The microRNA miR-133b functions to slow Duchenne muscular dystrophy pathogenesis.

KEY POINTS: Impairment of muscle biogenesis contributes to the progression of Duchenne muscular dystrophy (DMD). As a muscle enriched microRNA that has been implicated in muscle biogenesis, the role of miR-133b in DMD remains unknown. To assess miR-133b function in DMD-affected skeletal muscles, we genetically ablated miR-133b in the mdx mouse model of DMD. We show that deletion of miR-133b exacerbates the dystrophic phenotype of DMD-afflicted skeletal muscle by dysregulating muscle stem cells involved in muscle biogenesis, in addition to affecting signalling pathways related to inflammation and fibrosis. Our results provide evidence that miR-133b may underlie DMD pathology by affecting the proliferation and differentiation of muscle stem cells. ABSTRACT: Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle degeneration. No treatments are currently available to prevent the disease. While the muscle enriched microRNA miR-133b has been implicated in muscle biogenesis, its role in DMD remains unknown. To assess miR-133b function in DMD-affected skeletal muscles, we genetically ablated miR-133b in the mdx mouse model of DMD. In the absence of miR-133b, the tibialis anterior muscle of P30 mdx mice is smaller in size and exhibits a thickened interstitial space containing more mononucleated cells. Additional analysis revealed that miR-133b deletion influences muscle fibre regeneration, satellite cell proliferation and differentiation, and induces widespread transcriptomic changes in mdx muscle. These include known miR-133b targets as well as genes involved in cell proliferation and fibrosis. Altogether, our data demonstrate that skeletal muscles utilize miR-133b to mitigate the deleterious effects of DMD.

Genome-wide identification of enhancers and transcription factors regulating the myogenic differentiation of bovine satellite cells.

BACKGROUND: Satellite cells are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells into myotubes. RESULTS: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to identify genomic regions where lysine 27 of H3 histone is acetylated (H3K27ac), i.e., active enhancers, from bovine satellite cells before and during differentiation into myotubes. A total of 19,027 and 47,669 H3K27ac-marked enhancers were consistently identified from two biological replicates of before- and during-differentiation bovine satellite cells, respectively. Of these enhancers, 5882 were specific to before-differentiation, 35,723 to during-differentiation, and 13,199 common to before- and during-differentiation bovine satellite cells. Whereas most of the before- or during-differentiation-specific H3K27ac-marked enhancers were located distally to the transcription start site, the enhancers common to before- and during-differentiation were located both distally and proximally to the transcription start site. The three sets of H3K27ac-marked enhancers were associated with functionally different genes and enriched with different transcription factor binding sites. Specifically, many of the H3K27ac-marked enhancers specific to during-differentiation bovine satellite cells were associated with genes involved in muscle structure and development, and were enriched with binding sites for the MyoD, AP-1, KLF, TEAD, and MEF2 families of transcription factors. A positive role was validated for Fos and FosB, two AP-1 family transcription factors, in the differentiation of bovine satellite cells into myotubes by siRNA-mediated knockdown. CONCLUSIONS: Tens of thousands of H3K27ac-marked active enhancers have been identified from bovine satellite cells before or during differentiation. These enhancers contain binding sites not only for transcription factors whose role in satellite cell differentiation is well known but also for transcription factors whose role in satellite cell differentiation is unknown. These enhancers and transcription factors are valuable resources for understanding the complex mechanism that mediates gene expression during satellite cell differentiation. Because satellite cell differentiation is a key step in skeletal muscle growth, the enhancers, the transcription factors, and their target genes identified in this study are also valuable resources for identifying and interpreting skeletal muscle trait-associated DNA variants in cattle.

Major histocompatibility complex genes and locus organization in the Komodo dragon (Varanus komodoensis).

We performed a meta-analysis of the newly assembled Komodo dragon (Varanus komodoensis) genome to characterize the major histocompatibility complex (MHC) of the species. The MHC gene clusters of the Komodo dragon are gene dense, complex, and contain counterparts of many genes of the human MHC. Our analysis identified 20 contigs encompassing ~ 6.9 Mbp of sequence with 223 annotated genes of which many are predicted orthologs to the genes of the human MHC. These MHC contigs range in size from 13.2 kb to 21.5 Mbp, contain an average of one gene per 30 kb, and are thought to occur on at least two chromosomes. Eight contigs, each > 100 kb, could be aligned to the human MHC based on gene content, and these represent gene clusters found in each of the recognized mammalian MHC subregions. The MHC of the Komodo dragon shares organizational features of other non-mammalian taxa. Multiple class Iα and class IIß genes are indicated, with linkage between classical class I and immunoproteasome genes and between framework class I genes and genes associated with the mammalian class III subregion. These findings are supported in both Komodo genome assemblies and provide new insight into the MHC organization of these unique squamate reptiles.

