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.

Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation.

This study was conducted to further understand the mechanism that controls myoblast differentiation, a key step in skeletal muscle formation. RNA sequencing of primary bovine myoblasts revealed many genes encoding the ubiquitin-proteasome system were up-regulated during myoblast differentiation. This up-regulation was accompanied by increased proteasomal activity. Treating myoblasts with the proteasome-specific inhibitor lactacystin impeded myoblast differentiation. Adenovirus-mediated overexpression of inhibitor of DNA binding 1 (ID1) protein inhibited myoblast differentiation too. Further experiments were conducted to determine whether the proteasome promotes myoblast differentiation by degrading ID1 protein. Both ID1 protein and mRNA expression decreased during myoblast differentiation. However, treating myoblasts with lactacystin reversed the decrease in ID1 protein but not in ID1 mRNA expression. Surprisingly, this reversal was not observed when myoblasts were also treated with the mRNA translation inhibitor cycloheximide. Direct incubation of ID1 protein with proteasomes from myoblasts did not show differentiation stage-associated degradation of ID1 protein. Furthermore, ubiquitinated ID1 protein was not detected in lactacystin-treated myoblasts. Overall, the results of this study suggest that, during myoblast differentiation, the proteasomal activity is up-regulated to further myoblast differentiation and that the increased proteasomal activity improves myoblast differentiation partly by inhibiting the synthesis, not the degradation, of ID1 protein.-Leng, X., Ji, X., Hou, Y., Settlage, R., Jiang, H. Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation.

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.

Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.

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.

Hypothalamic differences in expression of genes involved in monoamine synthesis and signaling pathways after insulin injection in chickens from lines selected for high and low body weight.

Long-term selection for juvenile body weight from a common founder population resulted in two divergent chicken lines (low-weight selected line (LWS), high-weight selected line (HWS)) that display distinct food intake and blood glucose responses to exogenous neuropeptides and insulin. The objective of this study was to elucidate putative targets affecting food intake and energy homeostasis by sequencing hypothalamic RNA from LWS and HWS chickens after insulin injection. Ninety-day-old female LWS and HWS chickens were injected with either vehicle or insulin and hypothalamus collected at 1 h postinjection. Through RNA sequencing, a total of 361 differentially expressed genes (DEGs) were identified. There was greater expression of genes, mainly tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase (DDC), and vesicular monoamine transporter (VMAT), involved in serotonin and dopamine biosynthesis and signaling in LWS than in HWS vehicle-injected chickens. In contrast, after insulin injection, these genes were more highly expressed in HWS than in LWS. We identified 90 single nucleotide polymorphisms (SNPs) existing only in the HWS and 121 SNPs specific to LWS and 5119 SNPs close to fixation (with absolute frequency difference ≥0.9). Four were located in genes encoding enzymes associated with serotonergic and dopaminergic pathways, such as DDC, TH, and solute carrier family 18, member 2 (VMAT). These data implicate differences in biogenic amines such as serotonin and dopamine in hypothalamic physiology between the chicken lines, and these differences might be associated with polymorphisms during long-term selection. Changes in serotonergic and dopaminergic signaling pathways in response to insulin injection suggest a role in whole-body energy homeostasis.

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.

Fasting for 21days leads to changes in adipose tissue and liver physiology in juvenile checkered garter snakes (Thamnophis marcianus).

Snakes often undergo periods of prolonged fasting and, under certain conditions, can survive years without food. Despite this unique phenomenon, there are relatively few reports of the physiological adaptations to fasting in snakes. At post-prandial day 1 (fed) or 21 (fasting), brain, liver, and adipose tissues were collected from juvenile checkered garter snakes (Thamnophis marcianus). There was greater glycerol-3-phosphate dehydrogenase (G3PDH)-specific activity in the liver of fasted than fed snakes (P=0.01). The mRNA abundance of various fat metabolism-associated factors was measured in brain, liver, and adipose tissue. Lipoprotein lipase (LPL) mRNA was greater in fasted than fed snakes in the brain (P=0.04). Adipose triglyceride lipase (ATGL; P=0.006) mRNA was greater in the liver of fasted than fed snakes. In adipose tissue, expression of peroxisome proliferator-activated receptor (PPAR)γ (P=0.01), and fatty acid binding protein 4 (P=0.03) was greater in fed than fasted snakes. Analysis of adipocyte morphology revealed that cross-sectional area (P=0.095) and diameter (P=0.27) were not significantly different between fed and fasted snakes. Results suggest that mean adipocyte area can be preserved during fasting by dampening lipid biosynthesis while not changing rates of lipid hydrolysis. In the liver, however, extensive lipid remodeling may provide energy and lipoproteins to maintain lipid structural integrity during energy restriction. Because the duration of fasting was not sufficient to change adipocyte size, results suggest that the liver is important as a short-term provider of energy in the snake.

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.

Complete genome sequence of Brucella suis VBI22, isolated from bovine milk.

Brucella suis is the causative agent of swine brucellosis and is known to be able to infect several different hosts, including cattle, dogs, and horses, without causing disease symptoms. Here we report the complete genome sequence of Brucella suis VBI22, which was isolated from raw milk from an infected cow.