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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Profiling gene expression of the host response to a Plasmodium vivax irradiated sporozoite immunization and infectious challenge

The development of vaccines that provide sterile protection against human malaria is a major global public health priority requiring a better understanding of the mechanisms involved in natural and vaccine-induced sterile immunity. RNAseq was used to profile gene expression of peripheral blood samples from 12 Duffy positive (Fy+) (Plasmodium vivax susceptible) volunteers enrolled in a phase 2 trial who were vaccinated with radiation attenuated P. vivax sporozoites (RAS; 5 were protected, 7 not) and from 5 Fy- (P. vivax resistant) volunteers exposed to mosquitoes harboring live non-attenuated sporozoites. Blood samples were obtained before and after immunization as well as after controlled infection with live P vivax sporozoites. The most profound changes in gene expression were observed between baseline and post-challenge, with 97 distinct signatures differentiating protected and not protected Fy+ individuals. Differentiation was also observed between Fy- and Fy+ protected individuals, notably with downregulation of multiple inflammatory responses as well as extracellular matrix-related gene activity. Analysis of transcriptional modules shows that both B-cell and T-cell signaling are reduced while cell cycle regulation, interferon response, and other informative signatures are elevated in individuals who are not protected against malaria. An asymptomatic individual had an intermediate profile indicative of differential transcription associated with pathology and symptomology. Systems biology thus provides insight into how whole malaria-attenuated sporozoites prime the immune system to protect against malaria, as well as the transcription responses that are associated with sterile protection.\n\nAUTHOR SUMMARYTranscriptomic analysis has shed considerable light on the molecular mechanisms of vaccine efficacy against a variety of viral pathogens, but has not yet been used in relation to parasite vaccinology. Here we describe whole peripheral blood gene expression profiling to contrast the contributions of myeloid and lymphoid immune responses to vaccine-induced and natural protection against Plasmodium vivax- mediated malaria. Two modes of modular bioinformatic analyses are applied to RNAseq data from pre- and post-vaccination samples from 19 volunteer participants in a clinical trial investigating the efficacy of an irradiated sporozoite vaccine. During active infection, both B- and T-cell activity are reduced in individuals who are not protected by vaccination relative to those who are protected. Lack of the Fy antigen prevents entry of parasite into red blood cells, thereby providing natural clinical immunity, but we also document differences in T-cell and neutrophil activity in these individuals relative to those protected by irradiation of the parasite. One individual who was asymptomatic for malaria but had blood parasite despite vaccination showed an intermediate response indicating how systems biology approaches can distinguish gene expression associated with pathology and infection.

genomics↗

bioRxiv2018

Low-Density Lipoprotein Receptor-Mediated Lipidome-Transcriptome Reprogramming Impulses to Cisplatin Insensitivity

Platinum-based therapy remains the cornerstone for cancer patient management; however, its efficacy varies. Theis study demonstrated the differential expressions of low-density lipoprotein receptor (LDLR) in subtypes of epithelial ovarian carcinoma (EOC) determines cisplatin sensitivity. Its sensitive in serous EOCs (low LDLR), where insensitive in endometrioid and clear cell EOCs (high LDLR). Meanwhile, knocked-down or overexpressed LDLR in EOC could reversed the chemosensitivity pattern both in vitro and in vivo. Mechanistic dissection with transcriptome vs. lipidome trans-omics analyses elucidated the LDLR[->]LPC (Lyso-PhosphotidylCholine)[->]FAM83B (phospholipase-related)[->]FGFRs (cisplatin sensitivity and phospholipase-related) regulatory axis in cisplatin insensitivity. Implementing LPC-liposome encapsulated cisplatin could facilitate DNA-adduct formation via lipid droplets (LDs) delivery. Furthermore, Bioinformatics analyses found that the LDL/R[->]LD homeostasis alteration is critical for therapeutic prognosis. Lastly, using LPC-liposome-cisplatin improved cisplatin sensitivities in gastric cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, and pancreatic adenocarcinoma cells. In conclusion, this report discovered a LDL/R-reprogrammed transcriptome-lipidome network, by which impulses platinum insensitivity and disease outcome. The drug specific lipidome for liposome manufacture might be an efficienct pharmaceutics strategy for chemoagents.\n\nSignificanceLDLR reprograms cellular lipidome and transcriptome profiles to determines chemotherapy therapeutic efficacy. The LDLR-reduced LPC abundance disturbs phospholipids homeostasis of Lands cycle in LD, by which attenuates intracellular platinum transportation for DNA-adduct formation. Targeting LDLR-LD-lipidome with LPC-liposome-platinum could boost therapeutic efficacy for insensitivity.

cancer biology↗

bioRxiv2018

VIKNGS: A C++ VARIANT INTEGRATION KIT FOR NEXTGENERATION SEQUENCING ASSOCIATION ANALYSIS

MotivationIntegration of next generation sequencing data (NGS) across different research studies can improve the power of genetic association testing by increasing sample size and can obviate the need for sequencing controls. Unfortunately, if differential genotype uncertainty across studies is not accounted for, combining data sets can also produce spurious association results. The robust variance score statistic (RVS) for genetic association of rare and common variants has been shown to effectively adjust for bias caused by the differences in read depth in case-control genetic association studies when the two groups were sequenced using different experimental designs. To enable consortium research, the aggregation of several data sets for genetic association analysis of quantitative and binary traits with covariate adjustment is required, and we developed the Variant Integration Kit for NGS (VikNGS) that expands the functionality of RVS (vRVS) for this purpose.\n\nResultsVikNGS is a fast and computationally efficient cross-platform software package that provides an implementation for vRVS, as well as conventional rare and common variant genotype-based association analysis approaches. The package includes a graphical user interface that contains power simulation functionality and data visualization tools.\n\nAvailability and ImplementationThe VikNGS package can be downloaded at http://www.tcag.ca/tools/index.html\n\nDocumentation can be found at https://VikNGSdocs.readthedocs.io/en/latest/\n\nContactlisa.strug@sickkids.ca\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

genetics↗

bioRxiv2018

The Salivary Microbiome: Analysis of by Pyrosequencing and the Relationship with Helicobacter pylori Infection

