Transcriptional changes and preservation of bone mass in hibernating black bears.

Physical inactivity leads to losses of bone mass and strength in most mammalian species. In contrast, hibernating bears show no bone loss over the prolonged periods (4-6 months) of immobility during winter, which suggests that they have adaptive mechanisms to preserve bone mass. To identify transcriptional changes that underlie molecular mechanisms preventing disuse osteoporosis, we conducted a large-scale gene expression screening in the trabecular bone and bone marrow, comparing hibernating and summer active bears through sequencing of the transcriptome. Gene set enrichment analysis showed a coordinated down-regulation of genes involved in bone resorption, osteoclast differentiation and signaling, and apoptosis during hibernation. These findings are consistent with previous histological findings and likely contribute to the preservation of bone during the immobility of hibernation. In contrast, no significant enrichment indicating directional changes in gene expression was detected in the gene sets of bone formation and osteoblast signaling in hibernating bears. Additionally, we revealed significant and coordinated transcriptional induction of gene sets involved in aerobic energy production including fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, and mitochondrial metabolism. Mitochondrial oxidation was likely up-regulated by transcriptionally induced AMPK/PGC1α pathway, an upstream stimulator of mitochondrial function.

Endless Forms: Within-Host Variation in the Structure of the West Nile Virus RNA Genome during Serial Passage in Bird Hosts.

RNA viruses are infamous for their high rates of mutation, which produce swarms of genetic variants within individual hosts. To date, analyses of intrahost genetic diversity have focused on the primary genome sequence. However, virus phenotypes are shaped not only by primary sequence but also by the secondary structures into which this sequence folds. Such structures enable viral replication, translation, and binding of small RNAs, yet within-host variation at the structural level has not been adequately explored. We characterized the structural diversity of the 5' untranslated region (UTR) of populations of West Nile virus (WNV) that had been subject to five serial passages in triplicate in each of three bird species. Viral genomes were sampled from host serum samples at each passage (n = 45 populations) and subjected to next-generation sequencing. For populations derived from passages 1, 3, and 5 (n = 9 populations), we predicted the impact of each mutation occurring at a frequency of ≥1% on the secondary structure of the 5' UTR. As expected, mutations in double-stranded (DS) regions of the 5' UTR stem structures caused structural changes of significantly greater magnitude than did mutations in single-stranded (SS) regions. Despite the greater impact of mutations in DS regions, mutations in DS and SS regions occurred at similar frequencies, with no evidence of enhanced selection against mutation in DS regions. In contrast, mutations in two regions that mediate genome cyclization and thereby regulate replication and translation, the 5' cyclization sequence and the UAR flanking stem (UFS), were suppressed in all three hosts.IMPORTANCE The enzymes that copy RNA genomes lack proofreading, and viruses that possess RNA genomes, such as West Nile virus, rapidly diversify into swarms of mutant lineages within a host. Intrahost variation of the primary genomic sequence of RNA viruses has been studied extensively because the extent of this variation shapes key virus phenotypes. However, RNA genomes also form complex secondary structures based on within-genome nucleotide complementarity, which are critical regulators of the cyclization of the virus genome that is necessary for efficient replication and translation. We sought to characterize variation in these secondary structures within populations of West Nile virus during serial passage in three bird species. Our study indicates that the intrahost population of West Nile virus is a diverse assortment of RNA secondary structures that should be considered in future analyses of intrahost viral diversity, but some regions that are critical for genome cyclization are conserved within hosts. Besides potential impacts on viral replication, structural diversity can influence the efficacy of small RNA antiviral therapies.

Cranberry-derived proanthocyanidins induce a differential transcriptomic response within Candida albicans urinary biofilms.

Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.

Correction: Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

[This corrects the article DOI: 10.1371/journal.pone.0171345.].

Corrigendum: RNA-Seq Comparison of Larval and Adult Malpighian Tubules of the Yellow Fever Mosquito Aedes aegypti Reveals Life Stage-Specific Changes in Renal Function.

[This corrects the article on p. 283 in vol. 8, PMID: 28536536.].

Transcriptomic responses of Biomphalaria pfeifferi to Schistosoma mansoni: Investigation of a neglected African snail that supports more S. mansoni transmission than any other snail species.

