Faculty Bibliography

The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5 degrees C, CO(2) evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl(2) addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl(2) . Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5 degrees C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.

Fossils recently found in the Late Miocene of Sahabi, Libya, are attributed to the extinct species Semlikiichthys rhachirhinchus. This study enriches our knowledge of the anatomy of this species. The hyomandibula and, putatively, the operculum are for the first time attributed to Semliknchthys. The fragile laminar supraoccipital crest in the dorsicranium is preserved complete. The material from Sahabi described here extends the Late Miocene distribution of the species to the north and west to include Libya. The paleobiogeographic distribution of Semlikiichthys in Africa, and the freshwater faunal connections between different hydrographical basins in north central Africa, are discussed. (c) 2012 Elsevier Masson SAS. All rights reserved

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER), and the transcription factor X-box binding protein 1 spliced (XBP1S), a regulator of the UPR, is known to be important for ER stress (ERS)-mediated apoptosis and cell growth, but the molecular mechanism underlying these processes remains unexplored. Here, we report that knockdown of XBP1S by an siRNA silencing approach increased the expression of ERS-associated molecules. The overexpression of XBP1S stimulated, whereas its knockdown inhibited, cell proliferation in chondrocytes and chondrosarcoma cells; in addition, overexpression of XBP1S inhibited, while its repression enhanced, ERS-mediated apoptosis in chondrocytes and chondrosarcoma cells. Furthermore, XBP1S-mediated inhibition of apoptosis in response to ERS is through the Erk1/2 signaling pathway and down-regulation CHOP transcription factor. CHOP is one of the key downstream molecules known to be involved in ERS-mediated apoptosis. Collectively, these findings reveal a novel critical role of XBP1S in ERS-mediated apoptosis and the molecular mechanisms involved.

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-kappaB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-kappaB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.

Allergic asthma is a chronic disease of the lung characterized by underlying Th2- and IgE-mediated inflammation, structural alterations of the bronchial wall, and airway hyperresponsiveness. Initial allergic sensitization and later development of chronic disease are determined by close interactions between lung structural cells and the resident and migratory immune cells in the lung. Epithelial cells play a crucial role in allergic sensitization by directly influencing dendritic cells induction of tolerant or effector T cells and production of type 2 cytokines by innate immune cells. During chronic disease, the bronchial epithelium, stroma, and smooth muscle become structurally and functionally altered, contributing to the perpetuation of tissue remodeling. Thus, targeting tissue-driven pathology in addition to inflammation may increase the effectiveness of asthma treatment.

The heterogeneity of tumor samples is a major challenge in the analysis of high-throughput profiling of tumor biopsies and cell lines. The measured aggregate signals of multigenerational progenies often represent an average of several tumor subclones with varying genomic aberrations and different gene expression levels. The goal of the present study was to integrate copy number analyses from SNP-arrays and karyotyping, gene expression profiling, and pathway analyses to detect heterogeneity, identify driver mutations, and explore possible mechanisms of tumor evolution. We showed the heterogeneity of the studied samples, characterized the global copy number alteration profiles, and identified genes whose copy number status and expression levels were aberrant. In particular, we identified a recurrent association between two BRAF(V600E) and BRAF(V600K) mutations and changes in DKK1 gene expression levels, which might indicate an association between the BRAF and WNT pathways. These findings show that the integrated approaches used in the present study can robustly address the challenging issue of tumor heterogeneity in high-throughput profiling.

