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bioRxiv2019

Organism-wide single-cell transcriptomics of long-lived C. elegans daf-2 mutants reveals tissue-specific reprogramming of gene expression networks

A critical requirement for a systems-level understanding of complex biological processes such as aging is the ability to directly characterize interactions between cells and tissues within a multicellular organism. C. elegans nematodes harboring mutations in the insulin-like receptor daf-2 exhibit dramatically-increased lifespans. To identify tissue-specific biochemical mechanisms regulating aging plasticity, we single-cell sequenced 3-mRNA libraries generated from seven populations of whole day-one adult wild-type and daf-2-/- worms using the 10x ChromiumV1platform. The age-synchronized samples were bioinformatically merged into a single aligned dataset containing 40,000 age-synchronized wild-type and daf-2-/- cellular transcriptomes partitioned into 101 clusters, using unsupervised machine-learning algorithms to identify common cell types. Here we describe the basic features of the adult C. elegans single-cell transcriptome and summarize functional alterations observed in the gene expression profiles of long-lived daf-2-/- worms. Comprehensive methods and datasets are provided. This is the first study to directly quantify cell-specific differential gene expression between two age-synchronized, genetically-distinct populations of multicellular organisms. This novel approach answers fundamental questions regarding tissue-specific regulation of gene expression and helps to establish a foundation for a comprehensive C. elegans single-cell gene expression atlas.

cell biology↗

bioRxiv2019

Identifying small proteins by ribosome profiling with stalled initiation complexes

Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the true prevalence of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly-initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. The corresponding genes are not only intergenic, but are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.\n\nIMPORTANCEProteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the function of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.

microbiology↗

bioRxiv2019

Phage-encoded cationic antimicrobial peptide used for outer membrane disruption in lysis

Spanins are required for the last step in bacteriophage lysis: the disruption of the outer membrane. Bioinformatic analysis has shown that ~15% of phages lack a spanin gene, which suggests an alternate mechanism of outer membrane disruption. To address this, we selected virulent podophage {phi}KT as a spaninless exemplar and tested {phi}KT genes for outer membrane disruption during lysis. Hypothetical novel gene 28 causes outer membrane disruption when co-expressed with {phi}KT lysis genes and complements the lysis defect of a {lambda} spanin mutant. Gp28 is a 56 aa cationic peptide with predicted amphipathic helical structure and is associated with the particulate fraction after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic interaction with the membrane. Super high-resolution microscopy supports a primarily outer membrane localization for the peptide. Additionally, holin function is not required for gp28-mediated lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. In standard assays to measure bactericidal and inhibitory effects of antimicrobial peptides on bacterial cells, synthesized gp28 performed equivalently to LL-37. The studies presented here suggest that {phi}KT Gp28 disrupts bacterial outer membranes during lysis in a manner akin to antimicrobial peptides.\n\nSignificanceHere we provide evidence that {phi}KT produces an antimicrobial peptide for outer membrane disruption during lysis. The disruptin is a new paradigm for phage lysis, and has no similarities to other known lysis genes. Many mechanisms have been proposed for the function of antimicrobial peptides, however there is not a consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic selection system to support such studies. Therefore, the {phi}KT disruptin may represent the first genetically tractable antimicrobial peptide.

microbiology↗

bioRxiv2019

Environmental and epigenetic regulation of Rider retrotransposons in tomato

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Thus our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants.

plant biology↗

bioRxiv2019

Phylogenomics reveals major diversification rate shifts in the evolution of silk moths and relatives

The silkmoths and their relatives (Bombycoidea) are an ecologically and taxonomically diverse superfamily that includes some of the most charismatic species of all the Lepidoptera. Despite displaying some of the most spectacular forms and ecological traits among insects, relatively little attention has been given to understanding their evolution and the drivers of their diversity. We heavily sampled (both in taxa and loci) all major lineages of the Bombycoidea, producing a well-supported phylogeny that identified important evolutionary patterns (e.g., morphology, biogeography, and differences in speciation and extinction). Importantly, analysis of diversification rates highlights the stark increases that exist within the Sphingidae (hawkmoths) and Saturniidae (wild silkmoths). We postulate that these rate shifts are due to differences in the intense selective pressures from insectivorous bats. The study also introduces a new Bombycoidea-specific Anchored Hybrid Enrichment (AHE) probe set, a modified DNA extraction protocol for Lepidoptera specimens from natural history collections, and additional information on the existing AHE bioinformatics pipeline. Our research highlights the flexibility of AHE to generate genomic data from a wide range of museum specimens, both age and preservation method, and will allow researchers to tap into the wealth of biological data residing in natural history collections around the globe.

