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September 22, 2019

Identification of Burkholderia fungorum in the urine of an individual with spinal cord injury and augmentation cystoplasty using 16S sequencing: copathogen or innocent bystander?

People with neuropathic bladder (NB) secondary to spinal cord injury (SCI) are at risk for multiple genitourinary complications, the most frequent of which is urinary tract infection (UTI). Despite the high frequency with which UTI occurs, our understanding of the role of urinary microbes in health and disease is limited. In this paper, we present the first prospective case study integrating symptom reporting, urinalysis, urine cultivation, and 16S ribosomal ribonucleic acid (rRNA) sequencing of the urine microbiome.A 55-year-old male with NB secondary to SCI contributed 12 urine samples over an 8-month period during asymptomatic, symptomatic, and postantibiotic periods. All bacteria identified on culture were present on 16S rRNA sequencing, however, 16S rRNA sequencing revealed the presence of bacteria not isolated on culture. In particular, Burkholderia fungorum was present in three samples during both asymptomatic and symptomatic periods. White blood cells of =5-10/high power field and leukocyte esterase =2 on urinalysis was associated with the presence of symptoms.In this patient, there was a predominance of pathogenic bacteria and a lack of putative probiotic bacteria during both symptomatic and asymptomatic states. Urinalysis-defined inflammatory markers were present to a greater extent during symptomatic periods compared to the asymptomatic state, which may underscore a role for urinalysis or other inflammatory markers in differentiating asymptomatic bacteriuria from UTI in patients with NB. The finding of potentially pathogenic bacteria identified by sequencing but not cultivation, suggests a need for greater understanding of the relationships amongst bacterial species in the bacteriuric neuropathic bladder.

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September 22, 2019

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers. Copyright © 2015 Elsevier Ltd. All rights reserved.

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September 22, 2019

Alternative polyadenylation: methods, findings, and impacts.

Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3′ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

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September 22, 2019

The role of MHC-E in T cell immunity is conserved among humans, rhesus macaques, and cynomolgus macaques.

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology. Copyright © 2017 by The American Association of Immunologists, Inc.

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September 22, 2019

Rodent papillomaviruses.

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.

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September 22, 2019

Reduction in fecal microbiota diversity and short-chain fatty acid producers in Methicillin-resistant Staphylococcus aureus infected individuals as revealed by PacBio single molecule, real-time sequencing technology.

Methicillin-resistant Staphylococcus aureus (MRSA) may cause potentially lethal infections. Increasing evidence suggests that the gut microbiota is associated with human health. Yet, whether patients with MRSA infections carry specific signatures in their fecal microbiota composition has not been determined. Thus, this study aimed to compare the fecal microbiota profile of MRSA-positive patients (n=15) with individuals without MRSA infection (n=15) by using the PacBio single molecule, real-time (SMRT) DNA sequencing system and real-time quantitative polymerase chain reaction (qPCR). Mann-Whitney tests and unweighted UniFrac principal coordinate analysis (PCoA) showed that the profile of fecal microbiota was apparently different between the two populations. Both the community richness and diversity were reduced in the MRSA-positive group (p<0.050). The genera Acinetobacter and Enterococcus were highly enriched in the MRSA-positive group, whereas less short-chain fatty acid (SCFA)-producing bacteria, including Butyricimonas, Faecalibacterium, Roseburia, Ruminococcus, Megamonas and Phascolarctobacterium, were detected in the MRSA-positive group. At species level, the species Acinetobacter baumannii and Bacteroides thetaiotaomicron were prevalent in the MRSA-positive group, whereas opposite trends were observed in 17 other species, such as Faecalibacterium prausnitzii, Lactobacillus rogosae, Megamonas rupellensis and Phascolarctobacterium faecium. Positive correlations were observed between Acinetobacter baumannii and erythrocyte sedimentation rate (ESR) (R=0.554, p=0.001), as well as hypersensitive C reactive protein (hsCRP) (R=0.406, p=0.026). Faecalibacterium prausnitzii was negatively associated with ESR (R=-0.545, p=0.002), hsCRP (R=-0.401, p=0.028) and total bile acids (TBA) (R=-0.364, p=0.048). In conclusion, the fecal microbiota structure was different between MRSA-positive and -negative patients. The increase in potential pathogens with the reduction of beneficial populations, such as SCFA-producing bacteria, in MRSA-positive patients may affect prognosis.

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September 22, 2019

Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats.

