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September 22, 2019  |  

Fine mapping and candidate gene identification of the genic male-sterile gene ms3 in cabbage 51S.

The ms3 gene responsible for a male-sterile phenotype in cabbage was mapped to a 187.4-kb genomic fragment. The gene BoTPD1, a homolog of Arabidopsis TPD1, was identified as a strong candidate gene. Cabbage 51S is a spontaneous male-sterile mutant. Phenotypic investigation revealed defects in anther cell differentiation, with failure to form the tapetum layer and complete abortion of microsporocytes before the tetrad stage. Genetic analysis indicated that this male sterility was controlled by a single recessive gene, ms3. Using an F2 population, we mapped ms3 to a 187.4-kb interval. BoTPD1 was identified as a candidate from this interval. Sequence analysis revealed an intronic 182-bp insertion in 51S that interrupted the conserved motif at the 5′ splicing site of the third intron, possibly resulting in a truncated transcript. Analyses of BoTPD1 homologous proteins revealed evolutionarily conserved roles in anther cell fate determination during reproductive development. RT-PCR showed that BoTPD1 was expressed in various tissues, excluding the root, and high expression levels were detected in anthers and buds. A BoTPD1-specific marker based on the 182-bp insertion cosegregated with male sterility and can be used for marker-assisted selection.


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.


September 22, 2019  |  

Using PacBio long-read high-throughput microbial gene amplicon sequencing to evaluate infant formula safety.

Infant formula (IF) requires a strict microbiological standard because of the high vulnerability of infants to foodborne diseases. The current study used the PacBio single molecule real-time (SMRT) sequencing platform to generate full-length 16S rRNA-based bacterial microbiota profiles of thirty Chinese domestic and imported IF samples. A total of 600 species were identified, dominated by Streptococcus thermophilus, Lactococcus lactis and Lactococcus piscium. Distinctive bacterial profiles were observed between the two sample groups, as confirmed with both principal coordinate analysis and multivariate analysis of variance. Moreover, the product whey protein nitrogen index (WPNI), representing the degree of preheating, negatively correlated with the relative abundances of the Bacillus genus. Our study has demonstrated the application of the PacBio SMRT sequencing platform in assessing the bacterial contamination of IF products, which is of interest to the dairy industry for effective monitoring of microbial quality and safety during production.


September 22, 2019  |  

Genome-wide identification and analysis of the ALTERNATIVE OXIDASE gene family in diploid and hexaploid wheat.

A comprehensive understanding of wheat responses to environmental stress will contribute to the long-term goal of feeding the planet. ALERNATIVE OXIDASE (AOX) genes encode proteins involved in a bypass of the electron transport chain and are also known to be involved in stress tolerance in multiple species. Here, we report the identification and characterization of the AOX gene family in diploid and hexaploid wheat. Four genes each were found in the diploid ancestors Triticum urartu, and Aegilops tauschii, and three in Aegilops speltoides. In hexaploid wheat (Triticum aestivum), 20 genes were identified, some with multiple splice variants, corresponding to a total of 24 proteins for those with observed transcription and translation. These proteins were classified as AOX1a, AOX1c, AOX1e or AOX1d via phylogenetic analysis. Proteins lacking most or all signature AOX motifs were assigned to putative regulatory roles. Analysis of protein-targeting sequences suggests mixed localization to the mitochondria and other organelles. In comparison to the most studied AOX from Trypanosoma brucei, there were amino acid substitutions at critical functional domains indicating possible role divergence in wheat or grasses in general. In hexaploid wheat, AOX genes were expressed at specific developmental stages as well as in response to both biotic and abiotic stresses such as fungal pathogens, heat and drought. These AOX expression patterns suggest a highly regulated and diverse transcription and expression system. The insights gained provide a framework for the continued and expanded study of AOX genes in wheat for stress tolerance through breeding new varieties, as well as resistance to AOX-targeted herbicides, all of which can ultimately be used synergistically to improve crop yield.


September 22, 2019  |  

Long-term microbiota and virome in a Zürich patient after fecal transplantation against Clostridium difficile infection.

Fecal microbiota transplantation (FMT) is an emerging therapeutic option for Clostridium difficile infections that are refractory to conventional treatment. FMT introduces fecal microbes into the patient’s intestine that prevent the recurrence of C. difficile, leading to rapid expansion of bacteria characteristic of healthy microbiota. However, the long-term effects of FMT remain largely unknown. The C. difficile patient described in this paper revealed protracted microbiota adaptation processes from 6 to 42 months post-FMT. Ultimately, bacterial communities were donor similar, suggesting sustainable stool engraftment. Since little is known about the consequences of transmitted viruses during C. difficile infection, we also interrogated virome changes. Our approach allowed identification of about 10 phage types per sample that represented larger viral communities, and phages were found to be equally abundant in the cured patient and donor. The healthy microbiota appears to be characterized by low phage abundance. Although viruses were likely transferred, the patient established a virome distinct from the donor. Surprisingly, the patient had sequences of algal giant viruses (chloroviruses) that have not previously been reported for the human gut. Chloroviruses have not been associated with intestinal disease, but their presence in the oropharynx may influence cognitive abilities. The findings suggest that the virome is an important indicator of health or disease. A better understanding of the role of viruses in the gut ecosystem may uncover novel microbiota-modulating therapeutic strategies.© 2016 New York Academy of Sciences.


