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September 22, 2019  |  

Somatic APP gene recombination in Alzheimer’s disease and normal neurons.

The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer’s disease-related gene APP, which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant ‘genomic cDNAs’ (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal ‘retro-insertion’ of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer’s disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer’s disease that were absent from healthy neurons. Neuronal gene recombination may allow ‘recording’ of neural activity for selective ‘playback’ of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain.


September 22, 2019  |  

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.


September 22, 2019  |  

A manganese superoxide dismutase (MnSOD) from red lip mullet, Liza haematocheila: Evaluation of molecular structure, immune response, and antioxidant function.

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O2-) into less toxic hydrogen peroxide (H2O2) and oxygen (O2). Structural analysis of mullet MnSOD (MuMnSOD) was performed using different bioinformatics tools. Pairwise alignment revealed that the protein sequence matched to that derived from Larimichthys crocea with a 95.2% sequence identity. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. Multiple sequence alignment showed that a SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Quantitative real-time PCR showed that the highest MuMnSOD mRNA expression level was in blood cells. The highest expression level of MuMnSOD was observed in response to treatment with both Lactococcus garvieae and lipopolysaccharide (LPS) at 6?h post treatment in the head kidney and blood. Potential ROS-scavenging ability of the purified recombinant protein (rMuMnSOD) was examined by the xanthine oxidase assay (XOD assay). The optimum temperature and pH for XOD activity were found to be 25?°C and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. Activity of rMuMnSOD was inhibited by potassium cyanide (KCN) and N-N’-diethyl-dithiocarbamate (DDC). Moreover, expression of MuMnSOD resulted in considerable growth retardation of both gram-positive and gram-negative bacteria. Results of the current study suggest that MuMnSOD acts as an antioxidant enzyme and participates in the immune response in mullet. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.


September 22, 2019  |  

Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications.

Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date.Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA3, suggesting that post-transcriptional regulation might be also involved in the GA3 response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA3-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition.This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.


September 22, 2019  |  

Emergence, retention and selection: A trilogy of origination for functional de novo proteins from ancestral lncRNAs in primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.


September 22, 2019  |  

High-quality RNA isolation from wheat immature grains

Grain quality is one of the most important targets in wheat breeding. Transcriptome analyses of wheat developing grains and endosperm have been performed using the microarray and RNA sequencing (RNA-seq) approaches (Wan et al. 2008, 2009; Nemeth et al. 2010; Pellny et al. 2012; Dong et al. 2015). For the RNA-seq analysis of the grain transcriptome and precise quantification of each transcript in developing grain and endosperm, the high-quality RNA is essential. For the microarray analysis, =7.3 RIN (RNA integrity number) value for the RNA sample quality is required according to the Agilent microarray protocol. In the previous report for the transcriptome of wheat developing grains, the total RNA samples with =8.0 RIN values were used for the RNA-seq analysis based on the PacBio and Illumina platforms (Dong et al. 2015). Some RNA extraction buffers containing SDS, CTAB, or TRIzol® reagent (Thermo Fisher Scientific, Waltham, Massachusetts) and several commercial kits for RNA isolation have been used to isolate total RNA from wheat grain and endosperm (Kawakami et al. 1992; Wan et al. 2008; Kang et al. 2013). However, total RNA samples from the wheat developing and immature grains are often damaged due to high content of polysaccharides and high stickiness of the solution homogenized with the RNA extraction buffer, and thus extraction of the high-quality RNA with high RIN value is quite difficult. Here, we report a protocol for the wheat grain RNA extraction using Maxwell RSC Plant RNA Kit (Promega, Madison, Wisconsin).


September 22, 2019  |  

RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.

Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure, and hepatocellular carcinoma. Evidence suggests that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with an RNA interference (RNAi)-based therapeutic targeting HBV transcripts, ARC-520, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients who were HBeAg-negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). HBeAg positivity is associated with greater disease risk that may be moderately reduced upon HBeAg loss. The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome but also from transcripts arising from HBV DNA integrated into the host genome, which was the dominant source in HBeAg-negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the small interfering RNAs in ARC-520, explaining the reduced response in HBeAg-negative chimpanzees and, by extension, in HBeAg-negative patients. Our results uncover a heretofore underrecognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immunosurveillance. These results could alter trial design and endpoint expectations of new therapies for chronic HBV. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


September 22, 2019  |  

De novo transcriptome assembly of the Chinese pearl barley, adlay, by full-length isoform and short-read RNA sequencing.

Adlay (Coix lacryma-jobi) is a tropical grass that has long been used in traditional Chinese medicine and is known for its nutritional benefits. Recent studies have shown that vitamin E compounds in adlay protect against chronic diseases such as cancer and heart disease. However, the molecular basis of adlay’s health benefits remains unknown. Here, we generated adlay gene sets by de novo transcriptome assembly using long-read isoform sequencing (Iso-Seq) and short-read RNA-Sequencing (RNA-Seq). The gene sets obtained from Iso-seq and RNA-seq contained 31,177 genes and 57,901 genes, respectively. We confirmed the validity of the assembled gene sets by experimentally analyzing the levels of prolamin and vitamin E biosynthesis-associated proteins in adlay plant tissues and seeds. We compared the screened adlay genes with known gene families from closely related plant species, such as rice, sorghum and maize. We also identified tissue-specific genes from the adlay leaf, root, and young and mature seed, and experimentally validated the differential expression of 12 randomly-selected genes. Our study of the adlay transcriptome will provide a valuable resource for genetic studies that can enhance adlay breeding programs in the future.


