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September 22, 2019  |  

Researches on transcriptome sequencing in the study of traditional Chinese medicine

Due to its incomparable advantages, the application of transcriptome sequencing in the study of traditional Chinese medicine attracts more and more attention of researchers, which greatly promote the development of traditional Chinese medicine. In this paper, the applications of transcriptome sequencing in traditional Chinese medicine were summarized by reviewing recent related papers.

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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  |  

Analysis of the duodenal microbiotas of weaned piglet fed with epidermal growth factor-expressed Saccharomyces cerevisiae.

The bacterial community of the small intestine is a key factor that has strong influence on the health of gastrointestinal tract (GIT) in mammals during and shortly after weaning. The aim of this study was to analyze the effects of the diets of supplemented with epidermal growth factor (EGF)-expressed Saccharomyces cerevisiae (S. cerevisiae) on the duodenal microbiotas of weaned piglets.Revealed in this study, at day 7, 14 and 21, respectively, the compositional sequencing analysis of the 16S rRNA in the duodenum had no marked difference in microbial diversity from the phylum to species levels between the INVSc1(EV) and other recombinant strains encompassing INVSc1-EE(+), INVSc1-TE(-), and INVSc1-IE(+). Furthermore, the populations of potentially enterobacteria (e.g., Clostridium and Prevotella) and probiotic (e.g., Lactobacilli and Lactococcus) also remained unchanged among recombinant S. cerevisiae groups (P?>?0.05). However, the compositional sequencing analysis of the 16S rRNA in the duodenum revealed significant difference in microbial diversity from phylum to species levels between the control group and recombinant S. cerevisiae groups. In terms of the control group (the lack of S. cerevisiae), these data confirmed that dietary exogenous S. cerevisiae had the feasibility to be used as a supplement for enhancing potentially probiotic (e.g., Lactobacilli and Lactococcus) (P?

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September 22, 2019  |  

L_RNA_scaffolder: scaffolding genomes with transcripts.

Generation of large mate-pair libraries is necessary for de novo genome assembly but the procedure is complex and time-consuming. Furthermore, in some complex genomes, it is hard to increase the N50 length even with large mate-pair libraries, which leads to low transcript coverage. Thus, it is necessary to develop other simple scaffolding approaches, to at least solve the elongation of transcribed fragments.We describe L_RNA_scaffolder, a novel genome scaffolding method that uses long transcriptome reads to order, orient and combine genomic fragments into larger sequences. To demonstrate the accuracy of the method, the zebrafish genome was scaffolded. With expanded human transcriptome data, the N50 of human genome was doubled and L_RNA_scaffolder out-performed most scaffolding results by existing scaffolders which employ mate-pair libraries. In these two examples, the transcript coverage was almost complete, especially for long transcripts. We applied L_RNA_scaffolder to the highly polymorphic pearl oyster draft genome and the gene model length significantly increased.The simplicity and high-throughput of RNA-seq data makes this approach suitable for genome scaffolding. L_RNA_scaffolder is available at http://www.fishbrowser.org/software/L_RNA_scaffolder.

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September 22, 2019  |  

Transcriptome profiling using single-molecule direct RNA sequencing approach for in-depth understanding of genes in secondary metabolism pathways of Camellia sinensis.

Characteristic secondary metabolites, including flavonoids, theanine and caffeine, are important components of Camellia sinensis, and their biosynthesis has attracted widespread interest. Previous studies on the biosynthesis of these major secondary metabolites using next-generation sequencing technologies limited the accurately prediction of full-length (FL) splice isoforms. Herein, we applied single-molecule sequencing to pooled tea plant tissues, to provide a more complete transcriptome of C. sinensis. Moreover, we identified 94 FL transcripts and four alternative splicing events for enzyme-coding genes involved in the biosynthesis of flavonoids, theanine and caffeine. According to the comparison between long-read isoforms and assemble transcripts, we improved the quality and accuracy of genes sequenced by short-read next-generation sequencing technology. The resulting FL transcripts, together with the improved assembled transcripts and identified alternative splicing events, enhance our understanding of genes involved in the biosynthesis of characteristic secondary metabolites in C. sinensis.

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September 22, 2019  |  

Bacterial community structure in simultaneous nitrification, denitrification and organic matter removal process treating saline mustard tuber wastewater as revealed by 16S rRNA sequencing.

