Search

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Background: Groundbreaking studies have linked the gut microbiome with immune homeostasis and antitumor immune responses. Mounting evidence has also demonstrated an intratumoral microbiome, including in soft tissue sarcomas (STS), although detailed characterization of the STS intratumoral microbiome is limited. We sought to characterize the intratumoral microbiome in patients with STS undergoing preoperative radiotherapy and surgery, hypothesizing the presence of a distinct intratumoral microbiome with potentially clinically significant microbial signatures., Methods: We prospectively obtained tumor and stool samples from adult patients with non-metastatic STS using a strict sterile collection protocol to minimize contamination. Metagenomic classification was used to estimate abundance using genus and species taxonomic levels across all classified organisms, and data were analyzed with respect to clinicopathologic factors., Results: Fifteen patients were enrolled. Most tumors were located at an extremity (67%) and were histologic grade 3 (87%). 40% were well-differentiated/dedifferentiated liposarcoma histology. With a median follow-up of 24 months, 4 (27%) patients developed metastases, and 3 (20%) died. Despite overwhelming human DNA (>99%) intratumorally, we detected a small but consistent proportion of bacterial DNA (0.02-0.03%) in all tumors, including Proteobacteria, Bacteroidetes, and Firmicutes , as well as viral species. In the tumor microenvironment, we observed a strong positive correlation between viral relative abundance and natural killer (NK) infiltration, and higher NK infiltration was associated with superior metastasis-free and overall survival by immunohistochemical, flow cytometry, and multiplex immunofluorescence analyses., Conclusions: We prospectively demonstrate the presence of a distinct and measurable intratumoral microbiome in patients with STS at multiple time points. Our data suggest that the STS tumor microbiome has prognostic significance with viral relative abundance associated with NK infiltration and oncologic outcome. Additional studies are warranted to further assess the clinical impact of these findings., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Science education and research have the potential to drive profound change in low- and middle-income countries (LMICs) through encouraging innovation, attracting industry, and creating job opportunities. However, in LMICs, research capacity is often limited, and acquisition of funding and access to state-of-the-art technologies is challenging. The Alliance for Global Health and Science (the Alliance) was founded as a partnership between the University of California, Berkeley (USA) and Makerere University (Uganda), with the goal of strengthening Makerere University's capacity for bioscience research. The flagship program of the Alliance partnership is the MU/UCB Biosciences Training Program, an in-country, hands-on workshop model that trains a large number of students from Makerere University in infectious disease and molecular biology research. This approach nucleates training of larger and more diverse groups of students, development of mentoring and bi-directional research partnerships, and support of the local economy. Here, we describe the project, its conception, implementation, challenges, and outcomes of bioscience research workshops. We aim to provide a blueprint for workshop implementation, and create a valuable resource for bioscience research capacity strengthening in LMICs.

Self-transmissible multidrug resistance (MDR) plasmids are a major health concern because they can spread antibiotic resistance to pathogens. Even though most pathogens form biofilms, little is known about how MDR plasmids persist and evolve in biofilms. We hypothesize that (i) biofilms act as refugia of MDR plasmids by retaining them in the absence of antibiotics longer than well-mixed planktonic populations and that (ii) the evolutionary trajectories that account for the improvement of plasmid persistence over time differ between biofilms and planktonic populations. In this study, we evolved Acinetobacter baumannii with an MDR plasmid in biofilm and planktonic populations with and without antibiotic selection. In the absence of selection, biofilm populations were better able to maintain the MDR plasmid than planktonic populations. In planktonic populations, plasmid persistence improved rapidly but was accompanied by a loss of genes required for the horizontal transfer of plasmids. In contrast, in biofilms, most plasmids retained their transfer genes, but on average, plasmid, persistence improved less over time. Our results showed that biofilms can act as refugia of MDR plasmids and favor the horizontal mode of plasmid transfer, which has important implications for the spread of MDR., (© 2022. The Author(s).)

Objective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties., Design: A two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform., Results: We observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types., Conclusions: The knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ., (© 2022 The Authors.)

Social monogamy is a reproductive strategy characterized by pair living and defense of a common territory. Pair bonding, sometimes displayed by monogamous species, is an affective construct that includes preference for a specific partner, distress upon separation, and the ability of the partner to buffer against stress. Many seahorse species show a monogamous social structure in the wild, but their pair bond has not been well studied. We examined the gene expression of lined seahorses (Hippocampus erectus) during and after the process of pairing in the laboratory as well as color change (luminance), a potential form of social communication and behavioral synchrony between pair mates. When a seahorse of either sex was interacting with its pair mate, their changes in luminance ("brightness") were correlated and larger than when interacting with an opposite-sex stranger. At the conclusion of testing, subjects were euthanized, RNA was extracted from whole brains and analyzed via RNA sequencing. Changes in gene expression in paired males versus those that were unpaired included processes governing metabolic activity, hormones and cilia. Perhaps most interesting is the overlap in gene expression change induced by pairing in both male seahorses and male prairie voles, including components of hormone systems regulating reproduction. Because of our limited sample size, we consider our results and interpretations to be preliminary, and prompts for further exploration. Future studies will expand upon these findings and investigate the neuroendocrine and genetic basis of these behaviors., (© 2022 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Rett syndrome (RTT) is a regressive neurodevelopmental disorder in girls, characterized by multisystem complications including gut dysbiosis and altered metabolism. While RTT is known to be caused by mutations in the X-linked gene MECP2, the intermediate molecular pathways of progressive disease phenotypes are unknown. Mecp2 deficient rodents used to model RTT pathophysiology in most prior studies have been male. Thus, we utilized a patient-relevant mouse model of RTT to longitudinally profile the gut microbiome and metabolome across disease progression in both sexes. Fecal metabolites were altered in Mecp2e1 mutant females before onset of neuromotor phenotypes and correlated with lipid deficiencies in brain, results not observed in males. Females also displayed altered gut microbial communities and an inflammatory profile that were more consistent with RTT patients than males. These findings identify new molecular pathways of RTT disease progression and demonstrate the relevance of further study in female Mecp2 animal models., (© 2021. The Author(s).)

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen., Competing Interests: The authors declare that they have no competing interests.