HIV infection has been reported to alter cellular gene activity, but published studies have commonly assayed transformed cell lines and lab-adapted HIV strains, yielding inconsistent results. Here we carried out a deep RNA-Seq analysis of primary human T cells infected with the low passage HIV isolate HIV89.6.Seventeen percent of cellular genes showed altered activity 48 h after infection. In a meta-analysis including four other studies, our data differed from studies of HIV infection in cell lines but showed more parallels with infections of primary cells. We found a global trend toward retention of introns after infection, suggestive of a novel cellular response to infection. HIV89.6 infection was also associated with activation of several human endogenous retroviruses (HERVs) and retrotransposons, of interest as possible novel antigens that could serve as vaccine targets. The most highly activated group of HERVs was a subset of the ERV-9. Analysis showed that activation was associated with a particular variant of ERV-9 long terminal repeats that contains an indel near the U3-R border. These data also allowed quantification of >70 splice forms of the HIV89.6 RNA and specified the main types of chimeric HIV89.6-host RNAs. Comparison to over 100,000 integration site sequences from the same infected cell populations allowed quantification of authentic versus artifactual chimeric reads, showing that 5′ read-in, splicing out of HIV89.6 from the D4 donor and 3′ read-through were the most common HIV89.6-host cell chimeric RNA forms.Analysis of RNA abundance after infection of primary T cells with the low passage HIV89.6 isolate disclosed multiple novel features of HIV-host interactions, notably intron retention and induction of transcription of retrotransposons and endogenous retroviruses.

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September 22, 2019

A comprehensive analysis of alternative splicing in paleopolyploid maize.

Identifying and characterizing alternative splicing (AS) enables our understanding of the biological role of transcript isoform diversity. This study describes the use of publicly available RNA-Seq data to identify and characterize the global diversity of AS isoforms in maize using the inbred lines B73 and Mo17, and a related species, sorghum. Identification and characterization of AS within maize tissues revealed that genes expressed in seed exhibit the largest differential AS relative to other tissues examined. Additionally, differences in AS between the two genotypes B73 and Mo17 are greatest within genes expressed in seed. We demonstrate that changes in the level of alternatively spliced transcripts (intron retention and exon skipping) do not solely reflect differences in total transcript abundance, and we present evidence that intron retention may act to fine-tune gene expression across seed development stages. Furthermore, we have identified temperature sensitive AS in maize and demonstrate that drought-induced changes in AS involve distinct sets of genes in reproductive and vegetative tissues. Examining our identified AS isoforms within B73 × Mo17 recombinant inbred lines (RILs) identified splicing QTL (sQTL). The 43.3% of cis-sQTL regulated junctions are actually identified as alternatively spliced junctions in our analysis, while 10 Mb windows on each side of 48.2% of trans-sQTLs overlap with splicing related genes. Using sorghum as an out-group enabled direct examination of loss or conservation of AS between homeologous genes representing the two subgenomes of maize. We identify several instances where AS isoforms that are conserved between one maize homeolog and its sorghum ortholog are absent from the second maize homeolog, suggesting that these AS isoforms may have been lost after the maize whole genome duplication event. This comprehensive analysis provides new insights into the complexity of AS in maize.

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September 22, 2019

Extensive alternative splicing of KIR transcripts.

The killer-cell Ig-like receptors (KIR) form a multigene entity involved in modulating immune responses through interactions with MHC class I molecules. The complexity of the KIR cluster is reflected by, for instance, abundant levels of allelic polymorphism, gene copy number variation, and stochastic expression profiles. The current transcriptome study involving human and macaque families demonstrates that KIR family members are also subjected to differential levels of alternative splicing, and this seems to be gene dependent. Alternative splicing may result in the partial or complete skipping of exons, or the partial inclusion of introns, as documented at the transcription level. This post-transcriptional process can generate multiple isoforms from a single KIR gene, which diversifies the characteristics of the encoded proteins. For example, alternative splicing could modify ligand interactions, cellular localization, signaling properties, and the number of extracellular domains of the receptor. In humans, we observed abundant splicing for KIR2DL4, and to a lesser extent in the lineage III KIR genes. All experimentally documented splice events are substantiated by in silico splicing strength predictions. To a similar extent, alternative splicing is observed in rhesus macaques, a species that shares a close evolutionary relationship with humans. Splicing profiles of Mamu-KIR1D and Mamu-KIR2DL04 displayed a great diversity, whereas Mamu-KIR3DL20 (lineage V) is consistently spliced to generate a homolog of human KIR2DL5 (lineage I). The latter case represents an example of convergent evolution. Although just a single KIR splice event is shared between humans and macaques, the splicing mechanisms are similar, and the predicted consequences are comparable. In conclusion, alternative splicing adds an additional layer of complexity to the KIR gene system in primates, and results in a wide structural and functional variety of KIR receptors and its isoforms, which may play a role in health and disease.

