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September 22, 2019

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer.

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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September 22, 2019

Multiple regulatory networks are activated during cold stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.

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September 22, 2019

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

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September 22, 2019

Diverse antibiotic resistance genes in dairy cow manure.

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

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September 22, 2019

Multiplex amplicon sequencing for microbe identification in community-based culture collections.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

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September 22, 2019

Application of PacBio Single Molecule Real-Time (SMRT) sequencing in bacterial source tracking analysis during milk powder production

This work developed a 16S rRNA-PacBio Single Molecule Real-Time (SMRT) sequencing-based method to identify and track the bacterial community of milk powder (MP) from two kinds of production settings, i.e., small-scale production contained within an in-house environment (minimal milk storage before pasteurization, milk concentration, and spray drying) and a large-scale factory production (prolonged milk storage before direct spray drying). A total of 18 samples were analyzed at the species level. Comparing with the large-scale factory production, only relatively little changes were observed in the bacterial community during the in-house production process, without significant loss in the levels of bioactive minor proteins (namely, lactoferrin, immunoglobulin G, lactoperoxidase, and lysozyme). The two most prevalent species in the in-house production, Bacillus cereus and Bacillus flexus, were likely originated from the raw milk with only small changes in their relative abundances (from 25.97% to 26.40%–28.89% and 27.40%, respectively) throughout the processing (from raw milk to MP). In contrast, large-scale factory production resulted in more obvious variation in the microbial content. This microbial tracking approach is valuable in identifying the contamination source and the specific stage when contamination happens; the implementation of such technique may also enhance food quality assurance systems that are currently used in the dairy industry.

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September 22, 2019

SMRT sequencing of full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt).

This study was aimed at generating the full-length transcriptome of flea beetle Agasicles hygrophila (Selman and Vogt) using single-molecule real-time (SMRT) sequencing. Four developmental stages of A. hygrophila, including eggs, larvae, pupae, and adults were harvested for isolating total RNA. The mixed samples were used for SMRT sequencing to generate the full-length transcriptome. Based on the obtained transcriptome data, alternative splicing event, simple sequence repeat (SSR) analysis, coding sequence prediction, transcript functional annotation, and lncRNA prediction were performed. Total 9.45?Gb of clean reads were generated, including 335,045 reads of insert (ROI) and 158,085 full-length non-chimeric (FLNC) reads. Transcript clustering analysis of FLNC reads identified 40,004 consensus isoforms, including 31,015 high-quality ones. After removing redundant reads, 28,982 transcripts were obtained. Total 145 alternative splicing events were predicted. Additionally, 12,753 SSRs and 16,205 coding sequences were identified based on SSR analysis. Furthermore, 24,031 transcripts were annotated in eight functional databases, and 4,198 lncRNAs were predicted. This is the first study to perform SMRT sequencing of the full-length transcriptome of A. hygrophila. The obtained transcriptome may facilitate further exploration of the genetic data of A. hygrophila and uncover the interactions between this insect and the ecosystem.

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September 22, 2019

Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris

Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids, derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding a total of 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

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September 22, 2019

Atmospheric N deposition alters connectance, but not functional potential among saprotrophic bacterial communities.

The use of co-occurrence patterns to investigate interactions between micro-organisms has provided novel insight into organismal interactions within microbial communities. However, anthropogenic impacts on microbial co-occurrence patterns and ecosystem function remain an important gap in our ecological knowledge. In a northern hardwood forest ecosystem located in Michigan, USA, 20 years of experimentally increased atmospheric N deposition has reduced forest floor decay and increased soil C storage. This ecosystem-level response occurred concomitantly with compositional changes in saprophytic fungi and bacteria. Here, we investigated the influence of experimental N deposition on biotic interactions among forest floor bacterial assemblages by employing phylogenetic and molecular ecological network analysis. When compared to the ambient treatment, the forest floor bacterial community under experimental N deposition was less rich, more phylogenetically dispersed and exhibited a more clustered co-occurrence network topology. Together, our observations reveal the presence of increased biotic interactions among saprotrophic bacterial assemblages under future rates of N deposition. Moreover, they support the hypothesis that nearly two decades of experimental N deposition can modify the organization of microbial communities and provide further insight into why anthropogenic N deposition has reduced decomposition, increased soil C storage and accelerated phenolic DOC production in our field experiment. © 2015 John Wiley & Sons Ltd.

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September 22, 2019

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

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September 22, 2019

Metasecretome phage display.

Metasecretome is a collection of cell-surface and secreted proteins that mediate interactions between microbial communities and their environment. These include adhesins, enzymes, surface structures such as pili or flagella, vaccine targets or proteins responsible for immune evasion. Traditional approaches to exploring matasecretome of complex microbial communities via cultivation of microorganisms and screening of individual strains fail to sample extraordinary diversity in these communities, since only a limited fraction of microorganisms are represented by cultures. Advances in culture-independent sequence analysis methods, collectively referred to as metagenomics, offer an alternative approach that enables the direct analysis of collective microbial genomes (metagenome) recovered from environmental samples. This protocol describes a method, metasecretome phage display, which selectively displays the metasecretome portion of the metagenome. The metasecretome library can then be used for two purposes: (1) to sequence the entire metasecretome (using PacBio technology); (2) to identify metasecretome proteins that have a specific function of interest by affinity-screening (bio-panning) using a variety of methods described in other chapters of this volume.