evolutionary biology↗

bioRxiv2019

Grass silica mineralizer (GSM1) protein precipitates silica in sorghum silica cells

O_LISilicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume by unknown mechanism.\nC_LIO_LIUsing bioinformatics tools, we identified a previously uncharacterized protein in sorghum (Sorghum bicolor), which we named Siliplant1 (Slp1). Silica precipitation activity in vitro, expression profile, and activity in precipitating biosilica in vivo were characterized.\nC_LIO_LISlp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. A short peptide, repeating five times in the protein precipitated silica in vitro at a biologically relevant silicic acid concentration. Raman and NMR spectroscopies showed that the peptide attached the silica through lysine amine groups, forming a mineral-peptide open structure. We found Slp1 expression in immature leaf and inflorescence tissues. In the immature leaf active silicification zone, Slp1 was localized to the cytoplasm or near cell boundaries of silica cells. It was packed in vesicles and secreted to the paramural space. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types.\nC_LIO_LIOur results show that Slp1 precipitates silica in sorghum silica cells.\nC_LI

plant biology↗

bioRxiv2019

Comparative genomics analysis reveals high levels of differential DNA transposition among primates

Mobile elements generated via DNA transposition constitute [~]50% of the primate genomes. As a result of past and ongoing activity, DNA transposition is responsible for generating inter- and intra-species genomic variations, and it plays important roles in shaping genome evolution and impacting gene function. While limited analysis of mobile elements has been performed in many primate genomes, a large-scale comparative genomic analysis examining the impact of DNA transposition on primate evolution is still missing.\n\nUsing a bioinformatics comparative genomics approach, we performed analysis of species-specific mobile elements (SS-MEs) in eight primate genomes, which include human, chimpanzee, gorilla, orangutan, green monkey, crab-eating macaque, rhesus monkey, and baboon. These species have good representations for the top two primate families, Hominidae (great apes) and the Cercopithecidae (old world monkeys), for which draft genome sequences are available.\n\nOur analysis identified a total of 230,855 SS-MEs from the eight primate genomes, which collectively contribute to [~]82 Mbp genome sequences, ranging from 14 to 25 Mbp for individual genomes. Several new interesting observations were made based on these SS-MEs. First, the DNA transposition activity level reflected by the numbers of SS-MEs was shown to be drastically different across species with the highest (baboon genome) being more than 30 times higher than the lowest (crab-eating macaque genome). Second, the compositions of SS-MEs, as well as the top active ME subfamilies, also differ significantly across genomes. By the copy numbers of SS-MEs divided into major ME classes, SINE represents the dominant class in all genomes, but more so in the Cercopithecidae genomes than in the Hominidae genomes in general with the orangutan genome being the outliner of this trend by having LINE as the dominant class. While AluY represents the major SINE groups in the Hominidae genomes, AluYRa1 is the dominant SINE in the Cercopithecidae genomes. For LINEs, each Hominidae genome seems to have a unique most active L1 subfamily, but all Cercopithecidae genomes have L1RS2 as the most active LINEs. While genomes with a high number of SS-MEs all have one or more very active ME subfamilies, the crab-eating macaque genome, being the one with an extremely low level of DNA transposition, has no single ME class being very active, suggesting the existence of a genome-wide mechanism suppressing DNA transposition. Third, DNA transposons, despite being considered dead in primate genomes, were in fact shown to have a certain level of activity in all genomes examined with a total of [~]2,400 entries as SS-MEs. Among these SS-MEs, at least 23% locate to genic regions, including exons and regulatory elements, presenting significant potentials for their impact on gene function. Very interestingly, our data demonstrate that, among the eight primates included in this study, the human genome is shown to be the most actively evolving genome via DNA transposition as having the highest most recent activity of many ME subfamilies, notably the AluYa5/Yb8/Yb9, L1HS, and SVA-D subfamilies.\n\nRepresenting the first of its kind, our large-scale comparative genomics study has shown that mobile elements evolved quite differently among different groups and species of primates, indicating that differential DNA transposition has served as an important mechanism in primate evolution.

