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September 22, 2019

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

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September 22, 2019

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire.

Vertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. In particular, single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach.We generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5-6 and 21-25 amino acids. The average number of cysteine residues in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals.We utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation resulting in base insertions and deletions in CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues in bovine CDR3s provide further evidence that disulfide bond formation is likely of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.

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September 22, 2019

Analyses of intestinal microbiota: culture versus sequencing.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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September 22, 2019

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5′-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-?E4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy. © 2013 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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September 22, 2019

Resistance to ceftazidime-avibactam in Klebsiella pneumoniae due to porin mutations and the increased expression of KPC-3.

We reported the first clinical case of a ceftazidime-avibactam resistant KPC-3-producing Klebsiella pneumoniae (1), from a patient with no history of ceftazidime-avibactam therapy. We now present data documenting mechanisms of ceftazidime-avibactam resistance in this isolate. Whole-genome sequencing (WGS) was performed on two isolates: KP1245 (ceftazidime-avibactam MIC, 4 µg/ml; from blood on hospital day 1; referred to as isolate 1 in our previous report [1]) and KP1244 (ceftazidime-avibactam MIC, 32 µg/ml; from blood on hospital day 2; referred to as isolate 2 in our previous report [2]), using MiSeq (Illumina, San Diego, CA) and PacBio RSII (Menlo Park, CA) systems (2). The in silico multilocus sequence type (ST) was ST258. Single nucleotide polymorphism (SNP) analysis revealed 17 SNPs between KP1245 and KP1244, indicating that the isolates were related but that significant diversity existed in this patient (2). Nonsynonymous mutations are shown in Table 1; the most striking of these is in the OmpK36 porin gene. KP1244 contained a missense mutation predicted to encode a T333N mutation. Both isolates also harbored a mutation predicted to encode R191L in OmpK36 and had a nonfunctional OmpK35, due to a frameshift mutation that truncated the protein at amino acid 42, common to K. pneumoniae ST258 (3). Association between mutations in ompK36 and elevated ceftazidime-avibactam MICs has been shown previously (4). However, T333N, found in one of the ß-sheet domains of the OmpK36 subunit, has not been described in K. pneumoniae; as such, further validation is required to confirm the role of the OmpK36 mutation in this isolate’s ceftazidime-avibactam resistance phenotype.

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September 22, 2019

The human microbiome and understanding the 16S rRNA gene in translational nursing science.

As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings.The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science.Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

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September 22, 2019

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR.

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

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September 22, 2019

How far can mitochondrial DNA drive the disease?

Mitochondria are one of the dominant drivers for producing cellular energy to meet a large number of biological functions, of which the mitochondrial DNA (mtDNA) is the control center of energetic driving force and the dominant driver of mitochondrial molecular diversification. mtDNA transcription generates the necessary RNAs to regulate the extent and nature of mtRNA post-transcriptional modifications and the activity of nucleus-encoded enzymes. With a special focus on mtDNA, the current volume aims to overview the biology and structures of mtDNA, regulatory roles of mtDNA in lung diseases, or involvement of mtDNA in metabolism. We explore the significance of mtDNA sequencing, methylation, stability, and mutation in the pathogenesis of the diseases. Molecular mechanisms by which mtDNA contribute to the regulation of mitochondrial homeostasis and drug resistance are also discussed. We also point out the importance of mitochondrial ribosome, single cell biology, and gene editing in the understanding of the development of mitochondrial dysfunction in lung disease.

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September 22, 2019

Extensive horizontal gene transfer in cheese-associated bacteria.

Acquisition of genes through horizontal gene transfer (HGT) allows microbes to rapidly gain new capabilities and adapt to new or changing environments. Identifying widespread HGT regions within multispecies microbiomes can pinpoint the molecular mechanisms that play key roles in microbiome assembly. We sought to identify horizontally transferred genes within a model microbiome, the cheese rind. Comparing 31 newly sequenced and 134 previously sequenced bacterial isolates from cheese rinds, we identified over 200 putative horizontally transferred genomic regions containing 4733 protein coding genes. The largest of these regions are enriched for genes involved in siderophore acquisition, and are widely distributed in cheese rinds in both Europe and the US. These results suggest that HGT is prevalent in cheese rind microbiomes, and that identification of genes that are frequently transferred in a particular environment may provide insight into the selective forces shaping microbial communities.

