Generation and analysis of a mouse intestinal metatranscriptome through Illumina based RNA-sequencing

With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority from Bacteroides or Clostridium species. To place these HTS data within a systems context, we mapped the relative abundance of corresponding Escherichia coli homologs onto metabolic and protein-protein interaction networks. These maps identified bacterial processes with components that were well-represented in the datasets. In summary this study highlights the potential of exploiting the economy of HTS platforms for metatranscriptomics

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis

Systems-Based Analysis of the \u3cem\u3eSarcocystis neurona\u3c/em\u3e Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Genomic analysis of the causative agents of coccidiosis in domestic chickens

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro.

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways