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Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp. [ABSTRACT FROM AUTHOR]

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T. [ABSTRACT FROM AUTHOR]

Four full-sibling intact male Miniature Poodles were evaluated at 4–19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the potential effects of methyl donor supplementation of pregnant animals in the presence or absence of a concomitant lactation on the methylome of the offspring. Twenty Holstein cows, 10 nulliparous (non-lactating while pregnant) and 10 multiparous (lactating while pregnant) were blocked by parity and randomly assigned to an i.m. weekly injections of a placebo (CTRL) or a solution containing methyl donors (MET). After calving, 5 calves randomly selected from each treatment (two born to non-lactating and three to lactating dams) were blood-sampled to determine their full methylome. There were more than 2,000 CpG differentially methylated between calves born to CTRL and those born to MET, and also between calves born to lactating and non-lactating dams. Most of the differences affected genes involved in immune function, cell growth regulation and differentiation, kinase activity, and ion channeling. We conclude that the coexistence of pregnancy and lactation affects the methylome of the offspring, and that supplementation of methyl donors early in gestation has also consequences on the methylome. [ABSTRACT FROM AUTHOR]

Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG) challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3) and cell adhesion molecule 3 (Cadm3) were over-expressed and coiled-coil domain containing 162 (Ccdc162) and titin-cap (Tcap) were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9), interleukin 1 beta (Il1b) and kynurenine 3-monooxygenase (Kmo) were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1) and microtubule-actin crosslinking factor 1(Macf1). Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness symptoms, albeit lower than that observed in macrophages. The persistent transcriptome dysregulation in the microglia shared patterns with neurological disorders indicating that the associated persistent depressive symptoms share a common transcriptome basis. [ABSTRACT FROM AUTHOR]

The giant panda (Ailuropoda melanoleuca) is a native species to China. They are rare and endangered and are regarded as the ‘national treasure’ and ‘living fossil’ in China. For the time being, there are only about 2500 giant pandas in the world. Therefore, we still have to do much more efforts to protect the giant pandas. In captive wildlife, the cataract incidence of mammalian always increases with age. Currently, in China, the proportion of elderly giant pandas who suffering from cataract has reached 20%. The eye disorder thus has a strong influence on the physical health and life quality of the elderly giant pandas. To discover the genes associated with the pathogenesis of cataract in the elderly giant panda and achieve the goal of early assessment and diagnosis of cataract in giant pandas during aging, we performed whole genome methylation sequencing in 3 giant pandas with cataract and 3 healthy giant pandas using methylation-dependent restriction-site associated DNA sequencing (MethylRAD). In the present study, we obtained 3.62M reads, on average, for each sample, and identified 116 and 242 differentially methylated genes (DMGs) between the two groups under the context of CCGG and CCWGG on genome, respectively. Further KEGG and GO enrichment analyses determined a total of 110 DMGs that are involved in the biological functions associated with pathogenesis of cataract. Among them, 6 DMGs including EEA1, GARS, SLITRK4, GSTM3, CASP3, and EGLN3 have been linked with cataract in old age. [ABSTRACT FROM AUTHOR]

Gene-annotation enrichment is a common method for utilizing ontology-based annotations in gene and gene-product centric knowledgebases. Effective utilization of these annotations requires inferring semantic linkages by tracing paths through edges in the ontological graph, referred to as relations. However, some relations are semantically problematic with respect to scope, necessitating their omission or modification lest erroneous term mappings occur. To address these issues, we created the Gene Ontology Categorization Suite, or GOcats—a novel tool that organizes the Gene Ontology into subgraphs representing user-defined concepts, while ensuring that all appropriate relations are congruent with respect to scoping semantics. Here, we demonstrate the improvements in annotation enrichment by re-interpreting edges that would otherwise be omitted by traditional ancestor path-tracing methods. Specifically, we show that GOcats’ unique handling of relations improves enrichment over conventional methods in the analysis of two different gene-expression datasets: a breast cancer microarray dataset and several horse cartilage development RNAseq datasets. With the breast cancer microarray dataset, we observed significant improvement (one-sided binomial test p-value = 1.86E-25) in 182 of 217 significantly enriched GO terms identified from the conventional path traversal method when GOcats’ path traversal was used. We also found new significantly enriched terms using GOcats, whose biological relevancy has been experimentally demonstrated elsewhere. Likewise, on the horse RNAseq datasets, we observed a significant improvement in GO term enrichment when using GOcat’s path traversal: one-sided binomial test p-values range from 1.32E-03 to 2.58E-44. [ABSTRACT FROM AUTHOR]

It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3β RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3β RNA exon 1 and 2, which resulted in generation of N’-terminal truncated GSK3β protein. This event further caused decreased phosphorylation of β-catenin and subsequently increased β-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3β RNA splicing and canonical Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]

RNA methyltransferases post-transcriptionally add methyl groups to RNAs, which can regulate their fates and functions. Human BCDIN3D (Bicoid interacting 3 domain containing RNA methyltransferase) has been reported to specifically methylate the 5′-monophosphates of pre-miR-145 and cytoplasmic tRNAHis. Methylation of the 5′-monophosphate of pre-miR-145 blocks its cleavage by the miRNA generating enzyme Dicer, preventing generation of miR-145. Elevated expression of BCDIN3D has been associated with poor prognosis in breast cancer. However, the biological functions of BCDIN3D and its orthologs remain unknown. Here we studied the biological and molecular functions of CG1239, a Drosophila ortholog of BCDIN3D. We found that ovary-specific knockdown of Drosophila BCDIN3D causes female sterility. High-throughput sequencing revealed that miRNA and mRNA profiles are dysregulated in BCDIN3D knockdown ovaries. Pathway analysis showed that many of the dysregulated genes are involved in metabolic processes, ribonucleoprotein complex regulation, and translational control. Our results reveal BCDIN3D’s biological role in female fertility and its molecular role in defining miRNA and mRNA profiles in ovaries. [ABSTRACT FROM AUTHOR]