evolutionary biology↗

bioRxiv2019

Partitioning of microbial function among taxonomic ranks across the tree of life

Widely used microbial taxonomies, such as the NCBI taxonomy, are based on a combination of sequence homology among conserved genes and historically accepted taxonomies, which were developed based on observable traits such as morphology and physiology. A recently-proposed alternative taxonomy, the Genome Taxonomy Database (GTDB), incorporates only sequence homology of conserved genes and attempts to partition taxonomic ranks such that each rank implies the same amount of evolutionary distance, regardless of its position on the phylogenetic tree. This provides the first opportunity to completely separate taxonomy from traits, and therefore to quantify how taxonomic rank corresponds to traits across the microbial tree of life. We quantified the enrichment of clusters of orthologous gene functional categories (COG-FCs) as a proxy for traits within the lineages of 13,735 cultured and uncultured microbial lineages from a custom-curated genome database. On average, 41.4% of the variation in COG-FC enrichment is explained by taxonomic rank, with domain, phylum, class, order, family, and genus explaining, on average, 3.2%, 14.6%, 4.1%, 9.2%, 4.8%, and 5.5% of the variance, respectively (p<0.001 for all). To our knowledge, this is the first work to quantify the variance in metabolic potential contributed by individual taxonomic ranks. A qualitative comparison between the COG-FC enrichments and genus-level phylogenies, generated from published concatenated protein sequence alignments, further supports the idea that metabolic potential is taxonomically coherent at higher taxonomic ranks. The quantitative analyses presented here constrain the integral relationship between diversification of microbial lineages and the metabolisms which they host.\n\nImportanceRecently there has been great progress in defining a complete taxonomy of bacteria and archaea, which has been enabled by improvements in DNA sequencing technology and new bioinformatic techniques. A new, algorithmically-defined microbial tree of life describes those linkages relying solely on genetic data, which raises the question of how microbial traits relate to taxonomy. Here, we adopted cluster of orthologous group functional categories as a scheme to describe the genomic contents of microbes, which can be applied to any microbial lineage for which genomes are available. This simple approach allows quantitative comparisons between microbial genomes with different gene composition from across the microbial tree of life. Our observations demonstrate statistically significant patterns in cluster of orthologous group functional categories at the taxonomic levels spanning from domain to genus.

microbiology↗

bioRxiv2019

Regulation of Parent-of-Origin Allelic Expression in Arabidopsis thaliana endosperm

Genomic imprinting is an epigenetic phenomenon set in the gametes prior to fertilization that causes differential expression of parental alleles mainly in the endosperm of flowering plants. The overlap between previously identified panels of imprinted genes is limited. In order to achieve high resolution sequencing data we have used sequence capture technology to investigate imprinting. Here, we present a bioinformatics pipeline to assay parent-of-origin allele specific expression and report more than 300 loci with parental expression bias. We find that the level of expression from maternal and paternal alleles in most cases is not binary, instead favouring a differential dosage hypothesis for the evolution of imprinting in plants. To address imprinting regulation, we systematically employed mutations in regulative epigenetic pathways suggested to be major players in the process. We establish the mechanistic mode of imprinting for more than 50 loci regulated by DNA methylation and Polycomb-dependent histone methylation. However, the imprinting patterns of the majority of genes were not affected by these mechanisms. To this end we also demonstrate that the RNA-directed DNA methylation pathway alone does not influence imprinting patterns in a substantial manner, suggesting more complex epigenetic regulation pathways for the majority of identified imprinted genes.\n\nAuthor summaryExpression of gene copies only from the mother or the fathers genome, also termed imprinting, is a specialized epigenetic phenomenon that is found to be enriched at some genes expressed in the mammalian placenta and in the endosperm of the plant seed. Although several studies have reported on imprinted genes in plants, the identified loci are at large non-overlapping between reports. This motivated us to investigate in detail the expression pattern of imprinted genes in the endosperm and to determine how imprinting patterns are established at various imprinted loci. Although several underlying epigenetic regulation mechanisms have been demonstrated to establish imprinting patterns at certain genes, the majority of imprinted genes have not been linked to such mechanisms. In the present study we systematically investigated the mechanisms that are involved in establishing imprinting, by employing mutants of epigenetic regulators and high-throughput sequencing. In our high resolution study, we report more than 300 imprinted genes and demonstrate that the biological phenomenon imprinting involves gradual expression of parental gene copies rather than switching gene copies on or off. Notably, for the majority of imprinted genes, the mechanisms previously believed to be major to establish their imprinting patterns, are not responsible for mediating imprinting.