The Endoribonuclease RNase E Coordinates Expression of mRNAs and Small Regulatory RNAs and Is Critical for the Virulence of Brucella abortus.

RNases are key regulatory components in prokaryotes, responsible for the degradation and maturation of specific RNA molecules at precise times. Specifically, RNases allow cells to cope with changes in their environment through rapid alteration of gene expression. To date, few RNases have been characterized in the mammalian pathogen Brucella abortus In the present work, we sought to investigate several RNases in B. abortus and determine what role, if any, they have in pathogenesis. Of the 4 RNases reported in this study, the highly conserved endoribonuclease, RNase E, was found to play an integral role in the virulence of B. abortus Although rne, which encodes RNase E, is essential in B. abortus, we were able to generate a strain encoding a defective version of RNase E lacking the C-terminal portion of the protein, and this strain (rne-tnc) was attenuated in a mouse model of Brucella infection. RNA-sequencing analysis revealed massive RNA dysregulation in B. abortusrne-tnc, with 122 upregulated and 161 downregulated transcripts compared to the parental strain. Interestingly, several mRNAs related to metal homeostasis were significantly decreased in the rne-tnc strain. We also identified a small regulatory RNA (sRNA), called Bsr4, that exhibited significantly elevated levels in rne-tnc, demonstrating an important role for RNase E in sRNA-mediated regulatory pathways in Brucella Overall, these data highlight the importance of RNase E in B. abortus, including the role of RNase E in properly controlling mRNA levels and contributing to virulence in an animal model of infection.IMPORTANCE Brucellosis is a debilitating disease of humans and animals globally, and there is currently no vaccine to combat human infection by Brucella spp. Moreover, effective antibiotic treatment in humans is extremely difficult and can lead to disease relapse. Therefore, it is imperative that systems and pathways be identified and characterized in the brucellae so new vaccines and therapies can be generated. In this study, we describe the impact of the endoribonuclease RNase E on the control of mRNA and small regulatory RNA (sRNA) levels in B. abortus, as well as the importance of RNase E for the full virulence of B. abortus This work greatly enhances our understanding of ribonucleases in the biology and pathogenesis of Brucella spp.

The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.

BACKGROUND: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals. . RESULTS: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple ß-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon ß-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 ß-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome. CONCLUSIONS: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon.

Novel roles for scleraxis in regulating adult tenocyte function.

BACKGROUND: Tendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse. RESULTS: Equine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis. CONCLUSIONS: Our study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy.

Cutting Edge: Plasmacytoid Dendritic Cells in Late-Stage Lupus Mice Defective in Producing IFN-α.

Plasmacytoid dendritic cells (pDCs) are professional type I IFN producers believed to promote lupus. However, questions exist about whether they function at the same level throughout the course of lupus disease. We analyzed high-purity pDCs sorted from lupus mice. Although pDCs produced a large amount of IFN-α during disease initiation, those sorted from late-stage lupus mice were found to be defective in producing IFN-α. These pDCs expressed an increased level of MHC, suggesting a functional drift to Ag presentation. We examined the potential mechanism behind the defect and identified a novel transcriptional factor, Foxj2, which repressed the expression of several genes in pDCs, but not IFN-α. Dysregulation in pDCs appears to be predisposed, because they exhibited an altered transcriptional profile before the onset of clinical signs. Our results suggest that pDCs do not function the same throughout the disease course and lose the ability to produce IFN-α in late-stage lupus mice.

Targeted capture enrichment and sequencing identifies extensive nucleotide variation in the turkey MHC-B.

Variation in the major histocompatibility complex (MHC) is increasingly associated with disease susceptibility and resistance in avian species of agricultural importance. This variation includes sequence polymorphisms but also structural differences (gene rearrangement) and copy number variation (CNV). The MHC has now been described for multiple galliform species including the best defined assemblies of the chicken (Gallus gallus) and domestic turkey (Meleagris gallopavo). Using this sequence resource, this study applied high-throughput sequencing to investigate MHC variation in turkeys of North America (NA turkeys). An MHC-specific SureSelect (Agilent) capture array was developed, and libraries were created for 14 turkeys representing domestic (commercial bred), heritage breed, and wild turkeys. In addition, a representative of the Ocellated turkey (M. ocellata) and chicken (G. gallus) was included to test cross-species applicability of the capture array allowing for identification of new species-specific polymorphisms. Libraries were hybridized to ∼12 K cRNA baits and the resulting pools were sequenced. On average, 98% of processed reads mapped to the turkey whole genome sequence and 53% to the MHC target. In addition to the MHC, capture hybridization recovered sequences corresponding to other MHC regions. Sequence alignment and de novo assembly indicated the presence of several additional BG genes in the turkey with evidence for CNV. Variant detection identified an average of 2245 polymorphisms per individual for the NA turkeys, 3012 for the Ocellated turkey, and 462 variants in the chicken (RJF-256). This study provides an extensive sequence resource for examining MHC variation and its relation to health of this agriculturally important group of birds.

Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite.

BACKGROUND: A complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion. METHODS: The main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions. RESULTS: We report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity. CONCLUSIONS: The expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.

Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

Discretized Gaussian mixture for genotyping of microsatellite loci containing homopolymer runs.

MOTIVATION: Inferring lengths of inherited microsatellite alleles with single base pair resolution from short sequence reads is challenging due to several sources of noise caused by the repetitive nature of microsatellites and the technologies used to generate raw sequence data. RESULTS: We have developed a program, GenoTan, using a discretized Gaussian mixture model combined with a rules-based approach to identify inherited variation of microsatellite loci from short sequence reads without paired-end information. It effectively distinguishes length variants from noise including insertion/deletion errors in homopolymer runs by addressing the bidirectional aspect of insertion and deletion errors in sequence reads. Here we first introduce a homopolymer decomposition method which estimates error bias toward insertion or deletion in homopolymer sequence runs. Combining these approaches, GenoTan was able to genotype 94.9% of microsatellite loci accurately from simulated data with 40x sequence coverage quickly while the other programs showed <90% correct calls for the same data and required 5∼30× more computational time than GenoTan. It also showed the highest true-positive rate for real data using mixed sequence data of two Drosophila inbred lines, which was a novel validation approach for genotyping. AVAILABILITY: GenoTan is open-source software available at http://genotan.sourceforge.net.

ReviSTER: an automated pipeline to revise misaligned reads to simple tandem repeats.

MOTIVATION: Simple tandem repeats are highly variable genetic elements and widespread in genomes of many organisms. Next-generation sequencing technologies have enabled a robust comparison of large numbers of simple tandem repeat loci; however, analysis of their variation using traditional sequence analysis approaches still remains limiting and problematic due to variants occurring in repeat sequences confusing alignment programs into mapping sequence reads to incorrect loci when the sequence reads are significantly different from the reference sequence. RESULTS: We have developed a program, ReviSTER, which is an automated pipeline using a 'local mapping reference reconstruction method' to revise mismapped or partially misaligned reads at simple tandem repeat loci. RevisSTER estimates alleles of repeat loci using a local alignment method and creates temporary local mapping reference sequences, and finally remaps reads to the local mapping references. Using this approach, ReviSTER was able to successfully revise reads misaligned to repeat loci from both simulated data and real data. AVAILABILITY: ReviSTER is open-source software available at http://revister.sourceforge.net. CONTACT: garner@vbi.vt.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Next-generation sequencing strategies for characterizing the turkey genome.

The turkey genome sequencing project was initiated in 2008 and has relied primarily on next-generation sequencing (NGS) technologies. Our first efforts used a synergistic combination of 2 NGS platforms (Roche/454 and Illumina GAII), detailed bacterial artificial chromosome (BAC) maps, and unique assembly tools to sequence and assemble the genome of the domesticated turkey, Meleagris gallopavo. Since the first release in 2010, efforts to improve the genome assembly, gene annotation, and genomic analyses continue. The initial assembly build (2.01) represented about 89% of the genome sequence with 17X coverage depth (931 Mb). Sequence contigs were assigned to 30 of the 40 chromosomes with approximately 10% of the assembled sequence corresponding to unassigned chromosomes (ChrUn). The sequence has been refined through both genome-wide and area-focused sequencing, including shotgun and paired-end sequencing, and targeted sequencing of chromosomal regions with low or incomplete coverage. These additional efforts have improved the sequence assembly resulting in 2 subsequent genome builds of higher genome coverage (25X/Build3.0 and 30X/Build4.0) with a current sequence totaling 1,010 Mb. Further, BAC with end sequences assigned to the Z/W and MG18 (MHC) chromosomes, ChrUn, or not placed in the previous build were isolated, deeply sequenced (Hi-Seq), and incorporated into the latest build (5.0). To aid in the annotation and to generate a gene expression atlas of major tissues, a comprehensive set of RNA samples was collected at various developmental stages of female and male turkeys. Transcriptome sequencing data (using Illumina Hi-Seq) will provide information to enhance the final assembly and ultimately improve sequence annotation. The most current sequence covers more than 95% of the turkey genome and should yield a much improved gene level of annotation, making it a valuable resource for studying genetic variations underlying economically important traits in poultry.