Background: T cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19., Methods: Antigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes., Findings: High correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [T EMRA ]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had T EMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%-38% and 20%-23%, respectively, among COVID-19 patients., Conclusions: Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19., Funding: This study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation., Competing Interests: M.L.S., B.D.-J., M.D., L.R.B., A.H.L., X.J., M.W.K., and L.J. declare that they have no competing interests to disclose. D.R.W. has a financial interest in Quanterix Corporation, a company that develops an ultra-sensitive digital immunoassay platform. He is an inventor of the Simoa technology, founder of the company, and serves on its Board of Directors. The anti-SARS-CoV-2 Simoa assays in this publication have been licensed by Brigham and Women’s Hospital to Quanterix Corporation. T.G. receives royalty payments from Brigham and Women’s Hospital for the antibody assay technology. T.N.M. has a financial interest in neuAPC Therapeutics, a company that will develop AAC for the generation of nAPCs and serves as one of its scientific advisors. V.M., X.C., and T.N.M. have a provisional patent on the generation of immunogenic nAPCs and methods of use thereof., (© 2021 The Authors.)

Strains of Bacillus thuringiensis (Bt) are commonly commercialized as bioinoculants for insect pest control, but their benefits go beyond their insecticidal property: they can act as plant growth-promoters. Auxins play a major role in the plant growth promotion. However, the mechanism of auxin production by the Bacilli group, and more specifically by Bt strains, is unclear. In previous work, the plant growth-promoting rhizobacterium (PGPR) B. thuringiensis strain RZ2MS9 increased the corn roots. This drew our attention to the strain's auxin production trait, earlier detected in vitro. Here, we demonstrate that in its genome, RZ2MS9 harbours the complete set of genes required in two pathways that are used for Indole acetic acid (IAA) production. We also detected that the strain produces almost five times more IAA during the stationary phase. The bacterial application increased the shoot dry weight of the Micro-Tom (MT) tomato by 24%. The application also modified MT root architecture, with an increase of 26% in the average lateral root length and inhibition of the axial root. At the cellular level, RZ2MS9-treated MT plants presented elongated root cortical cells with intensified mitotic activity. Altogether, these are the best characterized auxin-associated phenotypes. Besides that, no growth alteration was detected in the auxin-insensitive diageotropic (dgt) plants either with or without the RZ2MS9 inoculation. Our results suggest that auxins play an important role in the ability of B. thuringiensis RZ2MS9 to promote MT growth and provide a better understanding of the auxin production mechanism by a Bt strain., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Evidence from natural systems suggests that hybridization between animal species is more common than traditionally thought, but the overall contribution of introgression to standing genetic variation within species remains unclear for most animal systems. Here, we use targeted exon capture to sequence thousands of nuclear loci and complete mitochondrial genomes from closely related chipmunk species in the Tamias quadrivittatus group that are distributed across the Great Basin and the central and southern Rocky Mountains of North America. This recent radiation includes six overlapping, ecologically distinct species (Tamias canipes, Tamias cinereicollis, Tamias dorsalis, T. quadrivittatus, Tamias rufus, and Tamias umbrinus) that show evidence for widespread introgression across species boundaries. Such evidence has historically been derived from a handful of markers, typically focused on mitochondrial loci, to describe patterns of introgression; consequently, the extent of introgression of nuclear genes is less well characterized. We conducted a series of phylogenomic and species-tree analyses to resolve the phylogeny of six species in this group. In addition, we performed several population-genomic analyses to characterize nuclear genomes and infer coancestry among individuals. Furthermore, we used emerging quartets-based approaches to simultaneously infer the species tree (SVDquartets) and identify introgression (HyDe). We found that, in spite of rampant introgression of mitochondrial genomes between some species pairs (and sometimes involving up to three species), there appears to be little to no evidence for nuclear introgression. These findings mirror other genomic results where complete mitochondrial capture has occurred between chipmunk species in the absence of appreciable nuclear gene flow. The underlying causes of recurrent massive cytonuclear discordance remain unresolved in this group but mitochondrial DNA appears highly misleading of population histories as a whole. Collectively, it appears that chipmunk species boundaries are largely impermeable to nuclear gene flow and that hybridization, while pervasive with respect to mtDNA, has likely played a relatively minor role in the evolutionary history of this group. [Cytonuclear discordance; hyridization; introgression, phylogenomics; SVDquartets; Tamias.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background & Aims: The pathogenesis of Wilson disease (WD) involves hepatic and brain copper accumulation resulting from pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior methylome investigations in human WD liver and blood and in the Jackson Laboratory (Bar Harbor, ME) C3He-Atp7b tx-j /J (tx-j) WD mouse model revealed an epigenetic signature of WD, including changes in histone deacetylase (HDAC) 5. We tested the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD., Methods: We investigated class IIa HDAC4 and HDAC5 and H3K9/H3K27 histone acetylation in tx-j mouse livers compared with C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet, D-penicillamine (copper chelator), and choline (methyl group donor). Experiments with copper-loaded hepatoma G2 cells were conducted to validate in vivo studies., Results: In 9-week tx-j mice, HDAC5 levels increased significantly after 8 days of a 60% kcal fat diet compared with chow. In 24-week tx-j mice, HDAC4/5 levels were reduced 5- to 10-fold compared with C3H, likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were affected by disease progression and accompanied by increased acetylation. D-penicillamine and choline partially restored HDAC4/5 and H3K9ac/H3K27ac to C3H levels. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development, which, in turn, were regulated by histone acetylation in tx-j mice compared with C3H mice, with Pparα and Pparγ among the most relevant targets., Conclusions: These results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation can regulate gene expression in key metabolic pathways in the pathogenesis of WD., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Naturally occurring disease in pet dogs is an untapped and unique resource for stem cell-based regenerative medicine translational research, given the many similarities and complexity such disease shares with their human counterparts. Canine-specific regulators of somatic cell reprogramming and pluripotency maintenance are poorly understood. While retroviral delivery of the four Yamanaka factors successfully reprogrammed canine embryonic fibroblasts, adult stromal cells remained resistant to reprogramming in spite of effective viral transduction and transgene expression. We hypothesized that adult stromal cells fail to reprogram due to an epigenetic barrier. Here, we performed assay for transposase-accessible chromatin using sequencing (ATAC-seq) on canine stromal and pluripotent stem cells, analyzing 51 samples in total, and establishing the global landscape of chromatin accessibility before and after reprogramming to induced pluripotent stem cells (iPSC). We also studied adult stromal cells that do not yield iPSC colonies to identify potential reprogramming barriers. ATAC-seq analysis identified distinct cell type clustering patterns and chromatin remodeling during embryonic fibroblast reprogramming. Compared with embryonic fibroblasts, adult stromal cells had a chromatin accessibility landscape that reflects phenotypic differentiation and somatic cell-fate stability. We ultimately identified 76 candidate genes and several transcription factor binding motifs that may be impeding somatic cell reprogramming to iPSC, and could be targeted for inhibition or activation, in order to improve the process in canines. These results provide a vast resource for better understanding of pluripotency regulators in dogs and provide an unbiased rationale for novel canine-specific reprogramming approaches., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Among people of European descent, the ability to digest lactose into adulthood arose via strong positive selection of a highly advantageous allele encompassing the lactase gene. Lactose-tolerant and intolerant individuals may have different disease risks due to the shared genetics of their haplotype block. Therefore, the overall objective of the study was to assess the genetic association of the lactase persistence haplotype to disease risk. Using data from the 1000Genomes project, we estimated the size of the lactase persistence haplotype block to be 1.9 Mbp containing up to 9 protein-coding genes and a microRNA. Based on the function of the genes and microRNA, we studied health phenotypes likely to be impacted by the lactase persistence allele: prostate cancer status, cardiovascular disease status, and bone mineral density. We used summary statistics from large genome-wide metanalyses-32,965 bone mineral density, 140,306 prostate cancer and 184,305 coronary artery disease subjects-to evaluate whether the lactase persistence allele was associated with these disease phenotypes. Despite the fact that previous work demonstrated that the lactase persistence haplotype block harbors increased deleterious mutations, these results suggest little effect on the studied disease phenotypes., (Copyright © 2020 Joslin, Durbin-Johnson, Britton, Settles, Korf and Lemay.)

