Faculty Bibliography

Epidermal melanocytes are constantly exposed to environmental stressors such as ultraviolet light (UV) and chemotoxins. Several evolutionarily conversed survival mechanisms are deployed to ensure melanocyte recovery after damage including the unfolded protein response (UPR) and integrated stress response (ISR). The UPR/ISR promote restoration of homeostasis, by modulating transcription and translation as well as activating Nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant activity. If repair fails, the UPR/ISR either stimulate cell death, or adaptation that can lead to survival of damaged cells and promote disease. For example, the UPR/ISR may support melanomagenesis by allowing UV-damaged, mutated cells to survive and adapt to a hostile tumor microenvironment that subjects cells to hypoxia, nutrient deprivation and sub-optimal pH. The UPR and ISR can also promote transcriptional changes that support tumor growth and/or metastasis. Furthermore, these pathways may also underlie acquisition of chemoresistance and modulation of protein expression that alters the efficacy of immunotherapies. UPR activation has also been implicated in the pathogenesis of vitiligo and may promote increased expression of chemokines such as interleukin 6 and interleukin 8 that trigger an autoimmune response against melanocytes. We herein review the potential roles of the UPR/ISR in the etiology of melanoma and vitiligo.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis^1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate^4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)^6-8.

Melanoma, the most lethal form of skin cancer, is rarely curable at its advanced stages. The early events of this disease, during which treatment would be beneficial, remain poorly elucidated. Melanocyte stem cells (McSCs) residing in the hair follicle niche have been proposed to be a cell-of-origin for melanoma. To understand the cellular and molecular mechanisms regulating the initiation and progression of McSC derived melanoma, we have established a novel c-Kit- CreER-driven melanoma mouse model that enabled us to \target McSCs and trace their oncogenic behaviors. Using this model, we showed that oncogenic McSCs first expand in the niche and then migrate to the epidermis to form epidermal melanoma that later invade into the underlying dermis and undergo metastasis. Furthermore, Wnt and Endothelin signals, secreted by epithelial niche cells during hair anagen onset promoted the malignant transformation of McSCs to melanoma. Finally, transcriptional profiling revealed a strong resemblance between murine McSC-derived melanoma and human melanoma in heterogeneity and gene signatures. These results suggest that follicular McSCs can be an origin of melanoma and that follicular niche can control McSC oncogenic transformation. The similarities of McSC derived melanoma with human melanoma in epidermal to dermal progression, heterogeneity and gene expression suggest the potential utilization of this mouse model as a pre-clinical model for human melanoma

Acyl-CoA:cholesterol acyltransferase (ACAT) mediates cellular cholesterol esterification. In atherosclerotic plaque macrophages, ACAT promotes cholesteryl ester accumulation, resulting in foam cell formation and atherosclerosis progression. Its complete inactivation in mice, however, showed toxic effects because of an excess of free cholesterol (FC) in macrophages, which can cause ER stress, cholesterol crystal formation, and inflammasome activation. Our previous studies showed that long-term partial ACAT inhibition, achieved by dietary supplementation with Fujirebio F1394, delays atherosclerosis progression in apoprotein E-deficient (Apoe^-/- ) mice by reducing plaque foam cell formation without inflammatory or toxic effects. Here, we determined whether short-term partial inhibition of ACAT, in combination with an enhanced systemic FC acceptor capacity, has synergistic benefits. Thus, we crossbred Apoe^-/- with human apoprotein A1-transgenic (APOA1^tg/tg ) mice, which have elevated cholesterol-effluxing high-density lipoprotein particles, and subjected Apoe^-/- and APOA1^tg/tg/Apoe^-/- mice to an atherogenic diet to develop advanced plaques. Then mice were either euthanized (baseline) or fed purified standard diet with or without F1394 for four more weeks. Plaques of APOA1^tg/tg/Apoe^-/- mice fed F1394 showed a 60% reduction of macrophages accompanied by multiple other benefits, such as reduced inflammation and favorable changes in extracellular composition, in comparison to Apoe^-/- baseline mice. In addition, there was no accumulation of cholesterol crystals or signs of toxicity. Overall, these results show that short-term partial ACAT inhibition, coupled to increased cholesterol efflux capacity, favorably remodels atherosclerosis lesions, supporting the potential of these combined therapies in the treatment of advanced atherosclerosis. Significance Statement Short-term pharmacological inhibition of ACAT-mediated cholesterol esterification, in combination with increased free cholesterol efflux acceptors, has positive effects in mice by (1) reducing the inflammatory state of the plaque macrophages, and (2) favoring compositional changes associated with plaque stabilization. These effects occur without toxicity, showing the potential of these combined therapies in the treatment of advanced atherosclerosis.