Improved variation calling via an iterative backbone remapping and local assembly method for bacterial genomes.

Sequencing data analysis remains limiting and problematic, especially for low complexity repeat sequences and transposon elements due to inherent sequencing errors and short sequence read lengths. We have developed a program, ReviSeq, which uses a hybrid method composed of iterative remapping and local assembly upon a bacterial sequence backbone. Application of this method to six Brucella suis field isolates compared to the newly revised B. suis 1330 reference genome identified on average 13, 15, 19 and 9 more variants per sample than STAMPY/SAMtools, BWA/SAMtools, iCORN and BWA/PINDEL pipelines, and excluded on average 4, 2, 3 and 19 variants per sample, respectively. In total, using this iterative approach, we identified on average 87 variants including SNVs, short INDELs and long INDELs per strain when compared to the reference. Our program outperforms other methods especially for long INDEL calling. The program is available at http://reviseq.sourceforge.net.

Aging alters mechanisms underlying voluntary movements in spinal motor neurons of mice, primates, and humans.

Spinal motor neurons have been implicated in the loss of motor function that occurs with advancing age. However, the cellular and molecular mechanisms that impair the function of these neurons during aging remain unknown. Here, we show that motor neurons do not die in old female and male mice, rhesus monkeys, and humans. Instead, these neurons selectively and progressively shed excitatory synaptic inputs throughout the soma and dendritic arbor during aging. Thus, aged motor neurons contain a motor circuitry with a reduced ratio of excitatory to inhibitory synapses that may be responsible for the diminished ability to activate motor neurons to commence movements. An examination of the motor neuron translatome (ribosomal transcripts) in male and female mice reveals genes and molecular pathways with roles in glia-mediated synaptic pruning, inflammation, axonal regeneration, and oxidative stress that are upregulated in aged motor neurons. Some of these genes and pathways are also found altered in motor neurons affected with amyotrophic lateral sclerosis (ALS) and responding to axotomy, demonstrating that aged motor neurons are under significant stress. Our findings show mechanisms altered in aged motor neurons that could serve as therapeutic targets to preserve motor function during aging.

The ABRF Proteomics Research Group studies: educational exercises for qualitative and quantitative proteomic analyses.

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.

Application of an end-to-end biomarker discovery platform to identify target engagement markers in cerebrospinal fluid by high resolution differential mass spectrometry.

The rapid identification of protein biomarkers in biofluids is important to drug discovery and development. Here, we describe a general proteomic approach for the discovery and identification of proteins that exhibit a statistically significant difference in abundance in cerebrospinal fluid (CSF) before and after pharmacological intervention. This approach, differential mass spectrometry (dMS), is based on the analysis of full scan mass spectrometry data. The dMS workflow does not require complex mixing and pooling strategies, or isotope labeling techniques. Accordingly, clinical samples can be analyzed individually, allowing the use of longitudinal designs and within-subject data analysis in which each subject acts as its own control. As a proof of concept, we performed multifactorial dMS analyses on CSF samples drawn at 6 time points from n = 6 cisterna magna ported (CMP) rhesus monkeys treated with 2 potent gamma secretase inhibitors (GSI) or comparable vehicle in a 3-way crossover study that included a total of 108 individual CSF samples. Using analysis of variance and statistical filtering on the aligned and normalized LC-MS data sets, we detected 26 features that were significantly altered in CSF by drug treatment. Of those 26 features, which belong to 10 distinct isotopic distributions, 20 were identified by MS/MS as 7 peptides from CD99, a cell surface protein. Six features from the remaining 3 isotopic distributions were not identified. A subsequent analysis showed that the relative abundance of these 26 features showed the same temporal profile as the ELISA measured levels of CSF A beta 42 peptide, a known pharmacodynamic marker for gamma-secretase inhibition. These data demonstrate that dMS is a promising approach for the discovery, quantification, and identification of candidate target engagement biomarkers in CSF.

Specific labeling of synaptic schwann cells reveals unique cellular and molecular features.