genetics↗

bioRxiv2019

Tetraploidy in rodent cardiac stem cells confers enhanced biological properties

Ploidy for cardiomyocytes is well described but remains obscure in cardiac interstitial cells (CICs). Ploidy of c-kit+CICs were assessed using a combination of confocal, karyotypic, and flow cytometric assessments coupled with molecular and bioinformatic analyses. Fundamental differences were found between cultured rodent (rat, mouse) c-kit+CICs possessing mononuclear tetraploid (4n) content versus large mammal (human, swine) with mononuclear diploid (2n) content. In-situ analysis, confirmed with fresh isolates, revealed diploid content in c-kit+CICs from human and a mixture of diploid and tetraploid nuclei in mouse. Molecular assessment of the p53 signaling pathway provides a plausible explanation for escape from replicative senescence in rodent but not human ckit+CICs. Single cell transcriptional profiling reveals distinctions between diploid versus tetraploid populations in mouse ckit+CICs, alluding to functional divergences. Collectively, these data reveal fundamental species-specific biological differences in c-kit+CICs that could account for challenges in extrapolation of myocardial preclinical studies from rodent to large animal models.

cell biology↗

bioRxiv2019

Identification of regulatory genes through global gene expression analysis of a Helicobacter pylori co-culture system

Helicobacter pylori is a gram-negative bacterium that establishes life-long infections by inducing immunoregulatory responses. We have developed a novel ex vivo H. pylori co-culture system to identify new regulatory genes based on expression kinetics overlapping with that of genes with known regulatory functions. Using this novel experimental platform, in combination with global transcriptomic analysis, we have identified five lead candidates, validated them using mouse models of H. pylori infection and in vitro co-cultures under pro-inflammatory conditions. Plexin domain containing 2 (Plxdc2) was selected as the top lead immunoregulatory target. Gene silencing and ligand-induced activation studies confirmed its predicted regulatory function. Our integrated bioinformatics analyses and experimental validation platform has enabled the discovery of new immunoregulatory genes. This pipeline can be used for the identification of genes with therapeutic applications for treating infectious, inflammatory, and autoimmune diseases.

immunology↗

bioRxiv2019

Transcriptome analysis of the Molecular Mechanism underlying Immunity- and Reproduction trade-off in Locusta migratoria Infected by Micrococcus luteus

Immune response and reproductive success are two of the main energy-consuming processes in living organisms. However, it is unclear which process is prioritized when both are required. Therefore, the present study was designed to examine this question using one of the worlds most destructive agricultural pests, the migratory locust Locusta migratoria. Transcripts from the ovaries and fat bodies of newly emerged locusts were analyzed, using RNA-seq based transcriptome and qualitative real-time PCR, at 4 h and 6 d after being infected with the gram-positive bacteria Microcroccus luteus, and changes in the main biological pathways involved in reproduction and immunization were analyzed using bioinformatics. At 4 h after infection, 348 and 133 transcripts were up- and down-regulated, respectively, whereas 5699 and 44 transcripts were up- and down-regulated, respectively, at 6 d after infection. Meanwhile, KEGG analysis indicated that vital pathways related with immunity and reproduction, such as Insulin resistance, FoxO signaling, Lysosome, mTOR signaling, and Toll-like receptor signaling pathways were up-regulated. Among the differentially expressed genes, 22 and 17 were related to immunity and reproduction, respectively, and the expression levels of PPO1 and antimicrobial peptide defensin 3 were increased (log2FC = 5.93 and 6.75, respectively), whereas those of VgA and VgB were reduced (log2FC = -17.82 and -18.13, respectively). These results indicated that that locusts allocate energy and resources to maintain their own survival by increasing immune response when dealing with both immune and reproductive processes. The present study provides the first report of expression levels for genes related with reproduction and immunity in locusts, thereby providing a reference for future studies, as well as theoretical guidance for investigations of locust control.