The transforming growth factor-β (TGFβ) family plays an important role in many developmental processes and when mutated often contributes to various diseases. Marfan syndrome is a genetic disease with an occurrence of approximately 1 in 5,000. The disease is caused by mutations in fibrillin, which lead to an increase in TGFβ ligand activity, resulting in abnormalities of connective tissues which can be life-threatening. Mutations in other components of TGFβ signaling (receptors, Smads, Schnurri) lead to similar diseases with attenuated phenotypes relative to Marfan syndrome. In particular, mutations in TGFβ receptors, most of which are clustered at the C-terminal end, result in Marfan-like (MFS-like) syndromes. Even though it was assumed that many of these receptor mutations would reduce or eliminate signaling, in many cases signaling is active. From our previous studies on receptor trafficking in C. elegans, we noticed that many of these receptor mutations that lead to Marfan-like syndromes overlap with mutations that cause mis-trafficking of the receptor, suggesting a link between Marfan-like syndromes and TGFβ receptor trafficking. To test this hypothesis, we introduced three of these key MFS and MFS-like mutations into the C. elegans TGFβ receptor and asked if receptor trafficking is altered. We find that in every case studied, mutated receptors mislocalize to the apical surface rather than basolateral surface of the polarized intestinal cells. Further, we find that these mutations result in longer animals, a phenotype due to over-stimulation of the nematode TGFβ pathway and, importantly, indicating that function of the receptor is not abrogated in these mutants. Our nematode models of Marfan syndrome suggest that MFS and MFS-like mutations in the type II receptor lead to mis-trafficking of the receptor and possibly provides an explanation for the disease, a phenomenon which might also occur in some cancers that possess the same mutations within the type II receptor (e.g. colon cancer). [ABSTRACT FROM AUTHOR]

Considering the divergent temperature habitats and morphological traits of four Percidae species: yellow perch (Perca flavescens), Eurasian perch (Perca fluviatilis), pike perch (Sander lucioperca), and ruffe (Gymnocephalus cernua), we stepped into the transcriptome level to discover genes and mechanisms that drive adaptation to different temperature environments and evolution in body shape. Based on 93,566 to 181,246 annotated unigenes of the four species, we identified 1,117 one-to-one orthologous genes and subsequently constructed the phylogenetic trees that are consistent with previous studies. Together with the tree, the ratios of nonsynonymous to synonymous substitutions presented decreased evolutionary rates from the D. rerio branch to the sub-branch clustered by P. flavescens and P. fluviatilis. The specific 93 fast-evolving genes and 57 positively selected genes in P. flavescens, compared with 22 shared fast-evolving genes among P. fluviatilis, G. cernua, and S. lucioperca, showed an intrinsic foundation that ensure its adaptation to the warmer Great Lakes and farther south, especially in functional terms like “Cul4-RING E3 ubiquitin ligase complex.” Meanwhile, the specific 78 fast-evolving genes and 41 positively selected genes in S. lucioperca drew a clear picture of how it evolved to a large and elongated body with camera-type eyes and muscle strength so that it could occupy the highest position in the food web. Overall, our results uncover genetic basis that support evolutionary adaptation of temperature and body shape in four Percid species, and could furthermore assist studies on environmental adaptation in fishes. [ABSTRACT FROM AUTHOR]

Insect growth is influenced by two major environmental factors: temperature and nutrient. These environmental factors are internally mediated by insulin/insulin-like growth factor signal (IIS) to coordinate tissue or organ growth. Maruca vitrata, a subtropical lepidopteran insect, migrates to different climate regions and feeds on various crops. The objective of this study was to determine molecular tools to predict growth rate of M. vitrata using IIS components. Four genes [insulin receptor (InR), Forkhead Box O (FOXO), Target of Rapamycin (TOR), and serine-threonine protein kinase (Akt)] were used to correlate their expression levels with larval growth rates under different environmental conditions. The functional association of IIS and larval growth was confirmed because RNA interference of these genes significantly decreased larval growth rate and pupal weight. Different rearing temperatures altered expression levels of these four IIS genes and changed their growth rate. Different nutrient conditions also significantly changed larval growth and altered expression levels of IIS components. Different local populations of M. vitrata exhibited significantly different larval growth rates under the same nutrient and temperature conditions along with different expression levels of IIS components. Under a constant temperature (25°C), larval growth rates showed significant correlations with IIS gene expression levels. Subsequent regression formulas of expression levels of four IIS components against larval growth rate were applied to predict growth patterns of M. vitrata larvae reared on different natural hosts and natural local populations reared on the same diet. All four formulas well predicted larval growth rates with some deviations. These results indicate that the IIS expression analysis explains the growth variation at the same temperature due to nutrient and genetic background. [ABSTRACT FROM AUTHOR]

The small GTPase RhoA plays a crucial role in the regulation of neuronal signalling to generate behaviour. In the developing nervous system RhoA is known to regulate the actin cytoskeleton, however the effectors of RhoA-signalling in adult neurons remain largely unidentified. We have previously shown that activation of the RhoA ortholog (RHO-1) in C. elegans cholinergic motor neurons triggers hyperactivity of these neurons and loopy locomotion with exaggerated body bends. This is achieved in part through increased diacylglycerol (DAG) levels and the recruitment of the synaptic vesicle protein UNC-13 to synaptic release sites, however other pathways remain to be identified. Dopamine, which is negatively regulated by the dopamine re-uptake transporter (DAT), has a central role in modulating locomotion in both humans and C. elegans. In this study we identify a new pathway in which RHO-1 regulates locomotory behaviour by repressing dopamine signalling, via DAT-1, linking these two pathways together. We observed an upregulation of dat-1 expression when RHO-1 is activated and show that loss of DAT-1 inhibits the loopy locomotion phenotype caused by RHO-1 activation. Reducing dopamine signalling in dat-1 mutants through mutations in genes involved in dopamine synthesis or in the dopamine receptor DOP-1 restores the ability of RHO-1 to trigger loopy locomotion in dat-1 mutants. Taken together, we show that negative regulation of dopamine signalling via DAT-1 is necessary for the neuronal RHO-1 pathway to regulate locomotion. [ABSTRACT FROM AUTHOR]