September 22, 2019  |  

Isoform sequencing provides a more comprehensive view of the Panax ginseng transcriptome.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng, we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana. Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng. In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.


September 22, 2019  |  

A workflow for studying specialized metabolism in nonmodel eukaryotic organisms

Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. © 2016 Elsevier Inc. All rights reserved.


September 22, 2019  |  

Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides.

Libraries of tens of thousands of transposon mutants generated from each of four human gut Bacteroides strains, two representing the same species, were introduced simultaneously into gnotobiotic mice together with 11 other wild-type strains to generate a 15-member artificial human gut microbiota. Mice received one of two distinct diets monotonously, or both in different ordered sequences. Quantifying the abundance of mutants in different diet contexts allowed gene-level characterization of fitness determinants, niche, stability, and resilience and yielded a prebiotic (arabinoxylan) that allowed targeted manipulation of the community. The approach described is generalizable and should be useful for defining mechanisms critical for sustaining and/or approaches for deliberately reconfiguring the highly adaptive and durable relationship between the human gut microbiota and host in ways that promote wellness. Copyright © 2015, American Association for the Advancement of Science.


September 22, 2019  |  

Single-Molecule Long-Read Sequencing of Zanthoxylum bungeanum Maxim. Transcriptome: Identification of Aroma-Related Genes

Zanthoxylum bungeanum Maxim. is an economically important tree species that is resistant to drought and infertility, and has potential medicinal and edible value. However, comprehensive genomic data are not yet available for this species, limiting its potential utility for medicinal use, breeding programs, and cultivation. Transcriptome sequencing provides an effective approach to remedying this shortcoming. Herein, single-molecule long-read sequencing and next-generation sequencingapproacheswereusedinparalleltoobtaintranscriptisoformstructureandgenefunctional informationinZ.bungeanum. Intotal, 282,101readsofinserts(ROIs)wereidentified, including134,074 full-length non-chimeric reads, among which 65,711 open reading frames (ORFs), 50,135 simple sequence repeats (SSRs), and 1492 long non-coding RNAs (lncRNAs) were detected. Functional annotation revealed metabolic pathways related to aroma components and color characteristics in Z. bungeanum. Unexpectedly, 30 transcripts were annotated as genes involved in regulating the pathogenesis of breast and colorectal cancers. This work provides a comprehensive transcriptome resource for Z. bungeanum, and lays a foundation for the further investigation and utilization of Zanthoxylum resources.


September 22, 2019  |  

Full-length transcriptome survey and expression analysis of Cassia obtusifolia to discover putative genes related to aurantio-obtusin biosynthesis, seed formation and development, and stress response.

The seed is the pharmaceutical and breeding organ of Cassia obtusifolia, a well-known medical herb containing aurantio-obtusin (a kind of anthraquinone), food, and landscape. In order to understand the molecular mechanism of the biosynthesis of aurantio-obtusin, seed formation and development, and stress response of C. obtusifolia, it is necessary to understand the genomics information. Although previous seed transcriptome of C. obtusifolia has been carried out by short-read next-generation sequencing (NGS) technology, the vast majority of the resulting unigenes did not represent full-length cDNA sequences and supply enough gene expression profile information of the various organs or tissues. In this study, fifteen cDNA libraries, which were constructed from the seed, root, stem, leaf, and flower (three repetitions with each organ) of C. obtusifolia, were sequenced using hybrid approach combining single-molecule real-time (SMRT) and NGS platform. More than 4,315,774 long reads with 9.66 Gb sequencing data and 361,427,021 short reads with 108.13 Gb sequencing data were generated by SMRT and NGS platform, respectively. 67,222 consensus isoforms were clustered from the reads and 81.73% (61,016) of which were longer than 1000 bp. Furthermore, the 67,222 consensus isoforms represented 58,106 nonredundant transcripts, 98.25% (57,092) of which were annotated and 25,573 of which were assigned to specific metabolic pathways by KEGG. CoDXS and CoDXR genes were directly used for functional characterization to validate the accuracy of sequences obtained from transcriptome. A total of 658 seed-specific transcripts indicated their special roles in physiological processes in seed. Analysis of transcripts which were involved in the early stage of anthraquinone biosynthesis suggested that the aurantio-obtusin in C. obtusifolia was mainly generated from isochorismate and Mevalonate/methylerythritol phosphate (MVA/MEP) pathway, and three reactions catalyzed by Menaquinone-specific isochorismate synthase (ICS), 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate (IPPS) might be the limited steps. Several seed-specific CYPs, SAM-dependent methyltransferase, and UDP-glycosyltransferase (UDPG) supplied promising candidate genes in the late stage of anthraquinone biosynthesis. In addition, four seed-specific transcriptional factors including three MYB Transcription Factor (MYB) and one MADS-box Transcription Factor (MADS) transcriptional factors) and alternative splicing might be involved with seed formation and development. Meanwhile, most members of Hsp20 genes showed high expression level in seed and flower; seven of which might have chaperon activities under various abiotic stresses. Finally, the expressional patterns of genes with particular interests showed similar trends in both transcriptome assay and qRT-PCR. In conclusion, this is the first full-length transcriptome sequencing reported in Caesalpiniaceae family, and thus providing a more complete insight into aurantio-obtusin biosynthesis, seed formation and development, and stress response as well in C. obtusifolia.


