesis are emerging.

BACKGROUND: In 2007, the World Health Organization (WHO) recommended scaling up voluntary medical male circumcision (VMMC) in priority countries with high HIV prevalence and low male circumcision (MC) prevalence. According to the Joint United Nations Programme on HIV/AIDS (UNAIDS), an estimated 5.8 million males had undergone VMMC by the end of 2013. Implementation experience has raised questions about the need to refocus VMMC programs on specific subpopulations for the greatest epidemiological impact and programmatic effectiveness. As Malawi prepared its national operational plan for VMMC, it sought to examine the impacts of focusing on specific subpopulations by age and region. METHODS: We used the Decision Makers' Program Planning Toolkit, Version 2.0, to study the impact of scaling up VMMC to different target populations of Malawi. National MC prevalence by age group from the 2010 Demographic and Health Survey was scaled according to the MC prevalence for each district and then halved, to adjust for over-reporting of circumcision. In-country stakeholders advised a VMMC unit cost of $100, based on implementation experience. We derived a cost of $451 per patient-year for antiretroviral therapy from costs collected as part of a strategic planning exercise previously conducted in- country by UNAIDS. RESULTS: Over a fifteen-year period, circumcising males ages 10-29 would avert 75% of HIV infections, and circumcising males ages 10-34 would avert 88% of infections, compared to the current strategy of circumcising males ages 15-49. The Ministry of Health's South West and South East health zones had the lowest cost per HIV infection averted. Moreover, VMMC met WHO's definition of cost-effectiveness (that is, the cost per disability-adjusted life-year [DALY] saved was less than three times the per capita gross domestic product) in all health zones except Central East. Comparing urban versus rural areas in the country, we found that circumcising men in urban areas would be both cost-effective and cost-saving, with a VMMC cost per DALY saved of $120 USD and with 15 years of VMMC implementation resulting in lifetime HIV treatment costs savings of $331 million USD. CONCLUSIONS: Based on the age analyses and programmatic experience, Malawi's VMMC operational plan focuses on males ages 10-34 in all districts in the South East and South West zones, as well as Lilongwe (an urban district in the Central zone). This plan covers 14 of the 28 districts in the country.

Recent developments in tissue clearing methods have provided investigators with an invaluable tool for visualizing and mapping three dimensional macromolecular structures and processes. By implementing a routine protocol, based on the passive clarity technique (PACT) method, for tissue clearing, the Research Histopathology Core at NYU Langone Medical Center seeks to provide investigators with a reliable, customizable service in conjunction with the immunohistochemistry (IHC) and Microscopy Cores in order to produce results in the most efficient way possible for both the investigators and the cores. The PACT method of clearing allows visualization of endogenous fluorescence and immunofluorescence labeling performed by the Core, or both. Stabilization through transparent hydrogel cross-linking, followed by delipidation in an sodium dodecyl sulfate buffer, results in a clear tissue sample that remains structurally sound with proteins, nucleic acids, and any associated labels in place. The clearing buffer can also be modified to allow simultaneous decalcification of bone specimens. Final clearing is achieved in a refractive index matching solution (RIMS buffer) which also serves as the microscopy medium. Cleared and labeled tissue can then be imaged on the Microscopy Core's Zeiss lightsheet microscope, allowing multichannel fluorescence from a range of angles and Z-stacking. The lightsheet microscope excites and detects only one thin optical section of the specimen at a time, making three dimensional imaging exceptionally light efficient. By honing proficiency in tissue clearing via the PACT method and working in close collaboration with neighboring core labs, the Histopathology Core can increase its breadth of expertise while relieving investigators of the time and cost intensive burden of protocol development and training.

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 degrees C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 A. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

Neurotrophins and glucocorticoids are robust synaptic modifiers, and deregulation of their activities is a risk factor for developing stress-related disorders. Low levels of brain-derived neurotrophic factor (BDNF) increase the desensitization of glucocorticoid receptors (GR) and vulnerability to stress, whereas higher levels of BDNF facilitate GR-mediated signaling and the response to antidepressants. However, the molecular mechanism underlying neurotrophic-priming of GR function is poorly understood. Here we provide evidence that activation of a TrkB-MAPK pathway, when paired with the deactivation of a GR-protein phosphatase 5 pathway, resulted in sustained GR phosphorylation at BDNF-sensitive sites that is essential for the transcription of neuronal plasticity genes. Genetic strategies that disrupted GR phosphorylation or TrkB signaling in vivo impaired the neuroplasticity to chronic stress and the effects of the antidepressant fluoxetine. Our findings reveal that the coordinated actions of BDNF and glucocorticoids promote neuronal plasticity and that disruption in either pathway could set the stage for the development of stress-induced psychiatric diseases.

BACKGROUND: Food intake of the adult fruit fly Drosophila melanogaster, an intermittent feeder, is attributed to several behavioral elements including foraging, feeding initiation and termination, and food ingestion. Despite the development of various feeding assays in fruit flies, how each of these behavioral elements, particularly food ingestion, is regulated remains largely uncharacterized. RESULTS: To this end, we have developed a manual feeding (MAFE) assay that specifically measures food ingestion of an individual fly completely independent of the other behavioral elements. This assay reliably recapitulates the effects of known feeding modulators, and offers temporal resolution in the scale of seconds. Using this assay, we find that fruit flies can rapidly assess the nutritional value of sugars within 20-30 s, and increase the ingestion of nutritive sugars after prolonged periods of starvation. Two candidate nutrient sensors, SLC5A11 and Gr43a, are required for discriminating the nutritive sugars, D-glucose and D-fructose, from their non-nutritive enantiomers, respectively. This suggests that differential sensing mechanisms play a key role in determining food nutritional value. CONCLUSIONS: Taken together, our MAFE assay offers a platform to specifically examine the regulation of food ingestion with excellent temporal resolution, and identifies a fast-acting neural mechanism that assesses food nutritional value and modulates food intake.