In humans, abnormal thickening of the left ventricle of the heart clinically defines hypertrophic cardiomyopathy (HCM), a common inherited cardiovascular disorder that can precede a sudden cardiac death event. The wide range of clinical presentations in HCM obscures genetic variants that may influence an individual's susceptibility to sudden cardiac death. Although exon sequencing of major sarcomere genes can be used to detect high-impact causal mutations, this strategy is successful in only half of patient cases. The incidence of left ventricular hypertrophy (LVH) in a managed research colony of rhesus macaques provides an excellent comparative model in which to explore the genomic etiology of severe HCM and sudden cardiac death. Because no rhesus HCM-associated mutations have been reported, we used a next-generation genotyping assay that targets 7 sarcomeric rhesus genes within 63 genomic sites that are orthologous to human genomic regions known to harbor HCM disease variants. Amplicon sequencing was performed on 52 macaques with confirmed LVH and 42 unrelated, unaffected animals representing both the Indian and Chinese rhesus macaque subspecies. Bias-reduced logistic regression uncovered a risk haplotype in the rhesus MYBPC3 gene, which is frequently disrupted in both human and feline HCM; this haplotype implicates an intronic variant strongly associated with disease in either homozygous or carrier form. Our results highlight that leveraging evolutionary genomic data provides a unique, practical strategy for minimizing population bias in complex disease studies.

Chinook salmon ( Oncorhynchus tshawytscha ) declines are widespread and may be attributed, at least in part, to warming river temperatures. Water temperatures in the Yukon River and tributaries often exceed 18°C, a threshold commonly associated with heat stress and elevated mortality in Pacific salmon. Untangling the complex web of direct and indirect physiological effects of heat stress on salmon is difficult in a natural setting with innumerable system challenges but is necessary to increase our understanding of both lethal and sublethal impacts of heat stress on populations. The goal of this study was to characterize the cellular stress response in multiple Chinook salmon tissues after acute elevated temperature challenges. We conducted a controlled 4-hour temperature exposure (control, 18°C and 21°C) experiment on the bank of the Yukon River followed by gene expression (GE) profiling using a 3'-Tag-RNA-Seq protocol. The full transcriptome was analysed for 22 Chinook salmon in muscle, gill and liver tissue. Both the 21°C and 18°C treatments induced greater activity in genes associated with protein folding (e.g. HSP70, HSP90 mRNA) processes in all tissues. Global GE patterns indicate that transcriptomic responses to heat stress were highly tissue-specific, underscoring the importance of analyzing multiple tissues for determination of physiological effect. Primary superclusters (i.e. groupings of loosely related terms) of altered biological processes were identified in each tissue type, including regulation of DNA damage response (gill), regulation by host of viral transcription (liver) and regulation of the force of heart contraction (muscle) in the 21°C treatment. This study provides insight into mechanisms potentially affecting adult Chinook salmon as they encounter warm water during their spawning migration in the Yukon River and suggests that both basic and more specialized cellular functions may be disrupted., (Published by Oxford University Press 2020.)

Here, we report the complete genome sequence of Bacillus sp. strain RZ2MS9, a plant growth-promoting bacterium isolated from the rhizosphere of guarana, a native crop from Amazonas, Brazil. The assembled genome comprises 5.35 Mbp, no plasmids, and a GC content of 35.22%., (Copyright © 2020 Bonatelli et al.)

Over the last decade, the cost of -omics data creation has decreased 10-fold, whereas the need for analytical support for those data has increased exponentially. Consequently, bioinformaticians face a second wave of challenges: novel applications of existing approaches ( e.g. , single-cell RNA sequencing), integration of -omics data sets of differing size and scale ( e.g. , spatial transcriptomics), as well as novel computational and statistical methods, all of which require more sophisticated pipelines and data management. Nonetheless, bioinformatics cores are often asked to operate under primarily a cost-recovery model, with limited institutional support. Seeing the need to assess bioinformatics core operations, the Association of Biomolecular Resource Facilities Genomics Bioinformatics Research Group conducted a survey to answer questions about staffing, services, financial models, and challenges to better understand the challenges bioinformatics core facilities are currently faced with and will need to address going forward. Of the respondent groups, we chose to focus on the survey data from smaller cores, which made up the majority. Although all cores indicated similar challenges in terms of changing technologies and analysis needs, small cores tended to have the added challenge of funding their operations largely through cost-recovery models with heavy administrative burdens., (© Association of Biomolecular Resource Facilities.)