September 22, 2019  |  

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.


September 22, 2019  |  

Bacterial microbiota composition of fermented fruit and vegetable juices (jiaosu) analyzed by single-molecule, real-time (SMRT) sequencing

Commercially manufactured ‘jiaosu’ (fermented fruit and vegetable juices) have gained popularity in Asia recently. Like other fermented products, they have a high microbial diversity and richness. However, no published study has yet described their microbiota composition. Thus, this work aimed to obtain the full-length 16S rRNA profiles of jiaosu using the PacBio single-molecule, real-time sequencing technology. We described the bacterial microbiota of three jiaosu products purchased from Taiwan and Japan. Bacterial sequences from all three samples distributed across seven different phyla, mainly Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Forty-three genera were identified (e.g. Ochrobactrum, Lactobacillus, Mycobacterium, and Acinetobacter). Fifty- five species were identified (e.g. Ochrobactrum lupini, Mycobacterium abscessus, Acinetobacter john- sonii, Lactobacillus paracasei, Lactobacillus delbrueckii, and Petrobacter succinatimandens). No patho- gen sequences were identified within the entire dataset. Moreover, only a low proportion of sequences represented common skin microflora and the food hygiene indicator Escherichia/ Shigella, suggesting overall acceptable sanitary conditions during the manufacturing process.


September 22, 2019  |  

Isoform evolution in primates through independent combination of alternative RNA processing events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875?bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


September 22, 2019  |  

A human-specific switch of alternatively spliced AFMID isoforms contributes to TP53 mutations and tumor recurrence in hepatocellular carcinoma.

Pre-mRNA splicing can contribute to the switch of cell identity that occurs in carcinogenesis. Here, we analyze a large collection of RNA-seq data sets and report that splicing changes in hepatocyte-specific enzymes, such as AFMID and KHK, are associated with HCC patients’ survival and relapse. The switch of AFMID isoforms is an early event in HCC development and is associated with driver mutations in TP53 and ARID1A The switch of AFMID isoforms is human-specific and not detectable in other species, including primates. Finally, we show that overexpression of the full-length AFMID isoform leads to a higher NAD+ level, lower DNA-damage response, and slower cell growth in HepG2 cells. The integrative analysis uncovered a mechanistic link between splicing switches, de novo NAD+ biosynthesis, driver mutations, and HCC recurrence.© 2018 Lin et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Extensively drug-resistant Escherichia coli sequence type 1642 carrying an IncX3 plasmid containing the blaKPC-2 gene associated with transposon Tn4401a.

Extensively drug-resistant (XDR) Enterobacteriaceae carrying the bla(KPC) gene have emerged as a major global therapeutic concern. The purpose of this study was to analyze the complete sequences of plasmids from KPC-2 carbapenemase-producing XDR Escherichia coli sequence type (ST) 1642 isolates.We performed antimicrobial susceptibility testing, PCR, multilocus sequence typing (MLST), and whole-genome sequencing to characterize the plasmid-mediated KPC-2-producing E. coli clinical isolates.The isolates were resistant to most available antibiotics, including meropenem, ampicillin, ceftriaxone, gentamicin, and ciprofloxacin, but susceptible to tigecycline and colistin. The isolates were identified as the rare ST1642 by MLST. The isolates carried four plasmids: the first 69-kb conjugative IncX3 plasmid harbors bla(KPC-2) within a truncated Tn4401a transposon and bla(SHV-11) with duplicated conjugative elements. The second 142-kb plasmid with a multireplicon consisting of IncQ, IncFIA, and IncIB carries bla(TEM-1b) and two class 1 integrons. This plasmid also harbors a wide variety of additional antimicrobial resistance genes including aadA5, dfrA17, mph(A), sul1, tet(B), aac(3′)-IId, strA, strB, and sul2.The complete sequence analysis of plasmids from an XDR E. coli strain related to persistent infection showed the coexistence of a bla(KPC-2)-carrying IncX3-type plasmid and a class 1 integron-harboring multireplicon, suggesting its potential to cause outbreaks. Of additional clinical significance, the rare ST1642, identified in a cat, could constitute the source of human infection.


September 22, 2019  |  

Genomic insights into the non-histamine production and proteolytic and lipolytic activities of Tetragenococcus halophilus KUD23.

Tetragenococcus halophilus KUD23, a non-histamine producer, was isolated from a traditional Korean high-salt fermented soybean paste, doenjang. The strain was safe in terms of antibiotic susceptibility, hemolytic activity and biofilm formation. It could grow on De Man-Rogosa-Sharpe agar containing 21% (w/v) NaCl, exhibited acid production at 15% NaCl, and had strain-specific proteolytic and lipolytic activities under salt stress. Complete genome analysis of T. halophilus KUD23 and comparative genomic analysis shed light on the genetic background behind these phenotypic characteristics, including non-production of histamine and proteolytic and lipolytic activities.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.


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