BACKGROUND: Biomphalaria pfeifferi is highly compatible with the widespread human-infecting blood fluke Schistosoma mansoni and transmits more cases of this parasite to people than any other snail species. For these reasons, B. pfeifferi is the world's most important vector snail for S. mansoni, yet we know relatively little at the molecular level regarding the interactions between B. pfeifferi and S. mansoni from early-stage sporocyst transformation to the development of cercariae. METHODOLOGY/PRINCIPAL FINDINGS: We sought to capture a portrait of the response of B. pfeifferi to S. mansoni as it occurs in nature by undertaking Illumina dual RNA-Seq on uninfected control B. pfeifferi and three intramolluscan developmental stages (1- and 3-days post infection and patent, cercariae-producing infections) using field-derived west Kenyan specimens. A high-quality, well-annotated de novo B. pfeifferi transcriptome was assembled from over a half billion non-S. mansoni paired-end reads. Reads associated with potential symbionts were noted. Some infected snails yielded fewer normalized S. mansoni reads and showed different patterns of transcriptional response than others, an indication that the ability of field-derived snails to support and respond to infection is variable. Alterations in transcripts associated with reproduction were noted, including for the oviposition-related hormone ovipostatin and enzymes involved in metabolism of bioactive amines like dopamine or serotonin. Shedding snails exhibited responses consistent with the need for tissue repair. Both generalized stress and immune factors immune factors (VIgLs, PGRPs, BGBPs, complement C1q-like, chitinases) exhibited complex transcriptional responses in this compatible host-parasite system. SIGNIFICANCE: This study provides for the first time a large sequence data set to help in interpreting the important vector role of the neglected snail B. pfeifferi in transmission of S. mansoni, including with an emphasis on more natural, field-derived specimens. We have identified B. pfeifferi targets particularly responsive during infection that enable further dissection of the functional role of these candidate molecules.

RNA-Seq Comparison of Larval and Adult Malpighian Tubules of the Yellow Fever Mosquito Aedes aegypti Reveals Life Stage-Specific Changes in Renal Function.

Introduction: The life history of Aedes aegypti presents diverse challenges to its diuretic system. During the larval and pupal life stages mosquitoes are aquatic. With the emergence of the adult they become terrestrial. This shifts the organism within minutes from an aquatic environment to a terrestrial environment where dehydration has to be avoided. In addition, female mosquitoes take large blood meals, which present an entirely new set of challenges to salt and water homeostasis. Methods: To determine differences in gene expression associated with these different life stages, we performed an RNA-seq analysis of the main diuretic tissue in A. aegypti, the Malpighian tubules. We compared transcript abundance in 4th instar larvae to that of adult females and analyzed the data with a focus on transcripts that encode proteins potentially involved in diuresis, like water and solute channels as well as ion transporters. We compared our results against the model of potassium- and sodium chloride excretion in the Malpighian tubules proposed by Hine et al. (2014), which involves at least eight ion transporters and a proton-pump. Results: We found 3,421 of a total number of 17,478 (19.6%) unique transcripts with a P < 0.05 and at least a 2.5 fold change in expression levels between the two groups. We identified two novel transporter genes that are highly expressed in the adult Malpighian tubules, which have not previously been part of the transport model in this species and may play important roles in diuresis. We also identified candidates for hypothesized sodium and chloride channels. Detoxification genes were generally higher expressed in larvae. Significance: This study represents the first comparison of Malpighian tubule transcriptomes between larval and adult A. aegypti mosquitoes, highlighting key differences in their renal systems that arise as they transform from an aquatic filter-feeding larval stage to a terrestrial, blood-feeding adult stage.

Zinc-Dependent Transcriptional Regulation in Paracoccus denitrificans.

Zinc homeostasis is critical for bacterial survival and is mediated largely at the transcriptional level by the regulation of zinc uptake and efflux genes. Here we use RNA-seq to assess transcriptional changes as a result of zinc limitation in the denitrifying bacterium Paracoccus denitrificans. The results identify the differential expression of 147 genes, most of which were upregulated in zinc-depleted medium. Included in this set of genes are a large number of transition metal transporters, several transcription factors, and hypothetical proteins. Intriguingly, genes encoding nitric oxide reductase (norCB) and nitrite reductase (nirS) were also upregulated. A Zur consensus binding motif was identified in the promoters of the most highly upregulated genes. The zinc uptake regulator (Zur) from this organism was also characterized and shown to bind to the Zur motif in a zinc-dependent manner. This work expands our current understanding of the transcriptional response of gram-negative bacteria to zinc limitation and identifies genes involved in denitrification as part of the Zur regulon.

Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses) may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal. METHODOLOGY/PRINCIPAL FINDINGS: We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses. CONCLUSION/SIGNIFICANCE: Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.

De novo assembly of a tadpole shrimp (Triops newberryi) transcriptome and preliminary differential gene expression analysis.

Next-generation sequencing techniques, such as RNA sequencing, have provided a wealth of genomic information for nonmodel species. Transcriptomic information can be used to quantify the patterns of gene expression, which can identify how environmental differences invoke organismal stress responses and provide a gauge in predicting species adaptability. In our study, we used RNA sequencing to characterize the first transcriptome from a naupliar tadpole shrimp (Triops newberryi) to identify the genes expressed during the early life history stages and which could be important for future genomic studies. RNA was extracted from naupliar T. newberryi that were reared in a laboratory-controlled setting and in two different water types, a native and a non-native condition. A total of six replicates, three per condition, were sequenced with the Illumina Hi-Seq 2000 achieving 365 M 50-nt reads. High-quality reads were produced and de novo assembly was used to construct a T. newberryi transcriptome that was approximately 24.8 M base pairs. More than 10 000 peptides were predicted from the assembly, and genes were sorted into gene ontology categories. The use of different water conditions allowed for a preliminary differential gene expression analysis in order to compare the changes in gene expression between conditions. There were 299 differentially expressed genes between water conditions that might serve as a focal point for future genomic studies of Triops acclimation to different environments. The Triops transcriptome could serve as vital genomic information for additional studies on Branchiopod crustaceans.

Complete Genome Sequence of Streptomyces venezuelae ATCC 15439, Producer of the Methymycin/Pikromycin Family of Macrolide Antibiotics, Using PacBio Technology.

Here, we report the complete genome sequence of Streptomyces venezuelae ATCC 15439, a producer of the methymycin/pikromycin family of macrolide antibiotics and a model host for natural product studies, obtained exclusively using PacBio sequencing technology. The 9.03-Mbp genome harbors 8,775 genes and 11 polyketide and nonribosomal peptide natural product gene clusters.

Small mosquitoes, large implications: crowding and starvation affects gene expression and nutrient accumulation in Aedes aegypti.

BACKGROUND: Environmental factors such as temperature, nutrient availability, and larval density determine the outcome of postembryonic development in mosquitoes. Suboptimal temperatures, crowding, and starvation during the larval phase reduce adult mosquito size, nutrient stores and affect vectorial capacity. METHODS: In this study we compared adult female Aedes aegypti, Rockefeller strain, raised under standard laboratory conditions (Large) with those raised under crowded and nutritionally deprived conditions (Small). To compare the gene expression and nutritional state of the major energy storage and metabolic organ, the fat body, we performed transcriptomics using Illumina based RNA-seq and metabolomics using GC/MS on females before and 24 hours following blood feeding. RESULTS: Analysis of fat body gene expression between the experimental groups revealed a large number of significantly differentially expressed genes. Transcripts related to immunity, reproduction, autophagy, several metabolic pathways; including amino acid degradation and metabolism; and membrane transport were differentially expressed. Metabolite profiling identified 60 metabolites within the fat body to be significantly affected between small and large mosquitoes, with the majority of detected free amino acids at a higher level in small mosquitoes compared to large. CONCLUSIONS: Gene expression and metabolites in the adult fat body reflect the individual post-embryonic developmental history of a mosquito larva. These changes affect nutritional storage and utilization, immunity, and reproduction. Therefore, it is apparent that changes in larval environment due to weather conditions, nutrition availability, vector control efforts, and other factors can affect adult vectorial capacity in the field.

Draft Genome Sequences of Vancomycin-Susceptible Staphylococcus aureus Related to Heterogeneous Vancomycin-Intermediate S. aureus.