animal behavior and cognition↗

bioRxiv2019

CTCF-mediated Chromatin Structures Dictate the Spatio-temporal Propagation of Replication Foci

Mammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically. To understand how replication is regulated at the sub-RD level, we directly visualized the spatio-temporal organization, morphology, and in situ epigenetic signatures of individual replication foci (RFi) across S-phase using super-resolution stochastic optical reconstruction microscopy (STORM). Importantly, we revealed a hierarchical radial pattern of RFi propagation that reverses its directionality from early to late S-phase, and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a CTCF-organized REplication Propagation (CoREP) model. The CoREP model suggests a non-random selection mechanism for replication activation mediated by CTCF at the sub-RD level, as well as the critical involvement of local chromatin environment in regulating replication in space and time.

cell biology↗

bioRxiv2019

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CICs interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, and also observed novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines predominantly at 3 and 5 untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CICs functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CICs potential roles in cancers. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts. Simple summaryCapicua (CIC) is a gene that is frequently mutated in several cancer types, including stomach cancers and certain subtypes of brain tumours and sarcomas. CIC, the protein encoded by the CIC gene, has been shown to play a multitude of roles in both normal and cancer cell functions; however, most studies exploring these roles focus on a single aspect of CIC function and may therefore overlook complex interconnected activities in which CIC is involved. In this study, we have used multiple complementary approaches to obtain a broader view of CICs complex functional networks. We observed novel interactions (genetic or physical) between CIC and genes/proteins involved in various aspects of cellular function, including regulation of cell division and processing of RNA molecules. Altogether, our work characterises the complexity of CICs functional network and expands our understanding of its potential roles in cancer.

cancer biology↗

bioRxiv2019

Reevaluating the Fusobacterium Virulence Factor Landscape

Fusobacterium are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including the oral pathogen Fusobacterium nucleatum being linked to the progression and severity of colorectal cancer. The global identification of virulence factors in Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the identification and correction of virulence factors from the Type 5 secreted autotransporter and FadA protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and the classic typed strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we employed protein sequence similarity networks to identify subsets of virulence factors, and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have defined protein subsets within the Type 5a effectors that are present in predicted invasive strains but are broadly lacking in passively invading strains; a key phenotype associated with Fusobacterium virulence. However, our data shows that prior bioinformatic analysis that predicted species of Fusobacterium to be non-{inverted exclamation}nvasive can indeed invade human cells, and that pure phylogenetic analysis to determine the virulence within this bacterial genus should be used cautiously and subsequently paired with experiments to validate these hypotheses. In addition, we provide data that show a complex interplay between autotransporters, MORN2 domain containing proteins, and FadA adhesins that we hypothesize synergistically contribute to host cell interactions and invasion. In summary, we report that accurate open reading frame annotations using complete Fusobacterium genomes, in combination with experimental validation of invasion, redefines the repertoire of virulence factors that could be contributing to the species specific pathology of multiple Fusobacterium induced infections and diseases. IMPORTANCEFusobacterium are emerging pathogens that contribute to the progression and severity of multiple mammalian and human infectious diseases, including colorectal cancer. Despite a validated connection with disease, a limited number of proteins have been characterized that define a direct molecular mechanism for pathogenesis in a diverse range of host tissue infections. We report a comprehensive examination of virulence associated protein families in multiple Fusobacterium species, and show that complete genomes facilitate the correction and identification of multiple, large Type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell invasion experiments to show that previously predicted non-invasive strains can indeed enter human cells, and that this is likely due to the expansion of specific virulence proteins that drive F. nucleatum infections and disease.

microbiology↗

bioRxiv2019

Systematic Analysis of Metabolic Pathway Distributions of Bacterial Energy Reserves