AbstractsO_ST_ABSBackgroundsC_ST_ABSThere have been reports of Helicobacter pylori (H. pylori) in the oral cavity and it has been suggested that the oral cavity may be a reservoir for H. pylori reflux from the stomach.\n\nObjectivesHigh-throughput pyrosequencing was used to assess the structure and composition of oral microbiota communities in individuals with or without confirmed H. pylori infection.\n\nMethodsSaliva samples were obtained from 34 H. pylori infected and 24 H. pylori uninfected subjects. Bacterial genomic DNA was extracted and examined by pyrosequencing by amplification of the 16S rDNA V3-V4 hypervariable regions followed by bioinformatics analysis. Saliva sampling was repeated from 22 of the 34 H. pylori infected subjects 2 months after H. pylori eradication.\n\nResultsHigh-quality sequences (2,812,659) clustered into 95,812 operational taxonomic units (OTUs; 97% identity), representing 440 independent species belonging to 138 genera, 68 families, 36 orders, 21 classes, and 11 phyla. Species richness (alpha diversity) of H. pylori infected subjects was similar to that of uninfected subjects. Eradication treatment decreased saliva bacterial diversity. Beta diversity analysis showed that the salivary microbial community structure differed between H. pylori infected and uninfected subjects both before and after H. pylori eradication.\n\nConclusionsSalivary microbiota diversity was similar in H. pylori infected and uninfected individuals. Antibiotic therapy was associated with a decline in salivary bacterial diversity. Both H. pylori infection and its eradication caused the oral microbiota alterations in community and structure. The present of H. pylori in oral cavity was not related with its infection status in stomach.\n\nTrial registrationClinicalTrials.gov, Identifier: NCT03730766\n\nImportanceThe oral cavity plays a vital role in Helicobacter pylori transmission among human. High-throughput pyrosequencing of the 16S rDNA V3-V4 hypervariable regions was used to assess the structure and composition of oral microbiota communities in individuals with or without confirmed Helicobacter pylori infection. We show that both Helicobacter pylori infection and eradication cause microbiota alterations in the oral microbiota. Prior studies report detection of Helicobacter pylori in the oral cavity by polymerase chain reaction. We show that the presence of Helicobacter pylori in the oral cavity is unrelated with its infection status in the stomach.

microbiology↗

bioRxiv2018

The Genomic Landscape of Centromeres in Various Cancers

Centromere genomics remain poorly characterized in cancer, due to technologic limitations in sequencing and bioinformatics methodologies that make high-resolution delineation of centromeric loci difficult to achieve. We here leverage a highly specific and targeted rapid PCR methodology to quantitatively assess the genomic landscape of centromeres in cancer cell lines and primary tissue. PCR-based profiling of centromeres revealed widespread heterogeneity of centromeric and pericentromeric sequences in cancer cells and tissue as compared to healthy counterparts. Quantitative reductions in select centromeric core and pericentromeric markers were observed in neoplastic samples as compared to healthy counterparts. Subsequent phylogenetic analysis of a pericentromeric endogenous retrovirus amplified by PCR revealed possible gene conversion events occurring at numerous pericentromeric loci in the setting of malignancy. Our findings collectively represent the first look into centromere genetics in the setting of malignancy, providing valuable insight into the evolution and reshuffling of centromeric sequences in cancer development and progression.

genomics↗

bioRxiv2019

Bacillus clarus sp. nov. is a new Bacillus cereus group species isolated from soil

Bacillus cereus group or B. cereus sensu lato (s.l.), is comprised of Gram-positive spore-forming, rod-like bacteria that are widespread in natural environments. Although the species in this group are known to be highly related in terms of phenotypic characteristics, they display different levels of pathogenicity. Biochemical assays are therefore considered to be insufficient for accurate taxonomic classification of B. cereus group species. To facilitate accurate taxonomic classification and associated prediction of pathogenic potential, we have conducted comparative genomic analyses of publicly available genome assemblies of B. cereus group isolates. Through that, we found that an isolate previously known as B. mycoides ATCC 21929 was sufficiently distant from valid and effective type strains to be considered a putative new species. We have conducted biochemical and bioinformatic characterization of strain ATCC 21929 that had been isolated from soil in Papua New Guinea. Strain ATCC 21929 most closely resembles B. paramycoides NH24A2T, producing ANIb and DDH values of 86.70% and 34.1%, respectively. Phenotypically, isolate ATCC 21929 does not possess cytochrome c oxidase activity, and is able to grow at a range of temperatures 15{degrees}C - 43{degrees}C and at a range of pH 6 - 9. With regards to fatty acid composition, this isolate has iso-C17:0 in highest abundance. We propose the strain ATCC 21929T (=PS00077AT = PS00077BT = PSU-0922T = BHPT) as a new species named Bacillus clarus sp. nov. to facilitate accurate taxonomic classification of B. cereus group isolates.

microbiology↗

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↗