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September 22, 2019

Next generation sequencing data of a defined microbial mock community.

Generating sequence data of a defined community composed of organisms with complete reference genomes is indispensable for the benchmarking of new genome sequence analysis methods, including assembly and binning tools. Moreover the validation of new sequencing library protocols and platforms to assess critical components such as sequencing errors and biases relies on such datasets. We here report the next generation metagenomic sequence data of a defined mock community (Mock Bacteria ARchaea Community; MBARC-26), composed of 23 bacterial and 3 archaeal strains with finished genomes. These strains span 10 phyla and 14 classes, a range of GC contents, genome sizes, repeat content and encompass a diverse abundance profile. Short read Illumina and long-read PacBio SMRT sequences of this mock community are described. These data represent a valuable resource for the scientific community, enabling extensive benchmarking and comparative evaluation of bioinformatics tools without the need to simulate data. As such, these data can aid in improving our current sequence data analysis toolkit and spur interest in the development of new tools.

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September 22, 2019

HIV-1 interacts with human endogenous retrovirus K (HML-2) envelopes derived from human primary lymphocytes.

Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication.Here, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deep-sequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins retain their ability to make a protein. Importantly, one of them, the envelope glycoprotein of HERV-K18, is incorporated into HIV-1 in an HIV matrix-specific fashion. The ramifications of such interactions are discussed, as the possibility of HIV-1 target tissue broadening and immune evasion are considered.

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September 22, 2019

Role of clinicogenomics in infectious disease diagnostics and public health microbiology.

Clinicogenomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The role of clinicogenomics in infectious disease diagnostics and public health microbiology was the topic of discussion during a recent symposium (session 161) presented at the 115th general meeting of the American Society for Microbiology that was held in New Orleans, LA. What follows is a collection of the most salient and promising aspects from each presentation at the symposium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing.

The evolution of mutations in the BCR-ABL1 fusion gene transcript renders CML patients resistant to tyrosine kinase inhibitor (TKI) based therapy. Thus screening for BCR-ABL1 mutations is recommended particularly in patients experiencing poor response to treatment. Herein we describe a novel approach for the detection and surveillance of BCR-ABL1 mutations in CML patients.To detect mutations in the BCR-ABL1 transcript we developed an assay based on the Pacific Biosciences (PacBio) sequencing technology, which allows for single-molecule long-read sequencing of BCR-ABL1 fusion transcript molecules. Samples from six patients with poor response to therapy were analyzed both at diagnosis and follow-up. cDNA was generated from total RNA and a 1,6 kb fragment encompassing the BCR-ABL1 transcript was amplified using long range PCR. To estimate the sensitivity of the assay, a serial dilution experiment was performed.Over 10,000 full-length BCR-ABL1 sequences were obtained for all samples studied. Through the serial dilution analysis, mutations in CML patient samples could be detected down to a level of at least 1%. Notably, the assay was determined to be sufficiently sensitive even in patients harboring a low abundance of BCR-ABL1 levels. The PacBio sequencing successfully identified all mutations seen by standard methods. Importantly, we identified several mutations that escaped detection by the clinical routine analysis. Resistance mutations were found in all but one of the patients. Due to the long reads afforded by PacBio sequencing, compound mutations present in the same molecule were readily distinguished from independent alterations arising in different molecules. Moreover, several transcript isoforms of the BCR-ABL1 transcript were identified in two of the CML patients. Finally, our assay allowed for a quick turn around time allowing samples to be reported upon within 2 days.In summary the PacBio sequencing assay can be applied to detect BCR-ABL1 resistance mutations in both diagnostic and follow-up CML patient samples using a simple protocol applicable to routine diagnosis. The method besides its sensitivity, gives a complete view of the clonal distribution of mutations, which is of importance when making therapy decisions.

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September 22, 2019

Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities.

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.

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September 22, 2019

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These “phasiRNAs” are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.© 2018 Kakrana et al.; Published by Cold Spring Harbor Laboratory Press.

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