Whole genome wide identification and annotation of genetic variations in camels is in its first steps. The aim of this study was the identification of genome wide variants, functional annotations of them and enrichment analysis of affected genes using whole genome sequencing data of three dromedary camels. The genomes of two Iranian female dromedary camels that mostly used to produce meat and milk were sequenced to 41.9-fold and 38.6-fold coverage. A total of 4,727,238 single-nucleotide polymorphisms (SNPs) and 692,908 indels (insertions and deletions) were found by mapping raw reads to the dromedary reference assembly (GenBank Accession: GCA_000767585.1). In-silico functional annotation of the discovered variants in under study samples revealed that most SNPs (2,305,738; 48.78%) and indels (339,756; 49.03%) were located in intergenic regions. A comparison of the identified SNPs with those of the African camel (BioProject Accession: PRJNA269274) indicated that they had 993,474 SNPs in common. We found 15,168 non-synonymous SNPs in the shared variants of the three camels that could affect gene function and protein structure. Obtained results revealed that there were 7085, 6271 and 4688 non-synonymous SNPs among the 3436, 3058 and 2882 genes in the specific gene sets of Yazd dromedary, Trod dromedary and African dromedary, respectively. The list of genes predicted to be affected by non-synonymous variants in different individuals was subjected to gene ontology (GO) enrichment analysis. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a serious neurodevelopmental disorder characterized by impaired or abnormal social interaction and communication and by restricted and repetitive behaviour. ASD is highly prevalent in Asia, Europe, and the United States, and the frequency of ASD is growing each year. Recent epidemiological studies have indicated that ASD may be caused or triggered by exposure to chemicals in the environment, such as those in the air or water. Thus, toxicological studies are needed to examine chemicals that might be implicated. However, the experimental efficiency of existing experimental models is limited, and many models represent challenges in terms of animal welfare. Thus, alternative ASD animal models are necessary. To address this, we examined the efficacy of the zebrafish embryo/larva as an alternative model of ASD. Specifically, we exposed zebrafish to valproic acid (0, 12.5, 25, 50, or 100 μM), which is a chemical known to induce autism-like effects. We then analysed subsequent developmental, behavioural, and transcriptomic changes. We found that 100 μM and 50 μM valproic acid decreased the hatching rate and locomotor activity of zebrafish embryos/larvae. Transcriptomic analysis revealed significant alterations in a number of genes associated with autism, such as adsl, mbd5, shank3, and tsc1b. Additionally, we found changes in gene ontology that were also reported in previous studies. Our findings indicate that zebrafish embryos/larvae and humans with ASD might have common physiological pathways, indicating that this animal model may represent an alternative tool for examining the causes of and potential treatments for this illness. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is common in both geriatric cats and aging humans, and is pathologically characterised by chronic tubulointerstitial inflammation and fibrosis in both species. Cats with CKD may represent a spontaneously occurring, non-rodent animal model of human disease, however little is known of feline renal cell biology. In other species, TGF-β1 signalling in the proximal tubular epithelium is thought to play a key role in the initiation and progression of renal fibrosis. In this study, we first aimed to isolate and characterise feline proximal tubular epithelial cells (FPTEC), comparing them to human primary renal epithelial cells (HREC) and the human proximal tubular cell line HK-2. Secondly, we aimed to examine and compare the effect of human recombinant TGF-β1 on cell proliferation, pro-apoptotic signalling and genes associated with epithelial-to-mesenchymal transition (EMT) in feline and human renal epithelial cells. FPTEC were successfully isolated from cadaverous feline renal tissue, and demonstrated a marker protein expression profile identical to that of HREC and HK-2. Exposure to TGF-β1 (0–10 ng/ml) induced a concentration-dependent loss of epithelial morphology and alterations in gene expression consistent with the occurrence of partial EMT in all cell types. This was associated with transcription of downstream pro-fibrotic mediators, growth arrest in FPTEC and HREC (but not HK-2), and increased apoptotic signalling at high concentrations of TGF- β1. These effects were inhibited by the ALK5 (TGF-β1RI) antagonist SB431542 (5 μM), suggesting they are mediated via the ALK5/TGF-β1RII receptor complex. Taken together, these results suggest that TGF-β1 may be involved in epithelial cell dedifferentiation, growth arrest and apoptosis in feline CKD as in human disease, and that cats may be a useful, naturally occurring model of human CKD. [ABSTRACT FROM AUTHOR]

The analysis of runs of homozygosity (ROH), using high throughput genomic data, has become a valuable and frequently used methodology to characterize the genomic and inbreeding variation of livestock and wildlife animal populations. However, this methodology has been scarcely used in highly inbred domestic animals. Here, we analyzed and characterized the occurrence of ROH fragments in highly inbred (HI; average pedigree-based inbreeding coefficient FPED = 0.164; 0.103 to 0.306) and outbred Retinta bulls (LI; average FPED = 0.008; 0 to 0.025). We studied the length of the fragments, their abundance, and genome distribution using high-density microarray data. The number of ROH was significantly higher in the HI group, especially for long fragments (>8Mb). In the LI group, the number of ROH continuously decreased with fragment length. Genome-wide distribution of ROH was highly variable between samples. Some chromosomes presented a larger number of fragments (BTA1, BTA19, BTA29), others had longer fragments (BTA4, BTA12, BTA17), while other ones showed an increased ROH accumulation over specific loci (BTA2, BTA7, BTA23, BTA29). Similar differences were observed in the analysis of 12 individuals produced by a similar inbred event (FPED3 = 0.125). The correlation between the fraction of the genome covered by ROH (FROH) and FPED was high (0.79), suggesting that ROH-based estimations are indicative of inbreeding levels. On the other hand, the correlation between FPED and the microsatellite-based inbreeding coefficient (FMIC) was only moderate (r = 0.44), suggesting that STR-based inbreeding estimations should be avoided. Similarly, we found a very low correlation (r = -0.0132) between recombination rate and ROH abundance across the genome. Finally, we performed functional annotation analyses of genome regions with significantly enriched ROH abundance. Results revealed gene clusters related to pregnancy-associated proteins and immune reaction. The same analysis performed for regions enriched with recently formed ROH (> 8 Mb) showed gene clusters related to flagellum assembly. In both cases, the processes were related to male and female reproductive functions, which may partially explain the reduced fertility associated with inbred populations. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is one of the most devastating pathogens of cultured shrimp, responsible for massive loss of its commercial products worldwide. The oriental river prawn Macrobrachium nipponense is an economically important species that is widely farmed in China and adult prawns can be infected by WSSV. However, the molecular mechanisms of the host pathogen interaction remain unknown. There is an urgent need to learn the host pathogen interaction between M. nipponense and WSSV which will be able to offer a solution in controlling the spread of WSSV. Next Generation Sequencing (NGS) was used in this study to determin the transcriptome differences by the comparison of control and WSSV-challenged moribund samples, control and WSSV-challenged survived samples of hepatopancreas in M. nipponense. A total of 64,049 predicted unigenes were obtained and classified into 63 functional groups. Approximately, 4,311 differential expression genes were identified with 3,308 genes were up-regulated when comparing the survived samples with the control. In the comparison of moribund samples with control, 1,960 differential expression genes were identified with 764 genes were up-regulated. In the contrast of two comparison libraries, 300 mutual DEGs with 95 up-regulated genes and 205 down-regulated genes. All the DEGs were performed GO and KEGG analysis, overall a total of 85 immune-related genes were obtained and these gene were groups into 13 functions and 4 KEGG pathways, such as protease inhibitors, heat shock proteins, oxidative stress, pathogen recognition immune receptors, PI3K/AKT/mTOR pathway, MAPK signaling pathway and Ubiquitin Proteasome Pathway. Ten genes that valuable in immune responses against WSSV were selected from those DEGs to furture discuss the response of host to WSSV. Results from this study contribute to a better understanding of the immune response of M. nipponense to WSSV, provide information for identifying novel genes in the absence of genome of M. nipponense. Furthermore, large number of transcripts obtained from this study could provide a strong basis for future genomic research on M. nipponense. [ABSTRACT FROM AUTHOR]