We report the draft genome sequences of three vancomycin-susceptible methicillin-resistant Staphylococcus aureus strains. S. aureus strain MV8 is a sequence type 8 (ST-8) staphylococcal cassette chromosome mec element type IV (SCCmec IV) derivative, while the other two strains (S. aureus MM25 and MM61) are ST-5 SCCmec II strains. MM61 is also closely related to the heterogeneous vancomycin-intermediate S. aureus strain MM66.

Draft Genomes of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strain MM66 and MM66 Derivatives with Altered Vancomycin Resistance Levels.

The draft genomes of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) strain MM66 and MM66 isolates demonstrating altered vancomycin resistance levels were produced in an effort to provide information on mutations contributing to the vancomycin resistance levels observed in these strains.

Improved Hybrid Genome Assemblies of Two Strains of Bacteroides xylanisolvens, SD_CC_1b and SD_CC_2a, Obtained Using Illumina and 454 Sequencing Technologies.

Bacteroides xlyanisolvens strains (SD_CC_1b, SD_CC_2a) isolated from human feces were grown on crystalline cellulose. Cellulolytic properties are not common in Bacteroides species. Here, we report improved genome sequences of both of the B. xlyanisolvens strains.

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence-related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Genome Sequence of Non-O1 Vibrio cholerae PS15.

The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V. cholerae.

Analysis of the transcriptomes downstream of Eyeless and the Hedgehog, Decapentaplegic and Notch signaling pathways in Drosophila melanogaster.

Tissue-specific transcription factors are thought to cooperate with signaling pathways to promote patterned tissue specification, in part by co-regulating transcription. The Drosophila melanogaster Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. We report a combinatorial approach, including mRNAseq and microarray analyses, to identify targets co-regulated by Eyeless and Hedgehog, Decapentaplegic or Notch. Multiple analyses suggest that the transcriptomes resulting from co-misexpression of Eyeless+signaling factors provide a more complete picture of eye development compared to previous efforts involving Eyeless alone: (1) Principal components analysis and two-way hierarchical clustering revealed that the Eyeless+signaling factor transcriptomes are closer to the eye control transcriptome than when Eyeless is misexpressed alone; (2) more genes are upregulated at least three-fold in response to Eyeless+signaling factors compared to Eyeless alone; (3) based on gene ontology analysis, the genes upregulated in response to Eyeless+signaling factors had a greater diversity of functions compared to Eyeless alone. Through a secondary screen that utilized RNA interference, we show that the predicted gene CG4721 has a role in eye development. CG4721 encodes a neprilysin family metalloprotease that is highly up-regulated in response to Eyeless+Notch, confirming the validity of our approach. Given the similarity between D. melanogaster and vertebrate eye development, the large number of novel genes identified as potential targets of Ey+signaling factors will provide novel insights to our understanding of eye development in D. melanogaster and humans.

Genome-wide association genetics of an adaptive trait in lodgepole pine.

Pine cones that remain closed and retain seeds until fire causes the cones to open (cone serotiny) represent a key adaptive trait in a variety of pine species. In lodgepole pine, there is substantial geographical variation in serotiny across the Rocky Mountain region. This variation in serotiny has evolved as a result of geographically divergent selection, with consequences that extend to forest communities and ecosystems. An understanding of the genetic architecture of this trait is of interest owing to the wide-reaching ecological consequences of serotiny and also because of the repeated evolution of the trait across the genus. Here, we present and utilize an inexpensive and time-effective method for generating population genomic data. The method uses restriction enzymes and PCR amplification to generate a library of fragments that can be sequenced with a high level of multiplexing. We obtained data for more than 95,000 single nucleotide polymorphisms across 98 serotinous and nonserotinous lodgepole pines from three populations. We used a Bayesian generalized linear model (GLM) to test for an association between genotypic variation at these loci and serotiny. The probability of serotiny varied by genotype at 11 loci, and the association between genotype and serotiny at these loci was consistent in each of the three populations of pines. Genetic variation across these 11 loci explained 50% of the phenotypic variation in serotiny. Our results provide a first genome-wide association map of serotiny in pines and demonstrate an inexpensive and efficient method for generating population genomic data.

Carrier testing for severe childhood recessive diseases by next-generation sequencing.

Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160x average target coverage, 93% of nucleotides had at least 20x coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.