Background: The proximal femoral head separation (FHS) or epiphysiolysis is a prevalent disorder affecting the chicken femur epiphysis, being considered a risk factor to infection which can cause bacterial chondronecrosis with osteomyelitis in broilers. To identify the genetic mechanisms involved in epiphysiolysis, differentially expressed (DE) genes in the femur of normal and FHS-affected broilers were identified using RNA-Seq technology. Femoral growth plate (GP) samples from 35-day-old commercial male broilers were collected from 4 healthy and 4 FHS-affected broilers. Sequencing was performed using an Illumina paired-end protocol. Differentially expressed genes were obtained using the edgeR package based on the False Discovery Rate (FDR < 0.05)., Results: Approximately 16 million reads/sample were generated with 2 × 100 bp paired-end reads. After data quality control, approximately 12 million reads/sample were mapped to the reference chicken genome (Galgal5). A total of 12,645 genes were expressed in the femur GP. Out of those, 314 were DE between groups, being 154 upregulated and 160 downregulated in FHS-affected broilers. In the functional analyses, several biological processes (BP) were overrepresented. Among them, those related to cell adhesion, extracellular matrix (ECM), bone development, blood circulation and lipid metabolism, which are more related to chicken growth, are possibly involved with the onset of FHS. On the other hand, BP associated to apoptosis or cell death and immune response, which were also found in our study, could be related to the consequence of the FHS., Conclusions: Genes with potential role in the epiphysiolysis were identified through the femur head transcriptome analysis, providing a better understanding of the mechanisms that regulate bone development in fast-growing chickens. In this study, we highlighted the importance of cell adhesion and extracellular matrix related genes in triggering FHS. Furthermore, we have shown new insights on the involvement of lipidemia and immune response/inflammation with FHS in broilers. Understanding the changes in the GP transcriptome might support breeding strategies to address poultry robustness and to obtain more resilient broilers.

Squamous cell carcinoma (SCC) is the most common periocular cancer in horses and the second most common tumor of the horse overall. A missense mutation in damage-specific DNA-binding protein 2 ( DDB2 , c.1012 C > T , p.Thr338Met) was previously found to be strongly associated with ocular SCC in Haflinger and Belgian horses, explaining 76% of cases across both breeds. To determine if this same variant in DDB2 contributes to risk for ocular SCC in the Arabian, Appaloosa, and Percheron breeds and to determine if the variant contributes to risk for oral or urogenital SCC, histologically confirmed SCC cases were genotyped for the DDB2 variant and associations were investigated. Horses with urogenital SCC that were heterozygous for the DDB2 risk allele were identified in the Appaloosa breed, but a significant association between the DDB2 variant and SCC occurring at any location in this breed was not detected. The risk allele was not identified in Arabians, and no Percherons were homozygous for the risk allele. High-throughput sequencing data from six Haflingers were analyzed to ascertain if any other variant from the previously associated 483 kb locus on ECA12 was more concordant with the SCC phenotype than the DDB2 variant. Sixty polymorphisms were prioritized for evaluation, and no other variant from this locus explained the genetic risk better than the DDB2 allele ( P = 3.39 × 10 -17 , n = 118). These data provide further support of the DDB2 variant contributing to risk for ocular SCC, specifically in the Haflinger and Belgian breeds., Competing Interests: Rebecca R. Bellone and Zachary T. Lounsberry are affiliated with the Veterinary Genetics Laboratory, which offers genetic diagnostic testing in horses., (Copyright © 2019 Moriel H. Singer-Berk et al.)

Amplification, sequencing, and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300-bp paired-end reads of higher quality than those produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-step PCR amplification protocol is also described that allows for targeting of different amplicon regions, and enhances amplification success from samples with low bacterial bioburden. IMPORTANCE Amplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high-throughput sequencing have made it a widely adopted approach, especially for projects that necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per-sample cost relative to those of the Illumina MiSeq platform without sacrificing amplicon length. To make this method more flexible for various amplicon-targeted regions as well as improve amplification from low-biomass samples, we also present and validate a 2-step PCR library preparation method.

White striping (WS) is one of the most common myopathies identified in broiler chickens leading to substantial production losses, where the incidence reaches 12% in commercial chickens. It occurs primarily in heavier chickens being a modification of the breast muscle characterized by the presence of pale parallel streaks in the same orientation of the muscle fibers. Since the WS etiology remains unclear, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of WS in affected and unaffected chicken breast muscles. A total of 11,177 genes were expressed in the pectoralis major muscle. Out of those, 1,441 genes were differentially expressed (FDR ≤ 0.01) between the two analyzed groups, being, respectively, 772 genes upregulated and 669 downregulated in the WS affected group. A total of 36 significantly overrepresented GO terms related to WS myopathy were enriched, and the most relevant biological processes were activation of immune system, angiogenesis, hypoxia, cell death, and striated muscle contraction. The unbalance of those biological processes may trigger the occurrence of the WS phenotype in broilers. The possible lack of capillary blood supply homogeneously in the muscle triggers the hypoxia, following the activation of glycolysis, calcium signaling and apoptosis related genes facilitating the tissue damage and WS incidence., (© 2018 Poultry Science Association Inc.)

The application of RNA sequencing in commercial poultry could facilitate a novel approach toward food safety with respect to identifying conditions in food production that mitigate transcription of genes associated with virulence and survivability. In this study, we evaluated the effects of disinfectant exposure on the transcriptomes of two field isolates of Salmonella Heidelberg (SH) isolated from a commercial broiler processing plant in 1992 and 2014. The isolates were each exposed separately to the following disinfectants commonly used in poultry processing: cetylpyridinium chloride (CPC), acidified calcium hypochlorite (aCH), and peroxyacetic acid (PAA). Exposure times were 8 s with CPC to simulate a poultry processing dipping station or 90 min with aCH and PAA to simulate the chiller tank in a poultry processing plant at 4°C. Based on comparison with a publicly available annotated SH reference genome with 5,088 genes, 90 genes were identified as associated with virulence, pathogenicity, and resistance (VPR). Of these 90 VPR genes, 9 (10.0%), 28 (31.1%), and 1 (1.1%) gene were upregulated in SH 2014 and 21 (23.3%), 26 (28.9%), and 2 (2.2%) genes were upregulated in SH 2014 challenged with CPC, aCH, and PAA, respectively. This information and previously reported MICs for the three disinfectants with both SH isolates allow researchers to make more accurate recommendations regarding control methods of SH and public health considerations related to SH in food production facilities where SH has been isolated. For example, the MICs revealed that aCH is ineffective for SH inhibition at regulatory levels allowed for poultry processing and that aCH was ineffective for inhibiting SH growth and caused an upregulation of VPR genes.