Background: Activation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples. Methods: For pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed. Results: Pharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats. Conclusion: GPNMB has been identified as a translatable target engagement biomarker for use in clinical studies. [ABSTRACT FROM AUTHOR]

Channel catfish (Ictalurus punctatus) is a highly adaptive species and has been used as a research model for comparative immunology, physiology, and toxicology among ectothermic vertebrates. It is also economically important for aquaculture. As such, its reference genome was generated and annotated with protein coding genes. However, the repetitive elements in the catfish genome are less well understood. In this study, over 417.8 Megabase (MB) of repetitive elements were identified and characterized in the channel catfish genome. Among them, the DNA/TcMar-Tc1 transposons are the most abundant type, making up ~20% of the total repetitive elements, followed by the microsatellites (14%). The prevalence of repetitive elements, especially the mobile elements, may have provided a driving force for the evolution of the catfish genome. A number of catfish-specific repetitive elements were identified including the previously reported Xba elements whose divergence rate was relatively low, slower than that in untranslated regions of genes but faster than the protein coding sequences, suggesting its evolutionary restrictions. [ABSTRACT FROM AUTHOR]

Acute stress responsiveness is a quantitative trait that varies in severity from one individual to another; however, the genetic component underlying the individual variation is largely unknown. Fischer 344 (F344) and Wistar Kyoto (WKY) rat strains show large differences in behavioral responsiveness to acute stress, such as freezing behavior in response to footshock during the conditioning phase of contextual fear conditioning (CFC). Quantitative trait loci (QTL) have been identified for behavioral responsiveness to acute stress in the defensive burying (DB) and open field test (OFT) from a reciprocal F2 cross of F344 and WKY rat strains. These included a significant QTL on chromosome 6 (Stresp10). Here, we hypothesized that the Stresp10 region harbors genes with sequence variation(s) that contribute to differences in multiple behavioral response phenotypes between the F344 and WKY rat strains. To test this hypothesis, first we identified differentially expressed genes within the Stresp10 QTL in the hippocampus, amygdala, and frontal cortex of F344 and WKY male rats using genome-wide microarray analyses. Genes with both expression differences and non-synonymous sequence variations in their coding regions were considered candidate quantitative trait genes (QTGs). As a proof-of-concept, the F344.WKY-Stresp10 congenic strain was generated with the Stresp10 WKY donor region into the F344 recipient strain. This congenic strain showed behavioral phenotypes similar to those of WKYs. Expression patterns of Gpatch11 (G-patch domain containing 11), Cdkl4 (Cyclin dependent kinase like 4), and Drc1 (Dynein regulatory complex subunit 1) paralleled that of WKY in the F344.WKY-Stresp10 strain matching the behavioral profiles of WKY as opposed to F344 parental strains. We propose that these genes are candidate QTGs for behavioral responsiveness to acute stress. [ABSTRACT FROM AUTHOR]

The with no lysine (WNK) protein kinase family is conserved among many species. Some mutations in human WNK gene are associated with pseudohypoaldosteronism type II, a form of hypertension, and hereditary sensory and autonomic neuropathy type 2A. In kidney, WNK regulates the activity of STE20/SPS1-related, proline alanine-rich kinase and/or oxidative-stress responsive 1, which in turn regulate ion co-transporters. The misregulation of this pathway is involved in the pathogenesis of pseudohypoaldosteronism type II. In the neural system, WNK is involved in the specification of the cholinergic neuron, but the pathogenesis of hereditary sensory and autonomic neuropathy type 2A is still unknown. To better understand the WNK pathway, we isolated WNK-associated genes using Drosophila. We identified Glycogen synthase kinase 3ß (GSK3ß)/Shaggy (Sgg) as a candidate gene that was shown to interact with the WNK signaling pathway in both Drosophila and mammalian cells. Furthermore, GSK3ß was involved in neural specification downstream of WNK. These results suggest that GSK3ß/Sgg functions as a positive effector in the WNK signaling pathway. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) mediates both cellular and humoral immune responses in insects. Its mediation of cellular immune responses uses eicosanoids as a downstream signal. However, the cross-talk with two immune mediators was not known in humoral immune responses. This study focuses on cross-talk between two immune mediators in inducing gene expression of anti-microbial peptides (AMPs) of a lepidopteran insect, Spodoptera exigua. Up-regulation of eight AMPs was observed in S. exigua against bacterial challenge. However, the AMP induction was suppressed by injection of an NO synthase inhibitor, L-NAME, while little expressional change was observed on injecting its enantiomer, D-NAME. The functional association between NO biosynthesis and AMP gene expression was further supported by RNA interference (RNAi) against NO synthase (SeNOS), which suppressed AMP gene expression under the immune challenge. The AMP induction was also mimicked by NO alone because injecting an NO analog, SNAP, without bacterial challenge significantly induced the AMP gene expression. Interestingly, an eicosanoid biosynthesis inhibitor, dexamethasone (DEX), suppressed the NO induction of AMP expression. The inhibitory activity of DEX was reversed by the addition of arachidonic acid, a precursor of eicosanoid biosynthesis. AMP expression of S. exigua was also controlled by the Toll/IMD signal pathway. The RNAi of Toll receptors or Relish suppressed AMP gene expression by suppressing NO levels and subsequently reducing PLA2 enzyme activity. These results suggest that eicosanoids are a downstream signal of NO mediation of AMP expression against bacterial challenge. [ABSTRACT FROM AUTHOR]

Glucocorticoids are potent inhibitors of angiogenesis in the rodent in vivo and in vitro but the mechanism by which this occurs has not been determined. Administration of glucocorticoids is used to treat a number of conditions in horses but the angiogenic response of equine vessels to glucocorticoids and, therefore, the potential role of glucocorticoids in pathogenesis and treatment of equine disease, is unknown. This study addressed the hypothesis that glucocorticoids would be angiostatic both in equine and murine blood vessels.The mouse aortic ring model of angiogenesis was adapted to assess the effects of cortisol in equine vessels. Vessel rings were cultured under basal conditions or exposed to: foetal bovine serum (FBS; 3%); cortisol (600 nM), cortisol (600nM) plus FBS (3%), cortisol (600nM) plus either the glucocorticoid receptor antagonist RU486 or the mineralocorticoid receptor antagonist spironolactone. In murine aortae cortisol inhibited and FBS stimulated new vessel growth. In contrast, in equine blood vessels FBS alone had no effect but cortisol alone, or in combination with FBS, dramatically increased new vessel growth compared with controls. This effect was blocked by glucocorticoid receptor antagonism but not by mineralocorticoid antagonism. The transcriptomes of murine and equine angiogenesis demonstrated cortisol-induced down-regulation of inflammatory pathways in both species but up-regulation of pro-angiogenic pathways selectively in the horse. Genes up-regulated in the horse and down-regulated in mice were associated with the extracellular matrix. These data call into question our understanding of glucocorticoids as angiostatic in every species and may be of clinical relevance in the horse. [ABSTRACT FROM AUTHOR]

