Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.     

On Oncogenes and Tumor Suppressor Genes in the Mammary Gland

Well-known oncogenes (e.g., MYC) and tumor suppressor genes (e.g., TP53) are involved in breast cancer. But the roles of newly identified genes, microRNAs, and other noncoding RNAs in the disease have yet to be defined.Breast cancer in humans is associated with genetic alterations of a number of oncogenes (ErbB2, MYC, PIK3CA) and tumor suppressors (TP53, BRCA1/2, RB1, PTEN), as outlined by Lee and Muller. The use of genetically engineered mouse models harboring deletions or mutations in these genes has provided insight into how such alterations drive tumor initiation, progression, and metastasis, and how they influence responses to anticancer agents. Beyond these well-characterized alterations, there has been a recent explosion in new information regarding the molecular pathogenesis of breast cancer, and therefore a need to define the functional roles of newly described potential breast cancer genes. For example, whole-genome sequencing has identified a large number of genes with recurrent sequence alterations in human breast cancer specimens (Wood et al. 2007). Moreover, gene copy number analyses have identified multiple regions of chromosomal gain or loss (Chin et al. 2006). Finally, microRNAs and other types of noncoding RNA have recently emerged as important modulators of cancer phenotypes (Ventura and Jacks 2009). Despite, this new information about cancer-associated molecular alterations, the full characterization of their impact on breast cancer biology in vivo remains incomplete. Therefore, a major current challenge is to functionally annotate these emerging groups of candidate breast cancer tumor suppressors/oncogenes.The generation of additional mouse models with engineered mutations or transgenic expression of new candidate genes will provide important information validating their role in cancer and elucidating their specific biological activities. However, it is important to point out that the existing mouse models do not recapitulate the estrogen receptor (ER)-positive histological subtype of breast cancer, which is an important subset in humans. Thus, current transgenic approaches do not provide a comprehensive platform for modeling the full spectrum of the human disease. An equally important issue is that the considerable time and expense of generating new engineered alleles and breeding to obtain compound mutant strains creates a bottleneck in fully annotating the breast cancer genome. Gain- and loss-of-function experiments in cancer cell lines and in immortalized breast epithelial cells (Weaver et al. 1995) can in many cases determine the roles of novel regulators of cellular transformation. However, factors contributing to cancer pathogenesis through such processes as control of cellular senescence, reprogramming of the cellular differentiation state, and interactions with the microenvironment may not be readily studied in such systems. Although xenografts of human cancer cell lines may be helpful in some cases, the functions of other cancer-relevant factors may be best uncovered by the genetic manipulation of primary cells and their subsequent growth as orthotopic implants (Heyer et al. 2010). By using isogenic cells, it is possible to the study tumor formation of such implants in the context of wild-type mice with a fully intact immune system, thereby fully recapitulating the microenvironment of spontaneous tumors. Breast progenitor/stem cells provide a valuable system in this regard, although the selection of other types of breast epithelial cells would likely influence the ensuing tumor phenotype and have utility in studying histological subtypes of breast cancer.An additional important question in annotating the breast cancer genome is whether a genetic lesion contributes to the maintenance of established tumors—rather than just to tumor initiation—and therefore points to a pathway whose deregulation represents a potential drug target. Lee and Muller (2011) refer to work with doxycline-inducible transgenics indicating that tumors induced by ErbB2 remain dependent on this factor for tumor maintenance. The generation of additional inducible overexpression systems and the use of inducible short hairpin RNA (shRNA) in the transplantable models discussed above will be important for defining additional factors required for tumor maintenance. The molecular and cellular alterations resulting from switching off a candidate oncogene in vivo also provides important benchmarks with which to evaluate new drugs that are designed to target such pathways.Noncoding RNAs represent an emerging class of cancer regulators in need of further study in vivo. Micro RNAs (miRNAs) are the most studied group of noncoding RNAs, and individual miRNAs have been shown to function as oncogenes and tumor suppressors in mouse models (Ventura and Jacks 2009). Deregulated miRNAs in breast cancer include miR-210 (Camps et al. 2008), a hypoxia-regulated miRNA that contributes to cancer growth by regulating energy metabolism. miR-335, -126, -10b, and miR-31 have all been shown to regulate invasion and metastasis (Ma et al. 2007; Tavazoie et al. 2008; Valastyan et al. 2009), and let7b and miR-200 negatively regulate breast cancer stem cell self-renewal (Iliopoulos et al. 2009; Shimono et al. 2009). The functional validation of these and other classes of noncoding RNAs in vivo is likely to yield novel observations about the molecular circuitry of breast cancer cells.The molecular pathogenesis of metastasis is a rapidly evolving area of research, and recent efforts have helped define activators and repressors of breast cancer metastasis as well as factors that influence the site of metastatic growth. Notably, it is now apparent that metastatic lesions have marked differences in mutational profiles compared to the primary tumors. Whole-genome sequencing of paired primary and metastatic basal-cell-like breast cancer, and xenografts of the same primary tumor tissue, identified striking similarities and some interesting differences in the prevalence of genetic mutations between the samples (Ding et al. 2010). Mutations detected in the metastatic tumor were highly concordant with those detected in the tumor xenograft. However, some of these mutations were not present in the primary tumor sample. This suggests that the evolutionary pressures driving metastasis and establishment of primary xenografts may be similar, and highlight the potential usefulness of functionally assessing mutations selectively enriched in both lesions. Whether these genetic changes influence tumor cell adaptability and secondary organ site selection are key questions in ongoing efforts to understand the mechanisms governing metastatic spread. In vivo studies have also established an important causative link between metastasis and the expression of specific micro RNAs (miRNA) and identified pro- and antimetastasis regulators (Ma et al. 2007; Tavazoie et al. 2008). The mechanisms by which the expression of these miRNAs are altered and key molecular targets and cellular functions of these miRNAs require further investigation.As new technologies provide an increasingly detailed view of mutational, gene-expression, and gene copy number profiles that define breast cancer, the potential of personalized medicine in cancer therapy becomes more attainable. The full realization of this goal will first require a broader characterization of novel candidate cancer regulators. It is clear that a multipronged approach using multiple model systems will be needed to translate genetic findings into a comprehensive picture of the underlying circuitry of distinct histological and genetic subsets of breast cancer.     