A simultaneous nitrification, denitrification and organic matter removal (SNDOR) process in sequencing batch biofilm reactor (SBBR) was established to treat saline mustard tuber wastewater (MTWW) in this study. An average COD removal efficiency of 86.48% and total nitrogen removal efficiency of 86.48% were achieved at 30gNaClL(-1) during 100days’ operation. The underlying mechanisms were investigated by PacBio SMRT DNA sequencing (V1-V9) to analyze the microbial community structures and its variation from low salinity at 10gNaClL(-1) to high salinity at 30gNaClL(-1). Results showed elevated salinity did not affect biological performance but reduced microbial diversity in SBBR, and halophilic bacteria gradually predominated by succession. Despite of high C/N, autotrophic ammonia-oxidizing bacteria (AOB) Nitrosomonas and ammonia-oxidizing archaea (AOA) Candidatus Nitrososphaera both contributed to ammonium oxidation. As salinity increasing, nitrite-oxidizing bacteria (NOB) were significantly inhibited, partial nitrification and denitrification (PND) process gradually contributed to nitrogen removal. Copyright © 2016 Elsevier Ltd. All rights reserved.

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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  |  

Antagonism between Staphylococcus epidermidis and Propionibacterium acnes and its genomic basis.

Propionibacterium acnes and Staphylococcus epidermidis live in close proximity on human skin, and both bacterial species can be isolated from normal and acne vulgaris-affected skin sites. The antagonistic interactions between the two species are poorly understood, as well as the potential significance of bacterial interferences for the skin microbiota. Here, we performed simultaneous antagonism assays to detect inhibitory activities between multiple isolates of the two species. Selected strains were sequenced to identify the genomic basis of their antimicrobial phenotypes.First, we screened 77 P. acnes strains isolated from healthy and acne-affected skin, and representing all known phylogenetic clades (I, II, and III), for their antimicrobial activities against 12?S. epidermidis isolates. One particular phylogroup (I-2) exhibited a higher antimicrobial activity than other P. acnes phylogroups. All genomes of type I-2 strains carry an island encoding the biosynthesis of a thiopeptide with possible antimicrobial activity against S. epidermidis. Second, 20?S. epidermidis isolates were examined for inhibitory activity against 25 P. acnes strains. The majority of S. epidermidis strains were able to inhibit P. acnes. Genomes of S. epidermidis strains with strong, medium and no inhibitory activities against P. acnes were sequenced. Genome comparison underlined the diversity of S. epidermidis and detected multiple clade- or strain-specific mobile genetic elements encoding a variety of functions important in antibiotic and stress resistance, biofilm formation and interbacterial competition, including bacteriocins such as epidermin. One isolate with an extraordinary antimicrobial activity against P. acnes harbors a functional ESAT-6 secretion system that might be involved in the antimicrobial activity against P. acnes via the secretion of polymorphic toxins.Taken together, our study suggests that interspecies interactions could potentially jeopardize balances in the skin microbiota. In particular, S. epidermidis strains possess an arsenal of different mechanisms to inhibit P. acnes. However, if such interactions are relevant in skin disorders such as acne vulgaris remains questionable, since no difference in the antimicrobial activity against, or the sensitivity towards S. epidermidis could be detected between health- and acne-associated strains of P. acnes.

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September 22, 2019  |  

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

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

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September 22, 2019  |  

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  |  

Analysis of microbial community structure of pit mud for Chinese strong-flavor liquor fermentation using next generation DNA sequencing of full-length 16S rRNA

The pit is the necessary bioreactor for brewing process of Chinese strong-flavor liquor. Pit mud in pits contains a large number of microorganisms and is a complex ecosystem. The analysis of bacterial flora in pit mud is of great significance to understand liquor fermentation mechanisms. To overcome taxonomic limitations of short reads in 16S rRNA variable region sequencing, we used high-throughput DNA sequencing of near full-length 16S rRNA gene to analyze microbial compositions of different types of pit mud that produce different qualities of strong-flavor liquor. The results showed that the main species in pit mud were Pseudomonas extremaustralis 14-3, Pseudomonas veronii, Serratia marcescens WW4, and Clostridium leptum in Ruminiclostridium. The microbial diversity of pit mud with different quality was significantly different. From poor to good quality of pit mud (thus the quality of liquor), the relative abundances of Ruminiclostridium and Syntrophomonas in Firmicutes was increased, and the relative abundance of Olsenella in Actinobacteria also increased, but the relative abundances of Pseudomonas and Serratia in Proteobacteria were decreased. The surprising findings of this study include that the diversity of intermediate level quality of N pit mud was the lowest, and the diversity levels of high quality pit mud G and poor quality pit mud B were similar. Correlation analysis showed that there were high positive correlations (r > 0.8) among different microbial groups in the flora. Based on the analysis of the microbial structures of pit mud in different quality, the good quality pit mud has a higher microbial diversity, but how this higher diversity and differential microbial compositions contribute to better quality of liquor fermentation remains obscure.

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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.

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