The pathobiology of vitiligo, characterized by the spread of depigmented skin patches due to localized melanocyte loss, is not fully understood. Oxidative stress is thought to play a role in disease onset with a subsequent autoimmune response underlying progression. We therefore sought to identify mechanisms that linked oxidative stress and autoimmune responses. Melanocytes at the periphery of vitiligo lesions have distended endoplasmic reticuli (ER). We hypothesized that oxidative stress disrupted homeostasis of the ER where oxidation/reduction reactions facilitate disulfide bond formation. As a result, misfolded peptides would accumulate, dilating the ER and activating the unfolded protein response (UPR). The UPR is a stress response pathway, initiated by three regulators (IRE1, PERK and ATF6). It first promotes cell survival, however sustained activation induces apoptosis. In order to identify a potential role for the UPR in vitiligo we dosed melanocytes with 4-tertiary butyl phenol (4- TBP) and monobenzyl ether of hydroquinone (MBEH), phenols known to trigger vitiligo. The phenols caused an increase in expression of IRE1 and PERK. PERK activation leads to enhancement of the antioxidant response by recruitment of the transcription factor NRF2 to the nucleus and increased expression of the antioxidant HMOX1. The IRE1 effector, X-box binding protein-1 (XBP1) was also activated by phenol treatment which led to increased production of interleukin-6 (IL6) and IL8, cytokines expressed at increased levels in perilesional skin in vitiligo. Treatment with XBP1 inhibitors reduced phenol-induced IL6 and IL8 production, while over-expression of active XBP1 increased their expression. Thus, chemicals known to cause vitiligo trigger a UPR-mediated increase in cytokine production. There are a number of potential roles for the UPR in vitiligo. The UPR may (i) induce apoptosis following sustained oxidative stress causing release of melanocyte specific antigens, (ii) be dysregulated resulting in a muted PERK-!

INTRODUCTION: Dendritic cells (DC) are professional antigen presenting cells (APC) that hold significant promise for the development of cancer vaccines. Endoplasmic reticular (ER)-stress can enhance the immunogenicity of APC. Since blockade of fatty acid synthesis increases ER-stress, we postulated that inhibition of fatty acid synthesis in DC would enhance their immune-stimulatory properties. METHODS: HumanmoDCwere generated fromPMBCby culture in GM-CSF/IL-4. Murine DC were generated from bone marrow using GM-CSF. TOFA-an acetyl-CoA carboxylase inhibitor-was used to block fatty acid synthesis. RESULTS: Blockade of fatty acid synthesis decreased DC development from precursors and mitigated DC proliferation in mice and humans. The mechanism was related to downregulation of Cyclin B-1-a pro-proliferative gene-and upregulation of Caspase 3 and PARP-apoptosis-related genes. Blockade of fatty acid synthesis enhanced DC activation and capacity for stimulation of immune effecter cells. TOFA-DC expressed elevated activated MAP-K, NF-kB, and PI3K/Akt intermediates and produced higher cytokines (Table). Peptide-pulsed TOFA-DC induced more vigorous proliferation of antigen-restricted CD4+ and CD8+ T cells, induced T cell IFN-g and TNF-a production, and enhanced CTL in vivo (Table). TOFA-DC induced higher NK expression of CD25 and production of IFN-g (Table). Accordingly, we found increased capacity for Notch signaling in TOFA-DC. TOFA-treated DC exhibited high ER-stress including increased expression of GRP-78, eIF2-a, p-eIF2-a and XBP-1, and blockade of ER-stress by a chaperone protein mitigated their enhanced immune-stimulation. (Table presented) CONCLUSIONS: Blockade of fatty acid synthesis markedly increases DC immunogenicity and capacity to induce CTL by augmenting ER stress. Our findings have significant implications for the design of immunotherapy vaccines

The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.

Regulation of hepatic very low density lipoprotein (VLDL) assembly and maturation is crucial in controlling lipid homeostasis and in the development of metabolic disorders, including obesity, hepatic steatosis, and insulin resistance. Cideb, a member of cell death-inducing DFF45-like effector (CIDE) protein family, has been previously shown to promote VLDL lipidation and maturation. However, the precise subcellular location of Cideb-mediated VLDL lipidation and the factors modulating its activity remain elusive. In addition to its localization to endoplasmic reticulum (ER) and lipid droplets (LD), we observed that Cideb was also localized to the Golgi apparatus. Mature and lipid-rich VLDL particles did not accumulate in the Golgi apparatus in Cideb(-/-) livers. Interestingly, we observed that hepatic perilipin 2/adipose differentiation-related protein (ADRP) levels were markedly increased in Cideb(-/-) mice. Liver-specific knockdown of perilipin 2 in Cideb(-/-) mice resulted in the reduced accumulation of hepatic triglycerides (TAG), increased VLDL-TAG secretion, and the accumulation of mature TAG-rich VLDL in the Golgi apparatus. These data reveal that Cideb and perilipin 2 play opposing roles in controlling VLDL lipidation and hepatic lipid homeostasis.