Beyond captions: linking figures with abstract sentences in biomedical articles

Although figures in scientific articles have high information content and concisely communicate many key research findings, they are currently under utilized by literature search and retrieval systems. Many systems ignore figures, and those that do not typically only consider caption text. This study describes and evaluates a fully automated approach for associating figures in the body of a biomedical article with sentences in its abstract. We use supervised methods to learn probabilistic language models, hidden Markov models, and conditional random fields for predicting associations between abstract sentences and figures. Three kinds of evidence are used: text in abstract sentences and figures, relative positions of sentences and figures, and the patterns of sentence/figure associations across an article. Each information source is shown to have predictive value, and models that use all kinds of evidence are more accurate than models that do not. Our most accurate method has an F1-score of 69% on a cross-validation experiment, is competitive with the accuracy of human experts, has significantly better predictive accuracy than state-of-the-art methods and enables users to access figures associated with an abstract sentence with an average of 1.82 fewer mouse clicks. A user evaluation shows that human users find our system beneficial. The system is available at http://FigureItOut.askHERMES.org.     

Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5

BACKGROUND: Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. METHODS: Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. RESULTS: In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. CONCLUSIONS: AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.     

Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in <Emphasis Type="Italic">Drosophila</Emphasis>

Background

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure.

Results

The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits.

Conclusions

We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

     

High-throughput sequence identification of gene coding variants within alcohol-related QTLs

Low initial response to alcohol has been shown to be among the best predictors of development of alcoholism. A similar phenotypic measure, difference in initial sensitivity to ethanol, has been used for the genetic selection of two mouse strains, the Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice, and for the subsequent identification of four quantitative trait loci (QTLs) for alcohol sensitivity. We now report the application of high throughput comparative gene sequencing in the search for genes underlying these four QTLs. To carry out this search, over 1.7 million bases of comparative DNA sequence were generated from 68 candidate genes within the QTL intervals, corresponding to a survey of over 36,000 amino acids. Eight central nervous system genes, located within these QTLs, were identified that contain a total of 36 changes in protein coding sequence. Some of these coding variants are likely to contribute to the phenotypic variation between ILS/ISS animals, including sensitivity to alcohol, providing specific new genetic targets potentially important to the neuronal actions of alcohol.