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September 22, 2019

Characterization of four C1q/TNF-related proteins (CTRPs) from red-lip mullet (Liza haematocheila) and their transcriptional modulation in response to bacterial and pathogen-associated molecular pattern stimuli.

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses. Copyright © 2018. Published by Elsevier Ltd.

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September 22, 2019

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform’s utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.

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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

Differential TGFß pathway targeting by miR-122 in humans and mice affects liver cancer metastasis.

Downregulation of a predominantly hepatocyte-specific miR-122 is associated with human liver cancer metastasis, whereas miR-122-deficient mice display normal liver function. Here we show a functional conservation of miR-122 in the TGFß pathway: miR-122 target site is present in the mouse but not human TGFßR1, whereas a noncanonical target site is present in the TGFß1 5’UTR in humans and other primates. Experimental switch of the miR-122 target between the receptor TGFßR1 and the ligand TGFß1 changes the metastatic properties of mouse and human liver cancer cells. High expression of TGFß1 in human primary liver tumours is associated with poor survival. We identify over 50 other miRNAs orthogonally targeting ligand/receptor pairs in humans and mice, suggesting that these are evolutionarily common events. These results reveal an evolutionary mechanism for miRNA-mediated gene regulation underlying species-specific physiological or pathological phenotype and provide a potentially valuable strategy for treating liver-associated diseases.

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September 22, 2019

Identification and characterization of a carboxypeptidase N1 from red lip mullet (Liza haematocheila); revealing its immune relevance.

Complement system orchestrates the innate and adaptive immunity via the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51?kDa. In silico analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the Maylandia zebra CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and Lactococcus garviae in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24?h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon L. garviae challenge were observed at 24?h post infection in head kidney tissue and 48?h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Limited effects of variable-retention harvesting on fungal communities decomposing fine roots in coastal temperate rainforests.

Fine root litter is the principal source of carbon stored in forest soils and a dominant source of carbon for fungal decomposers. Differences in decomposer capacity between fungal species may be important determinants of fine-root decomposition rates. Variable-retention harvesting (VRH) provides refuge for ectomycorrhizal fungi, but its influence on fine-root decomposers is unknown, as are the effects of functional shifts in these fungal communities on carbon cycling. We compared fungal communities decomposing fine roots (in litter bags) under VRH, clear-cut, and uncut stands at two sites (6 and 13 years postharvest) and two decay stages (43 days and 1 year after burial) in Douglas fir forests in coastal British Columbia, Canada. Fungal species and guilds were identified from decomposed fine roots using high-throughput sequencing. Variable retention had short-term effects on ß-diversity; harvest treatment modified the fungal community composition at the 6-year-postharvest site, but not at the 13-year-postharvest site. Ericoid and ectomycorrhizal guilds were not more abundant under VRH, but stand age significantly structured species composition. Guild composition varied by decay stage, with ruderal species later replaced by saprotrophs and ectomycorrhizae. Ectomycorrhizal abundance on decomposing fine roots may partially explain why fine roots typically decompose more slowly than surface litter. Our results indicate that stand age structures fine-root decomposers but that decay stage is more important in structuring the fungal community than shifts caused by harvesting. The rapid postharvest recovery of fungal communities decomposing fine roots suggests resiliency within this community, at least in these young regenerating stands in coastal British Columbia.IMPORTANCE Globally, fine roots are a dominant source of carbon in forest soils, yet the fungi that decompose this material and that drive the sequestration or respiration of this carbon remain largely uncharacterized. Fungi vary in their capacity to decompose plant litter, suggesting that fungal community composition is an important determinant of decomposition rates. Variable-retention harvesting is a forestry practice that modifies fungal communities by providing refuge for ectomycorrhizal fungi. We evaluated the effects of variable retention and clear-cut harvesting on fungal communities decomposing fine roots at two sites (6 and 13 years postharvest), at two decay stages (43 days and 1 year), and in uncut stands in temperate rainforests. Harvesting impacts on fungal community composition were detected only after 6 years after harvest. We suggest that fungal community composition may be an important factor that reduces fine-root decomposition rates relative to those of above-ground plant litter, which has important consequences for forest carbon cycling. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The third revolution in sequencing technology.

Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Computational identification of novel genes: current and future perspectives.

While it has long been thought that all genomic novelties are derived from the existing material, many genes lacking homology to known genes were found in recent genome projects. Some of these novel genes were proposed to have evolved de novo, ie, out of noncoding sequences, whereas some have been shown to follow a duplication and divergence process. Their discovery called for an extension of the historical hypotheses about gene origination. Besides the theoretical breakthrough, increasing evidence accumulated that novel genes play important roles in evolutionary processes, including adaptation and speciation events. Different techniques are available to identify genes and classify them as novel. Their classification as novel is usually based on their similarity to known genes, or lack thereof, detected by comparative genomics or against databases. Computational approaches are further prime methods that can be based on existing models or leveraging biological evidences from experiments. Identification of novel genes remains however a challenging task. With the constant software and technologies updates, no gold standard, and no available benchmark, evaluation and characterization of genomic novelty is a vibrant field. In this review, the classical and state-of-the-art tools for gene prediction are introduced. The current methods for novel gene detection are presented; the methodological strategies and their limits are discussed along with perspective approaches for further studies.

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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

Single-molecule long-read transcriptome profiling of Platysternon megacephalum mitochondrial genome with gene rearrangement and control region duplication.

Platysternon megacephalum is the sole living representative of the poorly studied turtle lineage Platysternidae. Their mitochondrial genome has been subject to gene rearrangement and control region duplication, resulting in a unique mitochondrial gene order in vertebrates. In this study, we sequenced the first full-length turtle (P. megacephalum) liver transcriptome using single-molecule real-time sequencing to study the transcriptional mechanisms of its mitochondrial genome. ND5 and ND6 anti-sense (ND6AS) forms a single transcript with the same expression in the human mitochondrial genome, but here we demonstrated differential expression of the rearranged ND5 and ND6AS genes in P. megacephalum. And some polycistronic transcripts were also reported in this study. Notably, we detected some novel long non-coding RNAs with alternative polyadenylation from the duplicated control region, and a novel ND6AS transcript composed of a long non-coding sequence, ND6AS, and tRNA-GluAS. These results provide the first description of a mtDNA transcriptome with gene rearrangement and control region duplication. These findings further our understanding of the fundamental concepts of mitochondrial gene transcription and RNA processing, and provide a new insight into the mechanism of transcription regulation of the mitochondrial genome.

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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

Influenza virus infection causes global RNAPII termination defects.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

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September 22, 2019

Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis).

Bamboo is one of the most important nontimber forestry products worldwide. However, a chromosome-level reference genome is lacking, and an evolutionary view of alternative splicing (AS) in bamboo remains unclear despite emerging omics data and improved technologies.Here, we provide a chromosome-level de novo genome assembly of moso bamboo (Phyllostachys edulis) using additional abundance sequencing data and a Hi-C scaffolding strategy. The significantly improved genome is a scaffold N50 of 79.90 Mb, approximately 243 times longer than the previous version. A total of 51,074 high-quality protein-coding loci with intact structures were identified using single-molecule real-time sequencing and manual verification. Moreover, we provide a comprehensive AS profile based on the identification of 266,711 unique AS events in 25,225 AS genes by large-scale transcriptomic sequencing of 26 representative bamboo tissues using both the Illumina and Pacific Biosciences sequencing platforms. Through comparisons with orthologous genes in related plant species, we observed that the AS genes are concentrated among more conserved genes that tend to accumulate higher transcript levels and share less tissue specificity. Furthermore, gene family expansion, abundant AS, and positive selection were identified in crucial genes involved in the lignin biosynthetic pathway of moso bamboo.These fundamental studies provide useful information for future in-depth analyses of comparative genome and AS features. Additionally, our results highlight a global perspective of AS during evolution and diversification in bamboo.

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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.

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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.

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