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September 22, 2019

Diversified microbiota of meconium is affected by maternal diabetes status.

This study was aimed to assess the diversity of the meconium microbiome and determine if the bacterial community is affected by maternal diabetes status.The first intestinal discharge (meconium) was collected from 23 newborns stratified by maternal diabetes status: 4 mothers had pre-gestational type 2 diabetes mellitus (DM) including one mother with dizygotic twins, 5 developed gestational diabetes mellitus (GDM) and 13 had no diabetes. The meconium microbiome was profiled using multi-barcode 16S rRNA sequencing followed by taxonomic assignment and diversity analysis.All meconium samples were not sterile and contained diversified microbiota. Compared with adult feces, the meconium showed a lower species diversity, higher sample-to-sample variation, and enrichment of Proteobacteria and reduction of Bacteroidetes. Among the meconium samples, the taxonomy analyses suggested that the overall bacterial content significantly differed by maternal diabetes status, with the microbiome of the DM group showing higher alpha-diversity than that of no-diabetes or GDM groups. No global difference was found between babies delivered vaginally versus via Cesarean-section. Regression analysis showed that the most robust predictor for the meconium microbiota composition was the maternal diabetes status that preceded pregnancy. Specifically, Bacteroidetes (phyla) and Parabacteriodes (genus) were enriched in the meconium in the DM group compared to the no-diabetes group.Our study provides evidence that meconium contains diversified microbiota and is not affected by the mode of delivery. It also suggests that the meconium microbiome of infants born to mothers with DM is enriched for the same bacterial taxa as those reported in the fecal microbiome of adult DM patients.

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September 22, 2019

Nasopharyngeal microbiome in premature infants and stability during rhinovirus infection.

The nasopharyngeal (NP) microbiota of newborns and infants plays a key role in modulating airway inflammation and respiratory symptoms during viral infections. Premature (PM) birth modifies the early NP environment and is a major risk factor for severe viral respiratory infections. However, it is currently unknown if the NP microbiota of PM infants is altered relative to full-term (FT) individuals.To characterize the NP microbiota differences in preterm and FT infants during rhinovirus (RV) infection.We determined the NP microbiota of infants 6 months to =2 years of age born FT (n=6) or severely PM<32 weeks gestation (n=7). We compared microbiota composition in healthy NP samples and performed a longitudinal analysis during naturally occurring RV infections to contrast the microbiota dynamics in PM versus FT infants.We observed significant differences in the NP bacterial community of PM versus FT. NP from PM infants had higher within-group dissimilarity (heterogeneity) relative to FT infants. Bacterial composition of NP samples from PM infants showed increased Proteobacteria and decreased in Firmicutes. There were also differences in the major taxonomic groups identified, including Streptococcus, Moraxella, and Haemophilus. Longitudinal data showed that these prematurity-related microbiota features persisted during RV infection.PM is associated with NP microbiota changes beyond the neonatal stage. PM infants have an NP microbiota with high heterogeneity relative to FT infants. These prematurity-related microbiota features persisted during RV infection, suggesting that the NP microbiota of PM may play an important role in modulating airway inflammatory and immune responses in this vulnerable group. Copyright © 2017 American Federation for Medical Research.

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September 22, 2019

Generation and comparative analysis of full-length transcriptomes in sweetpotato and its putative wild ancestor I. trifida.

Sweetpotato [Ipomoea batatas (L.) Lam.] is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither high-quality reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid are still lacking, which in turn impedes progress in research studies in sweetpotato functional genomics and molecular breeding. In this study, we apply a combination of second- and third-generation sequencing technologies to sequence full-length transcriptomes in sweetpotato and its putative ancestor I. trifida. In total, we obtained 53,861/51,184 high-quality transcripts, which includes 34,963/33,637 putative full-length cDNA sequences, from sweetpotato/I. trifida. Amongst, we identified 104,540/94,174 open reading frames, 1476/1475 transcription factors, 25,315/27,090 simple sequence repeats, 417/531 long non-coding RNAs out of the sweetpotato/I. trifida dataset. By utilizing public available genomic contigs, we analyzed the gene features (including exon number, exon size, intron number, intron size, exon-intron structure) of 33,119 and 32,793 full-length transcripts in sweetpotato and I. trifida, respectively. Furthermore, comparative analysis between our transcript datasets and other large-scale cDNA datasets from different plant species enables us assessing the quality of public datasets, estimating the genetic similarity across relative species, and surveyed the evolutionary pattern of genes. Overall, our study provided fundamental resources of large-scale full-length transcripts in sweetpotato and its putative ancestor, for the first time, and would facilitate structural, functional and comparative genomics studies in this important crop.

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