Metabolism of energy reserves are essential for bacterial functions such as pathogenicity, metabolic adaptation, and environmental persistence, etc. Previous bioinformatics studies have linked gain or loss of energy reserves such as glycogen and polyphosphate (polyP) with host-pathogen interactions and bacterial virulence based on a comparatively small number of bacterial genomes or proteomes. Thus, understanding the distribution patterns of energy reserves metabolism across bacterial species provides a shortcut route to look into bacterial lifestyle and physiology theoretically. So far, five major energy reserves have been identified in bacteria due to their effective capacity to support bacterial persistence under nutrient deprivation conditions, which include wax ester (WE), triacylglycerol (TAG), polyhydroxyalkanoates (PHA), polyphosphate, and glycogen. Although unknown pathways directly involved in energy reserves keep being discovered with the continuous endeavour of molecular microbiologists and it is currently rather clear about the enzymes related with the metabolism of energy reserves, there is a lack of systematic study of the pathway or key enzyme distributions of the five energy reserves in bacteria from an evolutionary point of view. With the fast development of sequencing technology, abundant bacterial proteomes are available in public database now. In this study, we sourced 8214 manually reviewed bacterial reference proteomes from UniProt database and used statistical models to search homologous sequences of key enzymes related with energy reserves. The distribution patterns of the pathways for energy reserves metabolism are visualized in taxonomy-based phylogenetic trees. According to the study, it was revealed that specific pathways and enzymes are associated with certain types of bacterial groups, which provides evolutionary insights into the understanding of their origins and functions. In addition, the study also confirmed that loss of energy reserves is correlated with bacterial genome reduction. Through this analysis, a much clearer picture about energy reserves metabolism in bacteria is present, which could serve a guide for further theoretical and experimental analyses of energy reserves metabolism in bacteria.

microbiology↗

bioRxiv2019

Conditional activation of immune-related signatures and prognostic significance: a pan-cancer analysis

BackgroundIt is becoming clear that tumor immune T cell infiltration and its functional orientation have substantial effect on cancer progression, influencing both response to therapy and prognosis. In this pan-cancer study, the previously described Immunologic Constant of Rejection (ICR) signature is used to define opposing immune phenotypes (i.e., immuneactive and immune-silent) across 31 different histologies. We systematically analyze the interconnections between the genetic programming of neoplasms and their immune orientation across different histologies, and the prognostic impact of such interplay. Moreover, we investigated the predictive value of ICR classification across various public datasets of immune checkpoint inhibition therapy. MethodsRNA-seq data of samples from a total of 9,282 patient tumor samples representing 31 cancer types were obtained from The Cancer Genome Atlas (TCGA). We classified each cancer type based on the expression of the ICR gene signature. Oncogenic pathway gene set enrichment and mutational status were analyzed in relation to ICR phenotypes. To explore whether tumorintrinsic attributes associate with the prognostic value of ICR across cancers, we compared mutational load, oncogenic alterations and expression of oncogenic pathways between cancer types using an integrative bioinformatic pipeline. ResultsOur analyses identified a distinct prognostic connotation of ICR depending on cancer histology. We identified several oncogenic pathways whose enrichment inversely correlated with ICR in multiple tumor types. We found several cancer specific pathways that were differentially enriched between tumors in which ICR had a prognostic impact versus the ones in which ICR did not bear any prognostic connotation such as proliferation and TGF-beta signaling. Importantly, this conditional impact of ICR was also validated in the context of immune checkpoint inhibition treatment. ConclusionsWe identified tumor-intrinsic attributes that correlate with immune phenotypes and potentially influence their development. In addition, a relationship was observed between the enrichment of oncogenic pathways and the prognostic significance of the ICR and its predictive value for patients treated with anti-CTLA4 immune checkpoint inhibition. Such information can be used to prioritize potential candidates for therapies aimed at converting immune-silent into immuneactive tumors and to refine stratification algorithms.

cancer biology↗

bioRxiv2019

Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data

Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-Seq, ATAC-Seq and NOMe-Seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However we also find a high proportion of assay specific NDRs that are often "called" by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-Seq and DNAse I-Seq provide a high NDR calling rate for relatively low sequencing costs in comparison to NOMe-Seq, NOMe-Seq singles out as it provides a multitude of information: it allows to not only detect NDRs but also endogenous DNA methylation, genome wide segmentation into heterochromatic A/B domains and local phasing of nucleosomes outside of NDRs. In summary our comparison strongly suggest to consider assay specific differences for the experimental desgin and for generalized and comparative functional interpretations.

genomics↗