Butana and Kenana are two types of zebu cattle found in Sudan. They are unique amongst African indigenous zebu cattle because of their high milk production. Aiming to understand their genome structure, we genotyped 25 individuals from each breed using the Illumina BovineHD Genotyping BeadChip. Genetic structure analysis shows that both breeds have an admixed genome composed of an even proportion of indicine (0.75 ± 0.03 in Butana, 0.76 ± 0.006 in Kenana) and taurine (0.23 ± 0.009 in Butana, 0.24 ± 0.006 in Kenana) ancestries. We also observe a proportion of 0.02 to 0.12 of European taurine ancestry in ten individuals of Butana that were sampled from cattle herds in Tamboul area suggesting local crossbreeding with exotic breeds. Signatures of selection analyses (iHS and Rsb) reveal 87 and 61 candidate positive selection regions in Butana and Kenana, respectively. These regions span genes and quantitative trait loci (QTL) associated with biological pathways that are important for adaptation to marginal environments (e.g., immunity, reproduction and heat tolerance). Trypanotolerance QTL are intersecting candidate regions in Kenana cattle indicating selection pressure acting on them, which might be associated with an unexplored level of trypanotolerance in this cattle breed. Several dairy traits QTL are overlapping the identified candidate regions in these two zebu cattle breeds. Our findings underline the potential to improve dairy production in the semi-arid pastoral areas of Africa through breeding improvement strategy of indigenous local breeds. [ABSTRACT FROM AUTHOR]

The ability of an organism to sense and adapt to environmental stressors is essential for proteome maintenance and survival. The highly conserved heat shock response is a survival mechanism employed by all organisms, including the nematode Caenorhabditis elegans, upon exposure to environmental extremes. Transcriptional control of the metazoan heat shock response is mediated by the heat shock transcription factor HSF-1. In addition to regulating global stress-responsive genes to promote stress-resistance and survival, HSF-1 has recently been shown to regulate stress-independent functions in controlling development, metabolism, and longevity. However, the indirect role of HSF-1 in coordinating stress-dependent and -independent processes through post-transcriptional regulation is largely unknown. MicroRNAs (miRNAs) have emerged as a class of post-transcriptional regulators that control gene expression through translational repression or mRNA degradation. To determine the role of HSF-1 in regulating miRNA expression, we have performed high-throughput small RNA-sequencing in C. elegans grown in the presence and absence of hsf-1 RNAi followed by treatment with or without heat shock. This has allowed us to uncover the miRNAs regulated by HSF-1 via heat-dependent and -independent mechanisms. Integrated miRNA/mRNA target-prediction analyses suggest HSF-1 as a post-transcriptional regulator of development, metabolism, and longevity through regulating miRNA expression. This provides new insight into the possible mechanism by which HSF-1 controls these processes. We have also uncovered oxidative stress response factors and insulin-like signaling factors as a common link between processes affected by HSF-1-regulated miRNAs in stress-dependent and -independent mechanisms, respectively. This may provide a role for miRNAs in regulating cross-talk between various stress responses. Our work therefore uncovers an interesting potential role for HSF-1 in post-transcriptionally controlling gene expression in C. elegans, and suggests a mechanism for cross-talk between stress responses. [ABSTRACT FROM AUTHOR]

Cystic echinococcosis (CE) is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz), a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 μM and 0.5 μM in protoscoleces and metacestodes, respectively (P < 0.05). After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin) and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch) in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16) together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases. [ABSTRACT FROM AUTHOR]

Oxidative and glycolytic myofibers have different structures and metabolic characteristics and their ratios are important in determining poultry meat quality. However, the molecular mechanisms underlying their differences are unclear. In this study, global gene expression profiling was conducted in oxidative skeletal muscle (obtained from the soleus, or SOL) and glycolytic skeletal muscle (obtained from the extensor digitorum longus, or EDL) of Chinese Qingyuan partridge chickens, using the Agilent Chicken Gene Expression Chip. A total of 1224 genes with at least 2-fold differences were identified (P < 0.05), of which 654 were upregulated and 570 were downregulated in SOL. GO, KEGG pathway, and co-expressed gene network analyses suggested that PRKAG3, ATP2A2, and PPARGC1A might play important roles in myofiber composition. The function of PPARGC1A gene was further validated. PPARGC1A mRNA expression levels were higher in SOL than in EDL muscles throughout the early postnatal development stages. In myoblast cells, shRNA knockdown of PPARGC1A significantly inhibited some muscle development and transition-related genes, including PPP3CA, MEF2C, and SM (P < 0.01 or P < 0.05), and significantly upregulated the expression of FWM (P < 0.05). Our study demonstrates strong transcriptome differences between oxidative and glycolytic myofibers, and the results suggest that PPARGC1A is a key gene involved in chicken myofiber composition and transition. [ABSTRACT FROM AUTHOR]

In mammals, a testis-specific isoform of the protein kinase LKB1 is required for spermiogenesis, but its exact function and specificity are not known. Human LKB1 rescues the functions of Drosophila Lkb1 essential for viability, but these males are sterile, revealing a new function for this genes in fly. We also identified a testis-specific transcript generated by an alternative promoter and that only differs by a longer 5’UTR. We show that dLKB1 is required in the germline for the formation of the actin cone, the polarized structure that allows spermatid individualization and cytoplasm excess extrusion during spermiogenesis. Three of the nine LKB1 classical targets in the Drosophila genome (AMPK, NUAK and KP78b) are required for proper spermiogenesis, but later than dLKB1. dLkb1 mutant phenotype is reminiscent of that of myosin V mutants, and both proteins show a dynamic localization profile before actin cone formation. Together, these data highlight a new dLKB1 function and suggest that dLKB1 posttranscriptional regulation in testis and involvement in spermatid morphogenesis are evolutionarily conserved features. [ABSTRACT FROM AUTHOR]

