Blair Bullard

Changes in Transcript Abundance in Chlamydomonas reinhardtii following Nitrogen Deprivation Predict Diversion of Metabolism

Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

Enhanced Uranium Immobilization and Reduction by Geobacter sulfurreducens Biofilms

ABSTRACT Biofilms formed by dissimilatory metal reducers are of interest to develop permeable biobarriers for the immobilization of soluble contaminants such as uranium. Here we show that biofilms of the model uranium-reducing bacterium Geobacter sulfurreducens immobilized substantially more U(VI) than planktonic cells and did so for longer periods of time, reductively precipitating it to a mononuclear U(IV) phase involving carbon ligands. The biofilms also tolerated high and otherwise toxic concentrations (up to 5 mM) of uranium, consistent with a respiratory strategy that also protected the cells from uranium toxicity. The enhanced ability of the biofilms to immobilize uranium correlated only partially with the biofilm biomass and thickness and depended greatly on the area of the biofilm exposed to the soluble contaminant. In contrast, uranium reduction depended on the expression of Geobacter conductive pili and, to a lesser extent, on the presence of the c cytochrome OmcZ in the biofilm matrix. The results support a model in which the electroactive biofilm matrix immobilizes and reduces the uranium in the top stratum. This mechanism prevents the permeation and mineralization of uranium in the cell envelope, thereby preserving essential cellular functions and enhancing the catalytic capacity of Geobacter cells to reduce uranium. Hence, the biofilms provide cells with a physically and chemically protected environment for the sustained immobilization and reduction of uranium that is of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium contamination.

Fibroblast growth factor receptor is a mechanistic link between visceral adiposity and cancer

Epidemiological evidence implicates excess adipose tissue in increasing cancer risk. Despite a steeply rising global prevalence of obesity, how adiposity contributes to transformation (stage a non-tumorigenic cell undergoes to become malignant) is unknown. To determine the factors in adipose tissue that stimulate transformation, we used a novel ex vivo system of visceral adipose tissue (VAT)-condition medium-stimulated epithelial cell growth in soft agar. To extend this system in vivo, we used a murine lipectomy model of ultraviolet light B-induced, VAT-promoted skin tumor formation. We found that VAT from mice and obese human donors stimulated growth in soft agar of non-tumorigenic epithelial cells. The difference in VAT activity was associated with fibroblast growth factor-2 (FGF2) levels. Moreover, human and mouse VAT failed to stimulate growth in soft of agar in cells deficient in FGFR-1 (FGF2 receptor). We also demonstrated that circulating levels of FGF2 were associated with non-melanoma tumor formation in vivo. These data implicate FGF2 as a major factor VAT releases to transform epithelial cells—a novel, potential pathway of VAT-enhanced tumorigenesis. Strategies designed to deplete VAT stores of FGF2 or inhibit FGFR-1 in abdominally obese individuals may be important cancer prevention strategies as well as adjuvant therapies for improving outcomes.

A BET bromodomain inhibitor suppresses adiposity-associated malignant transformation

Almost half a million of all new cancers have been attributed to obesity and epidemiologic evidence implicates visceral adipose tissue (VAT) and high-fat diets (HFD) in increasing cancer risk. We demonstrated that VAT-derived fibroblast growth factor 2 (FGF2) from mice fed an HFD or obese individuals stimulates the malignant transformation of epithelial cells. Mechanism-based strategies to prevent this VAT-enhanced tumorigenesis have not been explored. Clinical studies have indicated that bromodomain inhibitors have considerable potential as therapeutic agents for cancer by inhibiting the activity of several oncogenes, including c-Myc; however, their chemopreventive activity is unknown. We show herein that mice with visceral adiposity have elevated nuclear c-Myc expression in their epidermis. We hypothesized that the bromodomain inhibitor I-BET-762 (I-BET) would have efficacy in the prevention of malignant transformation by VAT and FGF2. We tested this hypothesis using our novel models of VAT-stimulated transformation in vitro and FGF2- stimulated tumor formation in vivo. We found that I-BET significantly attenuates VAT and FGF2-stimulated transformation and inhibits VAT-induced c-Myc protein expression in several skin and breast epithelial cell lines. Moreover, I-BET attenuated tumor growth significantly in FGF2-treated nude mice. Work is ongoing to determine the role of visceral adiposity in c-Myc activity in several tissues and determine the inhibitory effect of I-BET on VAT-promoted tumors in vivo. Cancer Prev Res; 11(3); 129–42. ©2017 AACR. See related editorial by Berger and Scacheri, p. 125

A role for FGF2 in visceral adiposity-associated mammary epithelial transformation

ABSTRACT Obesity is a leading risk factor for post-menopausal breast cancer, and this is concerning as 40% of cancer diagnoses in 2014 were associated with overweight/obesity. Despite this epidemiological link, the underlying mechanism responsible is unknown. We recently published that visceral adipose tissue (VAT) releases FGF2 and stimulates the transformation of skin epithelial cells. Furthermore, obesity is differentially associated with many epithelial cancers, and this mechanistic link could be translational. As FGF2 and FGFR1 are implicated in breast cancer progression, we hypothesize that VAT-derived FGF2 plays a translational role in promoting adiposity-associated mammary epithelial cell transformation. In this brief report, data suggest that FGF2/FGFR1 signaling is a potential mechanistic link in VAT-stimulated transformation of breast epithelial cells.

Abstract 2227: Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells

Background: Adiposity plays a crucial role in the pathogenesis and prognosis of different types of cancers. Epidemiological evidence suggests visceral adipose tissue (VAT) and high-fat diets (HFD) are associated with increased cancer risk however the mechanism is not understood. The aim of this study was to explore the factors in VAT that stimulate neoplastic transformation. Methods: We modeled visceral adiposity-stimulated neoplastic transformation using our novel ex vivo system of VAT-condition medium stimulated epithelial cell transformation (measured by growth in soft agar) and our in vivo murine lipectomy model of ultraviolet light B (UVB)-induced, VAT promoted skin tumor formation. FgfR1(-/-) stable cells were generated by using CRISPR-Cas9 technology and were used to investigate the role of fibroblast growth factor-2 (FGF2) and FGFR1 dependent signaling in neoplastic transformation, both in vitro and in vivo. Results: Only the VAT of obese mice fed a HFD [not VAT from low-fat diet (LFD) fed mice] stimulated neoplastic transformation of skin epithelial cells. Furthermore, human VAT stimulated both skin and mammary epithelial cell transformation. The differences in VAT activity between LFD and HFD fed mice and human donors were associated with the levels of FGF2. Circulating levels of FGF2 were associated with non-melanoma tumor formation in vivo. Human and mouse VAT failed to stimulate transformation in FgfR1(-/-) cells and do not form tumors when injected in Nude mice in vivo. Conclusion: Collectively, our data show FGF2 released from VAT and its interaction with FGFR1 is a novel and potential direct path of VAT-enhanced tumorigenesis. Blocking the FGFR1 induced signaling in VAT of abdominally obese individuals may be an important cancer prevention strategy as well as an adjuvant therapy for improving outcomes following cancer diagnosis. Citation Format: Debrup Chakraborty, Vanessa Benham, Elena Y. Demireva, Blair Bullard, Jamie J. Bernard. Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2017-2227