Female parenting is obligate in mammals, but fathering behavior among mammals is rare. Only 3-5% of mammalian species exhibit biparental care, including humans, and mechanisms of fathering behavior remain sparsely studied. However, in species where it does exist, paternal care is often crucial to the survivorship of offspring. The present study is the first to identify new gene targets linked to the experience of fathering behavior in a biparental species using RNA sequencing. In order to determine the pattern of gene expression within the medial preoptic area that is specifically associated with fathering behavior, we identified genes in male prairie voles ( Microtus ochrogaster) that experienced one of three social conditions: virgin males, pair bonded males, and males with fathering experience. A list of genes exhibiting different expression patterns in each comparison (i.e. Virgin vs Paired, Virgin vs Fathers, and Paired vs Fathers) was evaluated using the gene ontology enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes pathways analysis to reveal metabolic pathways associated with specific genes. Using these tools, we generated a filtered list of genes that exhibited altered patterns of expression in voles with different amounts of social experience. Finally, we used NanoString to quantify differences in the expression of these selected genes. These genes are involved in a variety of processes, with enrichment in genes associated with immune function, metabolism, synaptic plasticity, and the remodeling of dendritic spines. The identification of these genes and processes will lead to novel insights into the biological basis of fathering behavior.

Dysregulated bile acid (BA) synthesis is accompanied by dysbiosis, leading to compromised metabolism. This study analyzes the effect of epigallocatechin-3-gallate (EGCG) on diet-induced obesity through regulation of BA signaling and gut microbiota. The data revealed that EGCG effectively reduced diet-increased obesity, visceral fat, and insulin resistance. Gene profiling data showed that EGCG had a significant impact on regulating genes implicated in fatty acid uptake, adipogenesis, and metabolism in the adipose tissue. In addition, metabolomics analysis revealed that EGCG altered the lipid and sugar metabolic pathways. In the intestine, EGCG reduced the FXR agonist chenodeoxycholic acid, as well as the FXR-regulated pathway, suggesting intestinal FXR deactivation. However, in the liver, EGCG increased the concentration of FXR and TGR-5 agonists and their regulated signaling. Furthermore, our data suggested that EGCG activated Takeda G protein receptor (TGR)-5 based on increased GLP-1 release and elevated serum PYY level. EGCG and antibiotics had distinct antibacterial effects. They also differentially altered body weight and BA composition. EGCG, but not antibiotics, increased Verrucomicrobiaceae, under which EGCG promoted intestinal bloom of Akkermansia muciniphila. Excitingly, A. muciniphila was as effective as EGCG in treating diet-induced obesity. Together, EGCG shifts gut microbiota and regulates BA signaling thereby having a metabolic beneficial effect.-Sheng, L., Jena, P. K., Liu, H.-X., Hu, Y., Nagar, N., Bronner, D. N., Settles, M. L., Bäumler, A. J. Wan, Y.-J. Y. Obesity treatment by epigallocatechin-3-gallate-regulated bile acid signaling and its enriched Akkermansia muciniphila.

Shiga toxin-producing Escherichia coli (STEC) bacteria are zoonotic pathogens. We report here the high-quality complete genome sequences of three STEC O177:H- ( fliC H25 ) strains, SMN152SH1, SMN013SH2, and SMN197SH3. The assembled genomes consisted of one optical map-verified circular chromosome for each strain, plus two plasmids for SMN013SH2 and three plasmids for SMN152SH1 and SMN197SH3, respectively., (Copyright © 2018 Sheng et al.)

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures have advanced our understanding of Bd phylogenetics, genomic architecture and mechanisms of virulence. However, pure cultures are laborious to obtain and whole-genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus, we still know little about the genetic diversity of Bd in natural systems. The most common noninvasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field-collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole-genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, noninvasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations., (© 2017 John Wiley & Sons Ltd.)

Background: The human milk microbiome has been somewhat characterized, but little is known about changes over time and relations with maternal factors such as nutrient intake. Objective: We sought to characterize the human milk microbiome and described associations with maternal nutrient intake, time postpartum, delivery mode, and body mass index (BMI; in kg/m 2 ). Methods: Milk samples ( n = 104) and 24-h diet recalls were collected 9 times from 21 healthy lactating women from day 2 to 6 mo postpartum. Women were classified by BMI as healthy weight (<25) or overweight or obese (≥25). Bacterial taxa were characterized with the use of the high-throughput sequencing of the 16S ribosomal RNA gene. Results: The milk microbiome was relatively constant over time, although there were small changes in some of the lesser-abundant genera. Relative abundances of several taxa were associated with BMI, delivery mode, and infant sex. For instance, overweight and obese mothers produced milk with a higher relative abundance of Granulicatella than did healthy-weight women (1.8% ± 0.6% compared with 0.4% ± 0.2%, respectively; P < 0.05). Relative abundances of several bacterial taxa were also associated with variations in maternal dietary intake. For example, intakes of saturated fatty acids ( r s = -0.59; P = 0.005) and monounsaturated fatty acids ( r s = -0.46; P = 0.036) were inversely associated with the relative abundance of Corynebacterium ; total carbohydrates ( r s = -0.54; P = 0.011), disaccharides ( r s = -0.47; P = 0.031), and lactose ( r s = -0.51; P = 0.018) were negatively associated with Firmicutes; and protein consumption was positively correlated with the relative abundance of Gemella ( r s = 0.46; P = 0.037). Conclusions: Factors associated with variations in the human milk microbiome are complex and may include maternal nutrient intake, maternal BMI, delivery mode, and infant sex. Future studies designed to investigate the relation between maternal nutrient intake and the milk microbiome should strive to also evaluate dietary supplement usage and analyze the collected milk for its nutrient content., Competing Interests: Author disclosures: JEW, JMC, KAL, NFB, MAY, SLB, BS, WJP, MLS, MAM, and MKM, no conflicts of interest.