Molting is an important physiological process in the larval stage of Bombyx mori and is controlled by various hormones and peptides. The silkworm mutant that exhibits the phenotype of non-molting in the 2nd instar (nm2) is incapable of molting in the 2nd instar and dies after seven or more days. The ecdysone titer in the nm2 mutant is lower than that in the wildtype, and the mutant can be rescued by feeding with 20E and cholesterol. The results of positional cloning indicated that structural alteration of BmCPG10 is responsible for the phenotype of the nm2 mutant. To explore the possible relationship between BmCPG10 and the ecdysone titer as well as the genes affected by BmCPG10, digital gene expression (DGE) profile analysis was conducted in the nm2 mutant, with the wildtype strain C603 serving as the control. The results revealed 1727 differentially expressed genes, among which 651 genes were upregulated and 1076 were downregulated in nm2. BLASTGO analysis showed that these differentially expressed genes were involved in various biological processes, cellular components and molecular functions. KEGG analysis indicated an enrichment of these differentially expressed genes in 240 pathways, including metabolic pathways, pancreatic secretion, protein digestion and absorption, fat digestion and absorption and glycerolipid metabolism. To verify the accuracy of the DGE results, quantitative reverse transcription PCR (qRT-PCR) was performed, focusing on key genes in several related pathways, and the results were highly consistent with the DGE results. Our findings indicated significant differences in cuticular protein genes, ecdysone biosynthesis genes and ecdysone-related nuclear receptors genes, but no significant difference in juvenile hormone and chitin biosynthesis genes was detected. Our research findings lay the foundation for further research on the formation mechanism of the nm2 mutant. [ABSTRACT FROM AUTHOR]

Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage. [ABSTRACT FROM AUTHOR]

Scabies is a parasitic disease, caused by the mite Sarcoptes scabiei, and is considered one of the top 50 epidemic diseases and one the most common human skin disease, worldwide. Allergic dermatitis, including an intense itch, is a common symptom, however diagnosis is difficult and there is currently no effective vaccine. The goal of this study was to examine the immune interaction mechanism of both S. scabiei and infected hosts. mRNA-seq and microRNA-seq were conducted on the S. scabiei mite and on infected and uninfected hosts. We focused on differential expression of unigenes and microRNAs, as well as the real targets of unigenes in enriched immune signaling pathways. S. scabiei enhanced host immune function and decreased metabolism after infection, while the immune response of the host inhibited S. scabiei proliferation and metabolism signaling pathways. Differentially expressed unigenes of S. scabiei were enriched in the JAK-STAT signaling pathway and the Toll-like receptor signaling pathway. The differential expression analysis indicated that microRNAs of S. scabiei and hosts have major roles in regulating immune interactions between parasites and hosts. [ABSTRACT FROM AUTHOR]

TRAF family member-associated NF-κB activator (TANK)-binding kinase1 (TBK1) is a serine-threonine kinase at the crossroads of multiple interferon (IFN)-inducing signaling pathways in innate immunity. The importance of TBK1 in antiviral immunity is well established in mammal models, but in chicken, the molecular characterization and potential function of TBK1 remain unclear. In the present study, the open-reading frame (ORF) of chicken TBK1 (chTBK1) was cloned and characterized. The sequencing results revealed that the chTBK1 ORF consists of 2190 base pairs (bp) encoding a deduced protein of 729 amino acid residues. Multiple sequence alignment analysis demonstrated chTBK1 similarity to other birds and mammals, which indicates that it is evolutionarily conserved. Quantitative real-time PCR (qRT-PCR) results showed that chTBK1 was ubiquitously expressed in chicken tissues and expression was especially high in immune tissues. In addition, the expression of chTBK1 was significantly up-regulated by infection with avian leukosis virus subgroup J (ALV-J) both in vivo and in chicken embryo fibroblasts (CEFs) challenged with ALV-J or stimulated with poly I:C in vitro. Consistent with the activation of chTBK1, the interferon regulatory factor 3 (IRF3) and IFNβ gene in CEFs were also up-regulated after challenge with ALV-J or polyI:C. In contrast, the expression of IRF3 and IFNβ in CEFs was significantly reduced by siRNA targeting the chTBK1 gene compared with a negative control (NC) during ALV-J infection or polyI:C transfection. In conclusion, our results demonstrated that chTBK1 may be an important immunoregulator for IRF3 and IFNβ induction in response to viral stimulation in chicken. [ABSTRACT FROM AUTHOR]

Changes occurring during evolution in the cis-regulatory landscapes of individual members of multigene families might impart diversification in their spatiotemporal expression and function. The archetypal member of the echinoid hbox12/pmar1/micro1 family is hbox12-a, a homeobox-containing gene expressed exclusively by dorsal blastomeres, where it governs the dorsal/ventral gene regulatory network during embryogenesis of the sea urchin Paracentrotus lividus. Here we describe the inventory of the hbox12/pmar1/micro1 genes in P. lividus, highlighting that gene copy number variation occurs across individual sea urchins of the same species. We show that the various hbox12/pmar1/micro1 genes group into three subfamilies according to their spatiotemporal expression, which ranges from broad transcription throughout development to transient expression in either the animal hemisphere or micromeres of the early embryo. Interestingly, the promoter regions of those genes showing comparable expression patterns are highly similar, while differing from those of the other subfamilies. Strikingly, phylogenetic analysis suggests that the hbox12/pmar1/micro1 genes are species-specific, exhibiting extensive divergence in their noncoding, but not in their coding, sequences across three distinct sea urchin species. In spite of this, two micromere-specific genes of P. lividus possess a TCF/LEF-binding motif in a similar position, and their transcription relies on Wnt/β-catenin signaling, similar to the pmar1 and micro1 genes, which in other sea urchin species are involved in micromere specification. Altogether, our findings suggest that the hbox12/pmar1/micro1 gene family evolved rather rapidly, generating paralogs whose cis-regulatory sequences diverged following multiple rounds of duplication from a common ancestor. [ABSTRACT FROM AUTHOR]