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September 22, 2019

CRISPR/Cas9 deletions in a conserved exon of Distal-less generates gains and losses in a recently acquired morphological novelty in flies.

Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the upstream coding exon (Exon 2) produce losses or gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon skipping. Such contradictory phenotypes are also observed in butterflies, suggesting that mutations in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Cataloguing over-expressed genes in Epstein Barr Virus immortalized lymphoblastoid cell lines through consensus analysis of PacBio transcriptomes corroborates hypomethylation of chromosome 1

The ability of Epstein Barr Virus (EBV) to transform resting cell B-cells into immortalized lymphoblastoid cell lines (LCL) provides a continuous source of peripheral blood lymphocytes that are used to model conditions in which these lymphocytes play a key role. Here, the PacBio generated transcriptome of three LCLs from a parent-daughter trio (SRAid:SRP036136) provided by a previous study [1] were analyzed using a kmer-based version of YeATS (KEATS). The set of over-expressed genes in these cell lines were determined based on a comparison with the PacBio transcriptome of twenty tissues pro- vided by another study (hOPTRS) [2]. MIR155 long non-coding RNA (MIR155HG), Fc fragment of IgE receptor II (FCER2), T-cell leukemia/lymphoma 1A (TCL1A), and germinal center associated signaling and motility (GCSAM) were genes having the highest expression counts in the three LCLs with no expression in hOPTRS. Other over-expressed genes, having low expression in hOPTRS, were membrane spanning 4-domains A1 (MS4A1) and ribosomal protein S2 pseudogene 55 (RPS2P55). While some of these genes are known to be over-expressed in LCLs, this study provides a comprehensive cataloguing of such genes. A recent work involving a patient with EBV-positive large B-cell lymphoma was “unusually lacking various B-cell markers”, but over-expressing CD30 [3] – a gene ranked 79 among uniquely expressed genes here. Hypomethylation of chromosome 1 observed in EBV immortalized LCLs [4, 5] is also corroborated here by mapping the genes to chromosomes. Extending previous work identifying un-annotated genes [6], 80 genes were identified which are expressed in the three LCLs, not in hOPTRS, and missing in the GENCODE, RefSeq and RefSeqGene databases. KEATS introduces a method of determining expression counts based on a partitioning of the known annotated genes, has runtimes of a few hours on a personal workstation and provides detailed reports enabling proper debugging.

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September 22, 2019

Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations.

We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score = 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (~2%; 4/216) and TRAF7 (~2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

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September 22, 2019

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

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September 22, 2019

Next-generation approaches to advancing eco-immunogenomic research in critically endangered primates.

High-throughput sequencing platforms are generating massive amounts of genomic data from nonmodel species, and these data sets are valuable resources that can be mined to advance a number of research areas. An example is the growing amount of transcriptome data that allow for examination of gene expression in nonmodel species. Here, we show how publicly available transcriptome data from nonmodel primates can be used to design novel research focused on immunogenomics. We mined transcriptome data from the world’s most endangered group of primates, the lemurs of Madagascar, for sequences corresponding to immunoglobulins. Our results confirmed homology between strepsirrhine and haplorrhine primate immunoglobulins and allowed for high-throughput sequencing of expressed antibodies (Ig-seq) in Coquerel’s sifaka (Propithecus coquereli). Using both Pacific Biosciences RS and Ion Torrent PGM sequencing, we performed Ig-seq on two individuals of Coquerel’s sifaka. We generated over 150 000 sequences of expressed antibodies, allowing for molecular characterization of the antigen-binding region. Our analyses suggest that similar VDJ expression patterns exist across all primates, with sequences closely related to the human VH 3 immunoglobulin family being heavily represented in sifaka antibodies. Moreover, the antigen-binding region of sifaka antibodies exhibited similar amino acid variation with respect to haplorrhine primates. Our study represents the first attempt to characterize sequence diversity of the expressed antibody repertoire in a species of lemur. We anticipate that methods similar to ours will provide the framework for investigating the adaptive immune response in wild populations of other nonmodel organisms and can be used to advance the burgeoning field of eco-immunology. © 2014 John Wiley & Sons Ltd.

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September 22, 2019

Defining cell identity with single cell omics.

Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic, and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation-most notably in the emergence and evolution of tumors. Recent technical advances in single-cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a revolution in the study of multicellular systems. In this review, we discuss the technologies available to resolve the genomes, epigenomes, transcriptomes, proteomes, and metabolomes of single cells from a wide variety of living systems.© 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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