Most of the largest vertebrate genomes are found in salamanders, a clade of amphibians that includes 686 species. Salamander genomes range in size from 14 to 120 Gb, reflecting the accumulation of large numbers of transposable element (TE) sequences from all three TE classes. Although DNA loss rates are slow in salamanders relative to other vertebrates, high levels of TE insertion are also likely required to explain such high TE loads. Across the Tree of Life, novel TE insertions are suppressed by several pathways involving small RNA molecules. In most known animals, TE activity in the germline is primarily regulated by the Piwi-interacting RNA (piRNA) pathway. In this study, we test the hypothesis that salamanders' unusually high TE loads reflect the loss of the ancestral piRNA-mediated TE-silencing machinery. We characterized the small RNA pool in the female and male adult gonads, testing for the presence of small RNA molecules that bear the characteristics of TE-targeting piRNAs. We also analyzed the amino acid sequences of piRNA pathway proteins from salamanders and other vertebrates, testing whether the overall patterns of sequence divergence are consistent with conserved pathway function across the vertebrate clade. Our results do not support the hypothesis of piRNA pathway loss; instead, they suggest that the piRNA pathway is expressed in salamanders. Given these results, we propose hypotheses to explain how the extraordinary TE loads in salamander genomes could have accumulated, despite the expression of TE-silencing machinery.

The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or--more importantly--recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300 bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500 bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500 bp for 349 samples.

We report here the full mitochondrial genome sequence of Limonius californicus, a species of click beetle that is an agricultural pest in its larval form. The circular genome is 16.5 kb and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes., (Copyright © 2016 Gerritsen et al.)

We report herein the draft mitochondrial genome sequence of Naesiotus nux, a Galápagos endemic land snail species of the genus Naesiotus. The circular genome is 15 kb and encodes 13 protein-coding genes, 2 rRNA genes, and 21 tRNA genes., (Copyright © 2016 Hunter et al.)

Understanding the process of adaptation during rapid environmental change remains one of the central focal points of evolutionary biology. The recently formed White Sands system of southern New Mexico offers an outstanding example of rapid adaptation, with a variety of species having rapidly evolved blanched forms on the dunes that contrast with their close relatives in the surrounding dark soil habitat. In this study, we focus on two of the White Sands lizard species, Sceloporus cowlesi and Aspidoscelis inornata, for which previous research has linked mutations in the melanocortin-1 receptor gene (Mc1r) to blanched coloration. We sampled populations both on and off the dunes and used a custom sequence capture assay based on probed fosmid libraries to obtain >50 kb of sequence around Mc1r and hundreds of other random genomic locations. We then used model-based statistical inference methods to describe the demographic and adaptive history characterizing the colonization of White Sands. We identified a number of similarities between the two focal species, including strong evidence of selection in the blanched populations in the Mc1r region. We also found important differences between the species, suggesting different colonization times, different genetic architecture underlying the blanched phenotype and different ages of the beneficial alleles. Finally, the beneficial allele is dominant in S. cowlesi and recessive in A. inornata, allowing for a rare empirical test of theoretically expected patterns of selective sweeps under these differing models., (© 2015 John Wiley & Sons Ltd.)

Background: The fecal microbiota has been characterized in some adult populations, but little is known about its community structure during lactation., Objectives: We characterized the maternal fecal microbiome during lactation and explored possible mediating factors such as nutrition., Methods: Fecal samples were collected from 20 lactating women from 2 d to 6 mo postpartum, and bacterial taxa were characterized with the use of high-throughput sequencing. Bacterial community structure (at each taxonomic level) and relations between bacterial taxa and environmental and dietary variables were visualized and analyzed with the use of stacked bar charts, principal component analysis, and multivariate analyses such as nonmetric multidimensional scaling and canonical correlation analysis., Results: Complex bacterial community structure was somewhat similar to those previously published for other adult populations (although there were some notable differences), and there were no clear associations with time postpartum or anthropometric or environmental variables. However, Spearman rank correlations suggested that increased intake of pantothenic acid, riboflavin, vitamin B-6, and vitamin B-12 were related to increased relative abundance of Prevotella (r = 0.45, 0.39, 0.34, and 0.24, respectively; P ≤ 0.01) and decreased relative abundance of Bacteroides (r = -0.55, -0.46, -0.32, and -0.35, respectively; P ≤ 0.01). Intakes of copper, magnesium, manganese, and molybdenum were positively associated with Firmicutes (r = 0.33, 0.38, 0.44, and 0.51, respectively; P ≤ 0.01) and negatively associated with Bacteroidetes (r = -0.38, -0.44, -0.48, and -0.53, respectively; P ≤ 0.01). Overall, data consistently suggest that increased consumption of a more nutrient- and calorie-rich diet was positively associated with relative abundance of Firmicutes., Conclusions: The fecal microbiome of lactating women is relatively stable in the postpartum period and somewhat similar to that of other adult populations. Variation in dietary constituents may be related to that of relative abundance of individual bacterial taxa. Controlled dietary intervention studies will be required to determine whether these associations are causal in nature., (© 2015 American Society for Nutrition.)

Although vaginal microbial communities of some healthy women have high proportions of Atopobium vaginae, the genus Atopobium is more commonly associated with bacterial vaginosis, a syndrome associated with an increased risk of adverse pregnancy outcomes and the transmission of sexually transmitted diseases. Genetic differences within Atopobium species may explain why single species can be associated with both health and disease. We used 16S rRNA gene sequences from previously published studies to explore the taxonomic diversity of the genus Atopobium in vaginal microbial communities of healthy women. Although A. vaginae was the species most commonly found, we also observed three other Atopobium species in the vaginal microbiota, one of which, A. parvulum, was not previously known to reside in the human vagina. Furthermore, we found several potential novel species of the genus Atopobium and multiple phylogenetic clades of A. vaginae. The diversity of Atopobium found in our study, which focused only on samples from healthy women, is greater than previously recognized, suggesting that analysis of samples from women with BV would yield even more diversity. Classification of microbes only to the genus level may thus obfuscate differences that might be important to better understand health or disease., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We report the draft genome sequence of Pseudomonas sp. nov. H2, isolated from creek sediment in Moscow, ID, USA. The strain is most closely related to Pseudomonas putida. However, it has a slightly smaller genome that appears to have been impacted by horizontal gene transfer and poorly maintains IncP-1 plasmids., (Copyright © 2015 Loftie-Eaton et al.)