Perisynaptic Schwann cells (PSCs) are specialized, non-myelinating, synaptic glia of the neuromuscular junction (NMJ), that participate in synapse development, function, maintenance, and repair. The study of PSCs has relied on an anatomy-based approach, as the identities of cell-specific PSC molecular markers have remained elusive. This limited approach has precluded our ability to isolate and genetically manipulate PSCs in a cell specific manner. We have identified neuron-glia antigen 2 (NG2) as a unique molecular marker of S100ß+ PSCs in skeletal muscle. NG2 is expressed in Schwann cells already associated with the NMJ, indicating that it is a marker of differentiated PSCs. Using a newly generated transgenic mouse in which PSCs are specifically labeled, we show that PSCs have a unique molecular signature that includes genes known to play critical roles in PSCs and synapses. These findings will serve as a springboard for revealing drivers of PSC differentiation and function.

Fusobacterium Genomics Using MinION and Illumina Sequencing Enables Genome Completion and Correction.

Understanding the virulence mechanisms of human pathogens from the genus Fusobacterium has been hindered by a lack of properly assembled and annotated genomes. Here we report the first complete genomes for seven Fusobacterium strains, as well as resequencing of the reference strain Fusobacterium nucleatum subsp. nucleatum ATCC 25586 (total of seven species; total of eight genomes). A highly efficient and cost-effective sequencing pipeline was achieved using sample multiplexing for short-read Illumina (150 bp) and long-read Oxford Nanopore MinION (>80 kbp) platforms, coupled with genome assembly using the open-source software Unicycler. Compared to currently available draft assemblies (previously 24 to 67 contigs), these genomes are highly accurate and consist of only one complete chromosome. We present the complete genome sequence of F. nucleatum subsp. nucleatum ATCC 23726, a genetically tractable and biomedically important strain and, in addition, reveal that the previous F. nucleatum subsp. nucleatum ATCC 25586 genome assembly contains a 452-kb genomic inversion that has been corrected using our sequencing and assembly pipeline. To enable genomic analyses by the scientific community, we concurrently used these genomes to launch FusoPortal, a repository of interactive and downloadable genomic data, genome maps, gene annotations, and protein functional analyses and classifications. In summary, this report provides detailed methods for accurately sequencing, assembling, and annotating Fusobacterium genomes, while focusing on using open-source software to foster the availability of reproducible and open data. This resource will enhance efforts to properly identify virulence proteins that may contribute to a repertoire of diseases that includes periodontitis, preterm birth, and colorectal cancer.IMPORTANCEFusobacterium spp. are Gram-negative, oral bacteria that are increasingly associated with human pathologies as diverse as periodontitis, preterm birth, and colorectal cancer. While a recent surge in F. nucleatum research has increased our understanding of this human pathogen, a lack of complete genomes has hindered the identification and characterization of associated host-pathogen virulence factors. Here we report the first eight complete Fusobacterium genomes sequenced using an Oxford Nanopore MinION and Illumina sequencing pipeline and assembled using the open-source program Unicycler. These genomes are highly accurate, and seven of the genomes represent the first complete sequences for each strain. In summary, the FusoPortal resource provides a publicly available resource that will guide future genetic, bioinformatic, and biochemical experiments to characterize this genus of emerging human pathogens.

RNA-Seq Analysis of Developing Pecan (Carya illinoinensis) Embryos Reveals Parallel Expression Patterns among Allergen and Lipid Metabolism Genes.

The pecan nut is a nutrient-rich part of a healthy diet full of beneficial fatty acids and antioxidants, but can also cause allergic reactions in people suffering from food allergy to the nuts. The transcriptome of a developing pecan nut was characterized to identify the gene expression occurring during the process of nut development and to highlight those genes involved in fatty acid metabolism and those that commonly act as food allergens. Pecan samples were collected at several time points during the embryo development process including the water, gel, dough, and mature nut stages. Library preparation and sequencing were performed using Illumina-based mRNA HiSeq with RNA from four time points during the growing season during August and September 2012. Sequence analysis with Trinotate software following the Trinity protocol identified 133,000 unigenes with 52,267 named transcripts and 45,882 annotated genes. A total of 27,312 genes were defined by GO annotation. Gene expression clustering analysis identified 12 different gene expression profiles, each containing a number of genes. Three pecan seed storage proteins that commonly act as allergens, Car i 1, Car i 2, and Car i 4, were significantly up-regulated during the time course. Up-regulated fatty acid metabolism genes that were identified included acyl-[ACP] desaturase and omega-6 desaturase genes involved in oleic and linoleic acid metabolism. Notably, a few of the up-regulated acyl-[ACP] desaturase and omega-6 desaturase genes that were identified have expression patterns similar to the allergen genes based upon gene expression clustering and qPCR analysis. These findings suggest the possibility of coordinated accumulation of lipids and allergens during pecan nut embryogenesis.