Liquid-phase deposition of exfoliated 2D nanosheets is the basis for emerging technologies that include writable electronic inks, molecular barriers, selective membranes, and protective coatings against fouling or corrosion. These nanosheet thin films have complex internal structures that are discontinuous assemblies of irregularly tiled micron-scale sheets held together by van der Waals (vdW) forces. On stiff substrates, nanosheet vdW films are stable to many common stresses, but can fail by internal delamination under shear stress associated with handling or abrasion. This "re-exfoliation" pathway is an intrinsic feature of stacked vdW films and can limit nanosheet-based technologies. Here we investigate the shear stability of graphene oxide and MoSe₂ nanosheet vdW films through lap shear experiments on polymer-nanosheet-polymer laminates. These sandwich laminate structures fail in mixed cohesive and interfacial mode with critical shear forces from 40 - 140 kPa and fracture energies ranging from 0.2 - 6 J/m². Surprisingly these energies are higher than delamination energies reported for smooth peeling of ordered stacks of continuous 2D sheets, which we propose is due to energy dissipation and chaotic crack motion during nanosheet film disassembly at the crack tip. Experiment results also show that film thickness plays a key role in determining critical shear force (maximum load before failure) and dissipated energy for different nanosheet vdW films. Using a mechanical model with an edge crack in the thin nanosheet film, we propose a shear-to-tensile failure mode transition to explain a maximum in critical shear force for graphene oxide films but not MoSe₂ films. This transition reflects a weakening of the substrate confinement effect and increasing rotational deformation near the film edge as the film thickness increases. For graphene oxide, the critical shear force can be increased by electrostatic cross-linking achieved through interlayer incorporation of metal cations. These results have important implications for the stability of functional devices that employ 2D nanosheet coatings.

INTRODUCTION/BACKGROUND:Dalbavancin is a lipoglycopeptide antibiotic approved as a single- and two-dose regimen for adults with acute bacterial skin and skin structure infections (ABSSSI) caused by susceptible gram-positive organisms. We present nephrotoxicity rates for patients with ABSSSI who received dalbavancin in three pivotal clinical trials and compare the rates with vancomycin. METHODS:In a phase 3b clinical trial (DUR001-303), patients were randomized to dalbavancin single-dose (1500 mg intravenous [IV]) or two-dose regimen (1000 mg IV on day 1, 500 mg IV on day 8). In two phase 3 clinical trials (DISCOVER 1 and DISCOVER 2), patients were randomized to dalbavancin (two-dose regimen) or vancomycin 1 g (or 15 mg/kg) IV every 12 h for at least 3 days with an option to switch to orally administered linezolid 600 mg every 12 h for 10-14 days. Patients on dalbavancin with a creatinine clearance below 30 mL/min not on regular dialysis received a reduced dose of 1000 mg (single-dose arm) or 750 mg IV on day 1, 375 mg IV on day 8 (two-dose arm). Nephrotoxicity was defined as a 50% increase from baseline serum creatinine (SCr) or an absolute increase in SCr of 0.5 mg/dL at any time point. P values were obtained using the Cochran-Mantel-Haenszel test. RESULTS:In dalbavancin-treated patients, rates of nephrotoxicity were low. The safety population with available creatinine values included 1325/1347 patients on any regimen of dalbavancin, and 54/651 patients who received vancomycin intravenously for at least 10 days and were not switched to orally administered linezolid. Patients on any regimen of dalbavancin had a lower rate of nephrotoxicity compared with patients receiving vancomycin intravenously for at least 10 days (3.7% vs 9.3%, respectively; P = 0.039). CONCLUSIONS:Nephrotoxicity rates were lower in patients on dalbavancin relative to vancomycin for at least 10 days. On the basis of this experience, dalbavancin may be less nephrotoxic than intravenously administered vancomycin.

Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function¹. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown². Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)^1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation⁵ mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.

Vitiligo is an acquired condition that affects about 1% of the world's population and is defined by macular depigmentation of the skin that develops following melanocyte death. Vitiligo has a significant impact on both the physical and mental health of patients. While autoimmune-mediated destruction of melanocytes ultimately leads to depigmentation, the mechanisms that promote vitiligo onset remain poorly defined. We have been investigating the hypothesis that melanocytes from individuals genetically prone to develop vitiligo are less efficient in protecting against cellular traumas such as chemical exposure, which triggers an immune response against them. We delineated the response of melanocytes from normally pigmented individuals (NMs) to challenge with the topical agent monobenzone (monobenzyl ether of hydroquinone or MBEH). Three key stress response pathways were activated by MBEH exposure: the unfolded protein stress response (UPR), the NRF2-regulated antioxidant response and the nuclear factor-kappa B (NFkappaB) pathway. We established a key role for the UPR and NRF2 pathways in determining melanocyte viability and demonstrated disruption of their activity in melanocytes from individuals who developed vitiligo (VMs). We further showed that the NFkappaB pathway contributes to an increase in expression of IL6 and IL8 following NM exposure to MBEH and that expression of these chemokines is higher in VMs compared to NMs. These chemokines can promote an autoimmune response. We have now used transcriptome analysis to identify additional stress response pathways that are dysfunctional in vitiligo. Our data suggest that multiple signaling pathways that protect cells against trauma and facilitate a return to homeostasis are disrupted in VMs and may cause these cells to be targeted by the immune system