Unlabelled: Puberty is an important developmental stage wherein hormonal shifts mediate the physical and physiological changes that lead to menarche, but until now, the bacterial composition of vaginal microbiota during this period has been poorly characterized. We performed a prospective longitudinal study of perimenarcheal girls to gain insight into the timing and sequence of changes that occur in the vaginal and vulvar microbiota during puberty. The study enrolled 31 healthy, premenarcheal girls between the ages of 10 and 12 years and collected vaginal and vulvar swabs quarterly for up to 3 years. Bacterial composition was characterized by Roche 454 pyrosequencing and classification of regions V1 to V3 of 16S rRNA genes. Contrary to expectations, lactic acid bacteria, primarily Lactobacillus spp., were dominant in the microbiota of most girls well before the onset of menarche in the early to middle stages of puberty. Gardnerella vaginalis was detected at appreciable levels in approximately one-third of subjects, a notable finding considering that this organism is commonly associated with bacterial vaginosis in adults. Vulvar microbiota closely resembled vaginal microbiota but often exhibited additional taxa typically associated with skin microbiota. Our findings suggest that the vaginal microbiota of girls begin to resemble those of adults well before the onset of menarche., Importance: This study addresses longitudinal changes in vaginal and vulvar microbial communities prior to and immediately following menarche. The research is significant because microbial ecology of the vagina is an integral aspect of health, including resistance to infections. The physiologic changes of puberty and initiation of cyclic menstruation are likely to have profound effects on vaginal microbiota, but almost nothing is known about changes that normally occur during this time. Our understanding has been especially hampered by the lack of thorough characterization of microbial communities using techniques that do not rely on the cultivation of fastidious bacteria, as well as a dearth of studies on girls in the early to middle stages of puberty. This study improves our understanding of the normal development of vaginal microbiota during puberty and onset of menarche and may better inform clinical approaches to vulvovaginal care of adolescent girls., (Copyright © 2015 Hickey et al.)

Three undefined mixed cultures (activated sludge) from different municipal wastewater treatment plants were used as seeds in a novel lactic acid fermentation process fed with potato peel waste (PPW). Anaerobic sequencing batch fermenters were run under identical conditions to produce predominantly lactic acid. Illumina sequencing was used to examine the 16S rRNA genes of bacteria in the three seeds and fermenters. Results showed that the structure of microbial communities of three seeds were different. All three fermentation products had unique community structures that were dominated (>96%) by species of the genus Lactobacillus, while members of this genus constituted <0.1% in seeds. The species of Lactobacillus sp. differed among the three fermentations. Results of this study suggest the structure of microbial communities in lactic acid fermentation of PPW with undefined mixed cultures were robust and resilient, which provided engineering prospects for the microbial utilization of carbohydrate wastes to produce lactic acid., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the potential of using wood hydrolysates (enzymatically hydrolyzed from hybrid poplar) as substrate to produce polyhydroxybutyrate (PHB) using mixed microbial cultures. The optimal operational conditions for fed-batch bioreactors were 4d solid retention time with an organic loading rate of 2.5g/Ld. The maximum PHB accumulated was 27% of cell dry weight with a yield of 0.32g/g (g PHB produced per g sugars consumed). Microbial community analysis was done at the genus level by 16S rRNA sequencing on an Illumina system and community evolution was observed among different samples and initial seed. Actinobacteria, Alpha- and Beta-proteobacteria were found to be the dominant groups in all the bioreactors. Several PHB-storing microorganisms were characterized belonging to Alpha- and Beta-proteobacteria., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To investigate changes in female genital tract bacterial ecology associated with levonorgestrel intrauterine system (LNG IUS) use., Study Design: Study participants were regularly cycling Caucasian women without recent history of pregnancy or exogenous hormone use. Vaginal, cervical and uterine samples were obtained at nine time intervals, from 1 week before to 12 weeks after LNG IUS placement. Replicate paired vaginal and cervical specimens were collected to determine the consistency of specimen collection and processing., Results: A total of 406 samples from 11 women were available for analysis after DNA extraction, amplification and species identification. Of the 355 bacterial species or genera detected, Lactobacillus crispatus was most prevalent representing 48.9% of over 6 million total reads. L. crispatus reads of replicate vaginal samples were not significantly different [odds ratio (OR) 0.79, 0.36-1.73]. In addition, L. crispatus reads of vaginal and cervical samples from the same visit were not significantly different (OR 0.69, 0.31-1.51). Compared to sampling visits prior to LNG IUS placement, sampling visits after LNG IUS insertion were more likely to have L. crispatus reads greater than 50% of total reads (OR 2.13, 1.01-4.48). Of the 63 uterine samples demonstrating bacteria, Burkholderia genus proteobacteria, a common environmental contaminant, were most prevalent both before and after LNG IUS insertion accounting for 48.0% of all uterine sample reads., Conclusion: The vaginal microbiome changes very little in response to LNG IUS placement. Further study is needed to place this finding in context with clinical outcomes., Implications: There do not appear to be any clinically important differences in the vaginal microbiome in response to insertion of the LNG IUS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Exposure to environmental estrogens (xenoestrogens) may play a causal role in the increased breast cancer incidence which has been observed in Europe and the US over the last 50 years. The xenoestrogen bisphenol A (BPA) leaches from plastic food/beverage containers and dental materials. Fetal exposure to BPA induces preneoplastic and neoplastic lesions in the adult rat mammary gland. Previous results suggest that BPA acts through the estrogen receptors which are detected exclusively in the mesenchyme during the exposure period by directly altering gene expression, leading to alterations of the reciprocal interactions between mesenchyme and epithelium. This initiates a long sequence of altered morphogenetic events leading to neoplastic transformation. Additionally, BPA induces epigenetic changes in some tissues. To explore this mechanism in the mammary gland, Wistar-Furth rats were exposed subcutaneously via osmotic pumps to vehicle or 250 µg BPA/kg BW/day, a dose that induced ductal carcinomas in situ. Females exposed from gestational day 9 to postnatal day (PND) 1 were sacrificed at PND4, PND21 and at first estrus after PND50. Genomic DNA (gDNA) was isolated from the mammary tissue and immuno-precipitated using anti-5-methylcytosine antibodies. Detection and quantification of gDNA methylation status using the Nimblegen ChIP array revealed 7412 differentially methylated gDNA segments (out of 58207 segments), with the majority of changes occurring at PND21. Transcriptomal analysis revealed that the majority of gene expression differences between BPA- and vehicle-treated animals were observed later (PND50). BPA exposure resulted in higher levels of pro-activation histone H3K4 trimethylation at the transcriptional initiation site of the alpha-lactalbumin gene at PND4, concomitantly enhancing mRNA expression of this gene. These results show that fetal BPA exposure triggers changes in the postnatal and adult mammary gland epigenome and alters gene expression patterns. These events may contribute to the development of pre-neoplastic and neoplastic lesions that manifest during adulthood.

