Transcript and protein signatures derived from shared molecular interactions across cancers are associated with mortality.

BACKGROUND: Characterization of shared cancer mechanisms have been proposed to improve therapy strategies and prognosis. Here, we aimed to identify shared cell-cell interactions (CCIs) within the tumor microenvironment across multiple solid cancers and assess their association with cancer mortality. METHODS: CCIs of each cancer were identified by NicheNet analysis of single-cell RNA sequencing data from breast, colon, liver, lung, and ovarian cancers. These CCIs were used to construct a shared multi-cellular tumor model (shared-MCTM) representing common CCIs across cancers. A gene signature was identified from the shared-MCTM and tested on the mRNA and protein level in two large independent cohorts: The Cancer Genome Atlas (TCGA, 9185 tumor samples and 727 controls across 22 cancers) and UK biobank (UKBB, 10,384 cancer patients and 5063 controls with proteomics data across 17 cancers). Cox proportional hazards models were used to evaluate the association of the signature with 10-year all-cause mortality, including sex-specific analysis. RESULTS: A shared-MCTM was derived from five individual cancers. A shared gene signature was extracted from this shared-MCTM and the most prominent regulatory cell type, matrix cancer-associated fibroblast (mCAF). The signature exhibited significant expression changes in multiple cancers compared to controls at both mRNA and protein levels in two independent cohorts. Importantly, it was significantly associated with mortality in cancer patients in both cohorts. The highest hazard ratios were observed for brain cancer in TCGA (HR [95%CI] = 6.90[4.64-10.25]) and ovarian cancer in UKBB (5.53[2.08-8.80]). Sex-specific analysis revealed distinct risks, with a higher mortality risk associated with the protein signature score in males (2.41[1.97-2.96]) compared to females (1.84[1.44-2.37]). CONCLUSION: We identified a gene signature from a comprehensive shared-MCTM representing common CCIs across different cancers and revealed the regulatory role of mCAF in the tumor microenvironment. The pathogenic relevance of the gene signature was supported by differential expression and association with mortality on both mRNA and protein levels in two independent cohorts.

ProkSeq for complete analysis of RNA-Seq data from prokaryotes.

SUMMARY: Since its introduction, RNA-Seq technology has been used extensively in studies of pathogenic bacteria to identify and quantify differences in gene expression across multiple samples from bacteria exposed to different conditions. With some exceptions, tools for studying gene expression, determination of differential gene expression, downstream pathway analysis and normalization of data collected in extreme biological conditions is still lacking. Here, we describe ProkSeq, a user-friendly, fully automated RNA-Seq data analysis pipeline designed for prokaryotes. ProkSeq provides a wide variety of options for analysing differential expression, normalizing expression data and visualizing data and results. AVAILABILITY AND IMPLEMENTATION: ProkSeq is implemented in Python and is published under the MIT source license. The pipeline is available as a Docker container https://hub.docker.com/repository/docker/snandids/prokseq-v2.0, or can be used through Anaconda: https://anaconda.org/snandiDS/prokseq. The code is available on Github: https://github.com/snandiDS/prokseq and a detailed user documentation, including a manual and tutorial can be found at https://prokseqV20.readthedocs.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Elongator, a conserved complex required for wobble uridine modifications in eukaryotes.

Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm(5)) and 5-carbamoylmethyl (ncm(5)) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm(5) and mcm(5) side chains at U34 and their influence on Elongator activity.

RNA atlas of human bacterial pathogens uncovers stress dynamics linked to infection.

Bacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific 'universal stress responders', that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex).

Exploring a Drosophila Transcription Factor Interaction Network to Identify Cis-Regulatory Modules.

Multiple transcription factors (TFs) bind to specific sites in the genome and interact among themselves to form the cis-regulatory modules (CRMs). They are essential in modulating the expression of genes, and it is important to study this interplay to understand gene regulation. In the present study, we integrated experimentally identified TF binding sites collected from published studies with computationally predicted TF binding sites to identify Drosophila CRMs. Along with the detection of the previously known CRMs, this approach identified novel protein combinations. We determined high-occupancy target sites, where a large number of TFs bind. Investigating these sites revealed that Giant, Dichaete, and Knirp are highly enriched in these locations. A common TAG team motif was observed at these sites, which might play a role in recruiting other TFs. While comparing the binding sites at distal and proximal promoters, we found that certain regulatory TFs, such as Zelda, were highly enriched in enhancers. Our study has shown that, from the information available concerning the TF binding sites, the real CRMs could be predicted accurately and efficiently. Although we only may claim co-occurrence of these proteins in this study, it may actually point to their interaction (as known interaction proteins typically co-occur together). Such an integrative approach can, therefore, help us to provide a better understanding of the interplay among the factors, even though further experimental verification is required.

Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in Yersinia pseudotuberculosis.

RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN binding motif located at 103 inter- and intragenic sites on both sense and antisense strands. Deletion of rpoN had a large impact on gene expression, including downregulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the antisense strand commonly reduced expression of genes carried by the corresponding sense strand.IMPORTANCE The alternative sigma factor RpoN (σ54), which is widely distributed in eubacteria, has been implicated in controlling gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection, and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis, and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first study of its kind of RpoN in Y. pseudotuberculosis, revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA, providing important information about RpoN regulation in enterobacteria.

Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells.

Campylobacter jejuni is a prevalent human pathogen and a major cause of bacterial gastroenteritis in the world. In humans, C. jejuni colonizes the intestinal tract and its tolerance to bile is crucial for bacteria to survive and establish infection. C. jejuni produces outer membrane vesicles (OMVs) which have been suggested to be involved in virulence. In this study, the proteome composition of C. jejuni OMVs in response to low concentration of bile was investigated. We showed that exposure of C. jejuni to low concentrations of bile, similar to the concentration in cecum, induced significant changes in the protein profile of OMVs released during growth without affecting the protein profile of the bacteria. This suggests that bile influences a selective packing of the OMVs after bacterial exposure to low bile. A low concentration of bile was found to increase bacterial adhesion to intestinal epithelial cells, likely by an enhanced hydrophobicity of the cell membrane following exposure to bile. The increased bacterial adhesiveness was not associated with increased invasion, instead bile exposure decreased C. jejuni invasion. OMVs released from bacteria upon exposure to low bile showed to increase both adhesion and invasion of non-bile-exposed bacteria into intestinal epithelial cells. These findings suggest that C. jejuni in environments with low concentrations of bile produce OMVs that facilitates colonization of the bacteria, and this could potentially contribute to virulence of C. jejuni in the gut.

Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile.

INTRODUCTION: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified [Formula: see text], which in wild-type strains are modified with mcm5s2U, partially suppress phenotypes of an elp3Δ strain. OBJECTIVES: To identify metabolic alterations in an elp3Δ strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified [Formula: see text]. METHOD: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3Δ strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing [Formula: see text]. The temperature sensitive elp3Δ strain derivatives were cultivated at permissive (30 °C) or semi-permissive (34 °C) growth conditions. RESULTS: Culturing an elp3Δ strain at 30 or 34 °C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3Δ strain carrying the wild-type ELP3 gene. Overexpression of hypomodified [Formula: see text] suppressed a subset of the metabolic alterations observed in the elp3Δ strain. CONCLUSION: Our results suggest that the presence of ncm5- and mcm5-side chains on wobble uridines in tRNA are important for metabolic homeostasis.