Vertebrate sexual fate is decided primarily by the individual’s genotype (GSD), by the environmental temperature during development (TSD), or both. Turtles exhibit TSD and GSD, making them ideal to study the evolution of sex determination. Here we analyze temperature-specific gonadal transcriptomes (RNA-sequencing validated by qPCR) of painted turtles (Chrysemys picta TSD) before and during the thermosensitive period, and at equivalent stages in soft-shell turtles (Apalone spinifera—GSD), to test whether TSD’s and GSD’s transcriptional circuitry is identical but deployed differently between mechanisms. Our data show that most elements of the mammalian urogenital network are active during turtle gonadogenesis, but their transcription is generally more thermoresponsive in TSD than GSD, and concordant with their sex-specific function in mammals [e.g., upregulation of Amh, Ar, Esr1, Fog2, Gata4, Igf1r, Insr, and Lhx9 at male-producing temperature, and of β-catenin, Foxl2, Aromatase (Cyp19a1), Fst, Nf-kb, Crabp2 at female-producing temperature in Chrysemys]. Notably, antagonistic elements in gonadogenesis (e.g., β-catenin and Insr) were thermosensitive only in TSD early-embryos. Cirbp showed warm-temperature upregulation in both turtles disputing its purported key TSD role. Genes that may convert thermal inputs into sex-specific development (e.g., signaling and hormonal pathways, RNA-binding and heat-shock) were differentially regulated. Jak-Stat, Nf-κB, retinoic-acid, Wnt, and Mapk-signaling (not Akt and Ras-signaling) potentially mediate TSD thermosensitivity. Numerous species-specific ncRNAs (including Xist) were differentially-expressed, mostly upregulated at colder temperatures, as were unannotated loci that constitute novel TSD candidates. Cirbp showed warm-temperature upregulation in both turtles. Consistent transcription between turtles and alligator revealed putatively-critical reptilian TSD elements for male (Sf1, Amh, Amhr2) and female (Crabp2 and Hspb1) gonadogenesis. In conclusion, while preliminary, our data helps illuminate the regulation and evolution of vertebrate sex determination, and contribute genomic resources to guide further research into this fundamental biological process. [ABSTRACT FROM AUTHOR]

The early growth pattern, especially the age of peak growth, of broilers affects the time to market and slaughter weight, which in turn affect the profitability of the poultry industry. However, the underlying mechanisms regulating chicken growth and development have rarely been studied. This study aimed to identify candidate genes involved in chicken growth and investigated the potential regulatory mechanisms of early growth in chicken. RNA sequencing was applied to compare the transcriptomes of chicken muscle tissues at three developmental stages during early growth. In total, 978 differentially expressed genes (DEGs) (fold change ≥ 2; false discovery rate < 0.05) were detected by pairwise comparison. Functional analysis showed that the DEGs are mainly involved in the processes of cell growth, muscle development, and cellular activities (such as junction, migration, assembly, differentiation, and proliferation). Many of the DEGs are well known to be related to chicken growth, such as MYOD1, GH, IGF2BP2, IGFBP3, SMYD1, CEBPB, FGF2, and IGFBP5. KEGG pathway analysis identified that the DEGs were significantly enriched in five pathways (P < 0.1) related to growth and development: extracellular matrix–receptor interaction, focal adhesion, tight junction, insulin signaling pathway, and regulation of the actin cytoskeleton. A total of 42 DEGs assigned to these pathways are potential candidate genes inducing the difference in growth among the three developmental stages, such as MYH10, FGF2, FGF16, FN1, CFL2, MAPK9, IRS1, PHKA1, PHKB, and PHKG1. Thus, our study identified a series of genes and several pathways that may participate in the regulation of early growth in chicken. These results should serve as an important resource revealing the molecular basis of chicken growth and development. [ABSTRACT FROM AUTHOR]

We investigated the mechanisms of mushroom toxin resistance in the Drosophila Genetic Reference Panel (DGRP) fly lines, using genome-wide association studies (GWAS). While Drosophila melanogaster avoids mushrooms in nature, some lines are surprisingly resistant to α-amanitin—a toxin found solely in mushrooms. This resistance may represent a pre-adaptation, which might enable this species to invade the mushroom niche in the future. Although our previous microarray study had strongly suggested that pesticide-metabolizing detoxification genes confer α-amanitin resistance in a Taiwanese D. melanogaster line Ama-KTT, none of the traditional detoxification genes were among the top candidate genes resulting from the GWAS in the current study. Instead, we identified Megalin, Tequila, and widerborst as candidate genes underlying the α-amanitin resistance phenotype in the North American DGRP lines, all three of which are connected to the Target of Rapamycin (TOR) pathway. Both widerborst and Tequila are upstream regulators of TOR, and TOR is a key regulator of autophagy and Megalin-mediated endocytosis. We suggest that endocytosis and autophagy of α-amanitin, followed by lysosomal degradation of the toxin, is one of the mechanisms that confer α-amanitin resistance in the DGRP lines. [ABSTRACT FROM AUTHOR]

Birth defects are among the leading causes of infant mortality and contribute substantially to illness and long-term disability. Defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate. Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. In zebrafish, the scaffolding protein Wdr68 is required for edn1 expression and subsequent formation of the ventral Meckel’s cartilage as well as the dorsal Palatoquadrate. Here we report that wdr68 activity is required between the 17-somites and prim-5 stages, that edn1 functions downstream of wdr68, and that wdr68 activity restricts jag1b, hey1, and grem2 expression from ventral CNCC territory. Expression of dlx1a and dlx2a was also severely reduced in anterior dorsal and ventral 1st arch CNCC territory in wdr68 mutants. We also found that the BMP agonist isoliquiritigenin (ISL) can partially rescue lower jaw formation and edn1 expression in wdr68 mutants. However, we found no significant defects in BMP reporter induction or pSmad1/5 accumulation in wdr68 mutant cells or zebrafish. The Transforming Growth Factor Beta (TGF-β) signaling pathway is also known to be important for craniofacial development and can interfere with BMP signaling. Here we further report that TGF-β interference with BMP signaling was greater in wdr68 mutant cells relative to control cells. To determine whether interference might also act in vivo, we treated wdr68 mutant zebrafish embryos with the TGF-β signaling inhibitor SB431542 and found partial rescue of edn1 expression and craniofacial development. While ISL treatment failed, SB431542 partially rescued dlx2a expression in wdr68 mutants. Together these findings reveal an indirect role for Wdr68 in the BMP-Edn1-Jag1b signaling hierarchy and dorso-anterior expression of dlx1a/2a. [ABSTRACT FROM AUTHOR]

To better understand how the cellular response to DNA replication stress is regulated during embryonic development, we and others have established the early C. elegans embryo as a model system to study this important problem. As is the case in most eukaryotic cell types, the replication stress response is controlled by the ATR kinase in early worm embryos. In this report we use RNAi to systematically characterize ATR pathway components for roles in promoting cell cycle delay during a replication stress response, and we find that these genetic requirements vary, depending on the source of stress. We also examine how individual cell types within the embryo respond to replication stress, and we find that the strength of the response, as defined by duration of cell cycle delay, varies dramatically within blastomeres of the early embryo. Our studies shed light on how the replication stress response is managed in the context of embryonic development and show that this pathway is subject to developmental regulation. [ABSTRACT FROM AUTHOR]

Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research. [ABSTRACT FROM AUTHOR]

Next-generation sequencing and the collection of genome-wide single-nucleotide polymorphisms (SNPs) allow identifying fine-scale population genetic structure and genomic regions under selection. The spotted sea bass (Lateolabrax maculatus) is a non-model species of ecological and commercial importance and widely distributed in northwestern Pacific. A total of 22 648 SNPs was discovered across the genome of L. maculatus by paired-end sequencing of restriction-site associated DNA (RAD-PE) for 30 individuals from two populations. The nucleotide diversity (π) for each population was 0.0028±0.0001 in Dandong and 0.0018±0.0001 in Beihai, respectively. Shallow but significant genetic differentiation was detected between the two populations analyzed by using both the whole data set (FST = 0.0550, P < 0.001) and the putatively neutral SNPs (FST = 0.0347, P < 0.001). However, the two populations were highly differentiated based on the putatively adaptive SNPs (FST = 0.6929, P < 0.001). Moreover, a total of 356 SNPs representing 298 unique loci were detected as outliers putatively under divergent selection by FST-based outlier tests as implemented in BAYESCAN and LOSITAN. Functional annotation of the contigs containing putatively adaptive SNPs yielded hits for 22 of 55 (40%) significant BLASTX matches. Candidate genes for local selection constituted a wide array of functions, including binding, catalytic and metabolic activities, etc. The analyses with the SNPs developed in the present study highlighted the importance of genome-wide genetic variation for inference of population structure and local adaptation in L. maculatus. [ABSTRACT FROM AUTHOR]

Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment. [ABSTRACT FROM AUTHOR]

Improvement in feed conversion efficiency can improve the sustainability of beef cattle production, but genomic selection for feed efficiency affects many underlying molecular networks and physiological traits. This study describes the differences between steer progeny of two influential Angus bulls with divergent genomic predictions for residual feed intake (RFI). Eight steer progeny of each sire were phenotyped for growth and feed intake from 8 mo. of age (average BW 254 kg, with a mean difference between sire groups of 4.8 kg) until slaughter at 14–16 mo. of age (average BW 534 kg, sire group difference of 28.8 kg). Terminal samples from pituitary gland, skeletal muscle, liver, adipose, and duodenum were collected from each steer for transcriptome sequencing. Gene expression networks were derived using partial correlation and information theory (PCIT), including differentially expressed (DE) genes, tissue specific (TS) genes, transcription factors (TF), and genes associated with RFI from a genome-wide association study (GWAS). Relative to progeny of the high RFI sire, progeny of the low RFI sire had -0.56 kg/d finishing period RFI (P = 0.05), -1.08 finishing period feed conversion ratio (P = 0.01), +3.3 kg^0.75 finishing period metabolic mid-weight (MMW; P = 0.04), +28.8 kg final body weight (P = 0.01), -12.9 feed bunk visits per day (P = 0.02) with +0.60 min/visit duration (P = 0.01), and +0.0045 carcass specific gravity (weight in air/weight in air—weight in water, a predictor of carcass fat content; P = 0.03). RNA-seq identified 633 DE genes between sire groups among 17,016 expressed genes. PCIT analysis identified >115,000 significant co-expression correlations between genes and 25 TF hubs, i.e. controllers of clusters of DE, TS, and GWAS SNP genes. Pathway analysis suggests low RFI bull progeny possess heightened gut inflammation and reduced fat deposition. This multi-omics analysis shows how differences in RFI genomic breeding values can impact other traits and gene co-expression networks. [ABSTRACT FROM AUTHOR]

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR. [ABSTRACT FROM AUTHOR]

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. [ABSTRACT FROM AUTHOR]

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii. [ABSTRACT FROM AUTHOR]

Crucian carp are unusual among vertebrates in surviving extended periods in the complete absence of molecular oxygen. During this time cardiac output is maintained though these mechanisms are not well understood. Using a high-density cDNA microarray, we have defined the genome-wide gene expression responses of cardiac tissue after exposing the fish at two temperatures (8 and 13°C) to one and seven days of anoxia, followed by seven days after restoration to normoxia. At 8°C, using a false discovery rate of 5%, neither anoxia nor re-oxygenation elicited appreciable changes in gene expression. By contrast, at 13°C, 777 unique genes responded strongly. Up-regulated genes included those involved in protein turnover, the pentose phosphate pathway and cell morphogenesis while down-regulated gene categories included RNA splicing and transcription. Most genes were affected between one and seven days of anoxia, indicating gene regulation over the medium term but with few early response genes. Re-oxygenation for 7 days was sufficient to completely reverse these responses. Glycolysis displayed more complex responses with anoxia up-regulated transcripts for the key regulatory enzymes, hexokinase and phosphofructokinase, but with down-regulation of most of the non-regulatory genes. This complex pattern of responses in genomic transcription patterns indicates divergent cardiac responses to anoxia, with the transcriptionally driven reprogramming of cardiac function seen at 13°C being largely completed at 8°C. [ABSTRACT FROM AUTHOR]

The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of Wnk1 in angiogenesis was explored using the zebrafish model. There are two zebrafish wnk1 isoforms, wnk1a and wnk1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against wnk1a and wnk1b, we observed that wnk1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both wnk1a and wnk1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within wnk1 destroys this rescue capability. The rescue experiments provide evidence that wnk1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in wnk1a expression, as assessed by insitu hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of Wnk1 as well as transcriptional regulation of wnk1 expression. [ABSTRACT FROM AUTHOR]

The Drosophila melanogaster 91-R and 91-C strains are of common origin, however, 91-R has been intensely selected for dichlorodiphenyltrichloroethane (DDT) resistance over six decades while 91-C has been maintained as the non-selected control strain. These fly strains represent a unique genetic resource to understand the accumulation and fixation of mutations under laboratory conditions over decades of pesticide selection. Considerable research has been done to investigate the differential expression of genes associated with the highly DDT resistant strain 91-R, however, with the advent of whole genome sequencing we can now begin to develop an in depth understanding of the genomic changes associated with this intense decades-long xenobiotic selection pressure. Here we present the first whole genome sequencing analysis of the 91-R and 91-C fly strains to identify genome-wide structural changes within the open reading frames. Between-strain changes in allele frequencies revealed a higher percent of new alleles going to fixation for the 91-R strain, as compared to 91-C (P<0.0001). These results suggest that resistance to DDT in the 91-R laboratory strain could potentially be due primarily to new mutations, as well as being polygenic rather than the result of a few major mutations, two hypotheses that remain to be tested. [ABSTRACT FROM AUTHOR]

Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation. [ABSTRACT FROM AUTHOR]