Conditions during fetal development influence health and disease in adulthood, especially during critical windows of organogenesis. Fetal exposure to the endocrine disrupting chemical, bisphenol A (BPA) affects the development of multiple organ systems in rodents and monkeys. However, effects of BPA exposure on cardiac development have not been assessed. With evidence that maternal BPA is transplacentally delivered to the developing fetus, it becomes imperative to examine the physiological consequences of gestational exposure during primate development. Herein, we evaluate the effects of daily, oral BPA exposure of pregnant rhesus monkeys (Macaca mulatta) on the fetal heart transcriptome. Pregnant monkeys were given daily oral doses (400 µg/kg body weight) of BPA during early (50-100 ± 2 days post conception, dpc) or late (100 ± 2 dpc--term), gestation. At the end of treatment, fetal heart tissues were collected and chamber specific transcriptome expression was assessed using genome-wide microarray. Quantitative real-time PCR was conducted on select genes and ventricular tissue glycogen content was quantified. Our results show that BPA exposure alters transcription of genes that are recognized for their role in cardiac pathophysiologies. Importantly, myosin heavy chain, cardiac isoform alpha (Myh6) was down-regulated in the left ventricle, and 'A Disintegrin and Metalloprotease 12', long isoform (Adam12-l) was up-regulated in both ventricles, and the right atrium of the heart in BPA exposed fetuses. BPA induced alteration of these genes supports the hypothesis that exposure to BPA during fetal development may impact cardiovascular fitness. Our results intensify concerns about the role of BPA in the genesis of human metabolic and cardiovascular diseases.

We report the draft genome sequence of Pseudomonas moraviensis R28-S, isolated from the municipal wastewater treatment plant of Moscow, ID. The strain carries a native mercury resistance plasmid, poorly maintains introduced IncP-1 antibiotic resistance plasmids, and has been useful for studying the evolution of plasmid host range and stability.

Objective: To explore potential causes of male infertility by determining the composition and structure of commensal bacterial communities in seminal fluids., Design: Microscopy of Gram-stained semen samples and classification of 16S rRNA gene sequences to determine the species composition of semen bacterial communities., Setting: Clinical andrology laboratory and academic research laboratories., Patient(s): Nineteen sperm donors and 58 infertility patients., Intervention(s): None., Main Outcome Measure(s): Classification of 16S rRNA gene sequences, clustering of seminal microbial communities, and multiple statistical tests., Result(s): High numbers of diverse kinds of bacteria were present in most samples of both sperm donors and infertility patients. The bacterial communities varied widely among subjects, but they could be clustered into six groups based on similarities in composition and the rank abundances of taxa. Overall, there were no significant differences between sperm donors and infertility patients. However, multiple statistical tests showed a significant negative association between sperm quality and the presence of Anaerococcus. The results also indicated that many of the bacterial taxa identified in semen also occur in the vaginal communities of some women, especially those with bacterial vaginosis, which suggests that heterosexual sex partners may share bacteria., Conclusion(s): Diverse kinds of bacteria were present in the human semen, but there were no significant differences between sperm donors and infertility patients. The presence of Anaerococcus might be a biomarker for low sperm quality., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

With the advent of Next-Generation (NG) sequencing, it has become possible to sequence a entire genomes quickly and inexpensively. However, in some experiments one only needs to extract and assembly a portion of the sequence reads, for example when performing transcriptome studies, sequencing mitochondrial genomes, or characterizing exomes. With the raw DNA-library of a complete genome it would appear to be a trivial problem to identify reads of interest. But it is not always easy to incorporate well-known tools such as BLAST, BLAT, Bowtie, and SOAP directly into a bioinformatics pipelines before the assembly stage, either due to incompatibility with the assembler's file inputs, or because it is desirable to incorporate information that must be extracted separately. For example, in order to incorporate flowgrams from a Roche 454 sequencer into the Newbler assembler it is necessary to first extract them from the original SFF files. We present SlopMap, a bioinformatics software utility that allows quickly identification similar to the provided reference reads from either Roche 454 or Illumnia DNA library. With simple and intuitive command-line interface along with file output formats compatible to assembly programs, SlopMap can be directly embedded to biological data processing pipeline without any additional programming work. In addition, SlopMap preserves flowgram information needed for Roche 454 assembler.

The potential benefits of selenium (Se) supplementation are currently under investigation for prevention of certain cancers and treatment of neurological disorders. However, little is known concerning the response of the brain to increased dietary Se under conditions of Se sufficiency, despite the majority of Se supplementation trials occurring in healthy, Se sufficient subjects. We evaluated the transcriptional response of Se-dependent genes, selenoproteins and the genes necessary for their synthesis (the selenoproteome), in the zebrafish (Danio rerio) brain to supplementation with nutritionally relevant levels of dietary Se (sodium selenite) during conditions of assumed Se sufficiency. We first used a microarray approach to analyze the response of the brain selenoproteome to dietary Se supplementation for 14 days and then assessed the immediacy and time-scale transcriptional response of the brain selenoproteome to 1, 7, and 14 days of Se supplementation by quantitative real-time PCR (qRT-PCR). The microarray approach did not indicate large-scale influences of Se on the brain transcriptome as a whole or the selenoproteome specifically; only one nonselenoproteome gene (si:ch73-44m9.2) was significantly differentially expressed. Our qRT-PCR results, however, indicate that increases of dietary Se cause small, but significant transcriptional changes within the brain selenoproteome, even after only 1 day of supplementation. These responses were dynamic over a short period of supplementation in a manner highly dependent on sex and the duration of Se supplementation. In nutritional intervention studies, it may be necessary to utilize methods such as qRT-PCR, which allow larger sample sizes, for detecting subtle transcriptional changes in the brain.