September 22, 2019  |  

Gut microbiota, nitric oxide, and microglia as prerequisites for neurodegenerative disorders.

Regulating fluctuating endogenous nitric oxide (NO) levels is necessary for proper physiological functions. Aberrant NO pathways are implicated in a number of neurological disorders, including Alzheimer’s disease (AD) and Parkinson’s disease. The mechanism of NO in oxidative and nitrosative stress with pathological consequences involves reactions with reactive oxygen species (e.g., superoxide) to form the highly reactive peroxynitrite, hydrogen peroxide, hypochloride ions and hydroxyl radical. NO levels are typically regulated by endogenous nitric oxide synthases (NOS), and inflammatory iNOS is implicated in the pathogenesis of neurodegenerative diseases, in which elevated NO mediates axonal degeneration and activates cyclooxygenases to provoke neuroinflammation. NO also instigates a down-regulated secretion of brain-derived neurotrophic factor, which is essential for neuronal survival, development and differentiation, synaptogenesis, and learning and memory. The gut-brain axis denotes communication between the enteric nervous system (ENS) of the GI tract and the central nervous system (CNS) of the brain, and the modes of communication include the vagus nerve, passive diffusion and carrier by oxyhemoglobin. Amyloid precursor protein that forms amyloid beta plaques in AD is normally expressed in the ENS by gut bacteria, but when amyloid beta accumulates, it compromises CNS functions. Escherichia coli and Salmonella enterica are among the many bacterial strains that express and secrete amyloid proteins and contribute to AD pathogenesis. Gut microbiota is essential for regulating microglia maturation and activation, and activated microglia secrete significant amounts of iNOS. Pharmacological interventions and lifestyle modifications to rectify aberrant NO signaling in AD include NOS inhibitors, NMDA receptor antagonists, potassium channel modulators, probiotics, diet, and exercise.


September 22, 2019  |  

Long-read, Single Molecule, Real-Time (SMRT) DNA Sequencing for metagenomic applications

In this chapter, we describe applications of single molecule, real-time (SMRT) DNA sequencing toward metagenomic research. The long sequence reads, combined with a lack of bias with respect to DNA sequence context or GC content, facilitate a more comprehensive analysis of the genomic constitution of microbial communities. Full-length 16S RNA gene sequencing at high (>99%) accuracy allows for species-level characterization of community members concomitant with the determination of community structure. The application of SMRT sequencing to whole-community shotgun microbial metagenomics has also been discussed.


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.


September 22, 2019  |  

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed =13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-a, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.


September 22, 2019  |  

Transgenerational attenuation of opioid self-administration as a consequence of adolescent morphine exposure.

The United States is in the midst of an opiate epidemic, with abuse of prescription and illegal opioids increasing steadily over the past decade. While it is clear that there is a genetic component to opioid addiction, there is a significant portion of heritability that cannot be explained by genetics alone. The current study was designed to test the hypothesis that maternal exposure to opioids prior to pregnancy alters abuse liability in subsequent generations. Female adolescent Sprague Dawley rats were administered morphine at increasing doses (5-25 mg/kg, s.c.) or saline for 10 days (P30-39). During adulthood, animals were bred with drug-naïve colony males. Male and female adult offspring (F1 animals) were tested for morphine self-administration acquisition, progressive ratio, extinction, and reinstatement at three doses of morphine (0.25, 0.75, 1.25 mg/kg/infusion). Grand offspring (F2 animals, from the maternal line) were also examined. Additionally, gene expression changes within the nucleus accumbens were examined with RNA deep sequencing (PacBio) and qPCR. There were dose- and sex-dependent effects on all phases of the self-administration paradigm that indicate decreased morphine reinforcement and attenuated relapse-like behavior. Additionally, genes related to synaptic plasticity, as well as myelin basic protein (MBP), were dysregulated. Some, but not all, effects persisted into the subsequent (F2) generation. The results demonstrate that even limited opioid exposure during adolescence can have lasting effects across multiple generations, which has implications for mechanisms of the transmission of drug abuse liability in humans. Copyright © 2016 Elsevier Ltd. All rights reserved.


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