Search

Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig. [ABSTRACT FROM AUTHOR]

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]

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]

In humans and mice, the detailed phenotypic and functional characterization of peripheral blood monocytes allows for identification of three monocyte subsets. There are also evidences of monocyte phenotypic heterogeneity in other species, including cattle, sheep, pig and horse. However, little is known about such variability in dogs. The aim of the study was to determine whether and how peripheral blood monocytes of healthy dogs differ in the presence of MHCII and CD4 and in the basal production of reactive oxygen species (ROS). Three distinct subsets of CD11b+CD14+ monocytes were found in peripheral blood samples of healthy dogs, based on the variations in the density of MHCII and CD4 surface molecules: MHCII+CD4– (Mo1), MHCII+CD4+ (Mo2) and MHCII–CD4+ (Mo3). The Mo2 and Mo3 were significantly lower in percentage than Mo1 but their basal ROS production was higher. Within the Mo2 and Mo3 subsets, the percentage of cells producing ROS was significantly higher comparing to cells lacking this activity. Canine peripheral blood monocytes vary in the expression of MHCII and CD4 and in the activity suggesting that cells within the three identified subsets carry out different functions. The higher production of ROS in non-activated cells within small subsets of Mo2 and Mo3 monocytes might indicate their immunomodulatory potential. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase receptor type Z (PTPRZ) is preferentially expressed in the central nervous system as two transmembrane receptor isoforms PTPRZ-A/B and one secretory isoform PTPRZ-S. Ptprz-knockout mice lacking the expression of all three isoforms show behavioral, learning, and neurological abnormalities, including increased exploratory activities to novelty, deficits in spatial and contextual learning, and reduced responses to methamphetamine, relative to wild-type mice. To investigate whether PTPRZ isoforms play distinct physiological roles, we herein performed behavioral studies on two knock-in mouse lines: One expresses the catalytically inactive Cys-1930 to Ser (CS) mutants of PTPRZ-A/B, while the other generated in the present study expresses catalytically active mutants of PTPRZ-A/B lacking the negative regulatory PTP-D2 domain and C-terminal PDZ-binding motif (ΔD2) instead of wild-type PTPRZ-A/-B. In contrast to Ptprz-knockout mice, neither increased responses to novelty in the open field nor memory impairments in the inhibitory-avoidance task were observed in Ptprz-CS or Ptprz-ΔD2 mice. However, the effects of methamphetamine on locomotor activity were significantly weaker in Ptprz-KO mice and CS mutant mice than in wild-type mice, but were normal in ΔD2 mutant mice. Furthermore, microdialysis experiments revealed that methamphetamine-evoked dopamine release in the nucleus accumbens was reduced in Ptprz-KO mice and CS mutant mice. These results suggest that the extracellular region of PTPRZ, including the secretory isoform, is crucial for behavioral responses to novelty and the formation of aversive memories, whereas the PTPase activities of PTPRZ receptor isoforms are involved in regulating the dopaminergic system. [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]

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32–36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function. [ABSTRACT FROM AUTHOR]

Background: Canine hemangiosarcoma (cHSA) is a highly metastatic mesenchymal cancer that disseminates by hematogenous and direct implantation routes. Therapies for cHSA are generally ineffective, in part due to advanced clinical disease stage at the time of diagnosis. The validation of conventional molecular methods for detecting novel biomarkers preferentially expressed by cHSA could lead to more timely diagnosis, earlier therapeutic interventions, and improved outcomes. In humans, prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed by prostate carcinoma and tumor-associated endothelium of various solid cancer histologies. Importantly, the preferential overexpression of PSMA by certain cancers has been leveraged for the development of diagnostic molecular imaging reagents and targeted therapeutics. Recently, PSMA has been qualitatively demonstrated to be expressed in cHSA cell lines, however, quantitative PSMA expressions and the potential utility of PSMA transcript identification in biologic fluids to support the presence of microscopic cHSA burden has not been reported. Therefore, this study sought to characterize the differential quantitative expressions of PSMA between cHSA and non-malignant tissues, and to determine the potential diagnostic utility of PCR-generated PSMA amplicons as a surrogate of rare cHSA cells dwelling within peritoneal and pericardial cavities. Methods: Quantitative gene and protein expressions for PSMA were compared between one normal endothelial and six cHSA cell lines by RT-PCR, western blot analysis, and fluorescent microscopy. Additionally, gene and protein expressions of PSMA in normal canine tissues were characterized. Graded expressions of PSMA were determined in spontaneously-arising cHSA tumor samples and the feasibility of qualitative PCR as a molecular diagnostic to detect PSMA transcripts in whole blood from healthy dogs and hemorrhagic effusions from cHSA-bearing dogs were evaluated. Results: PSMA gene and protein expressions were elevated (up to 6-fold) in cHSA cells compared with non-malignant endothelium. By immunohistochemistry, protein expressions of PSMA were detectable in all cHSA tissue samples evaluated. As predicted by human protein atlas data, PSMA’s expression was comparably identified at substantial levels in select normal canine tissues including kidney, liver, and intestine. In young healthy pet dogs, PSMA amplicons could not be identified in circulating whole blood yet were detectable in hemorrhagic effusions collected from pet dogs with confirmed cHSA or PSMA-expressing cancer. Conclusions: PSMA is quantitatively overexpressed in cHSA compared to normal endothelium, but its protein expression is not restricted to only cHSA tumor tissues, as specific visceral organs also substantively express PSMA. Optimized qualitative PCR methods failed to amplify PSMA amplicons sufficiently for visible detection in circulating whole blood derived from healthy young dogs, yet PSMA transcripts were readily identifiable in hemorrhagic effusions collected from pet dogs with histologically confirmed cHSA or PSMA-expressing cancer. While preliminary, findings derived from a limited cohort of normal and diseased pet dogs provocatively raise the potential value of PSMA amplicon detection as an ancillary molecular diagnostic test for supporting the presence of microscopic cHSA disease burden within hemorrhagic body cavity effusions. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR), the culprit behind the genetic disease cystic fibrosis (CF), is a phosphorylation-activated, but ATP-gated anion channel. Studies of human CFTR over the past two decades have provided an in-depth understanding of how CFTR works as an ion channel despite its structural resemblance to ABC transporters. Recently-solved cryo-EM structures of unphosphorylated human and zebrafish CFTR (hCFTR and zCFTR), as well as phosphorylated ATP-bound zebrafish and human CFTR offer an unprecedented opportunity to understand CFTR’s function at a molecular level. Interestingly, despite millions of years of phylogenetic distance between human and zebrafish, the structures of zCFTR and hCFTR exhibit remarkable similarities. In the current study, we characterized biophysical and pharmacological properties of zCFTR with the patch-clamp technique, and showed surprisingly very different functional properties between these two orthologs. First, while hCFTR has a single-channel conductance of 8.4 pS with a linear I-V curve, zCFTR shows an inwardly-rectified I-V relationship with a single-channel conductance of ~3.5 pS. Second, single-channel gating behaviors of phosphorylated zCFTR are very different from those of hCFTR, featuring a very low open probability Po (0.03 ± 0.02, vs. ~0.50 for hCFTR) with exceedingly long closed events and brief openings. In addition, unlike hCFTR where each open burst is clearly defined with rare short-lived flickery closures, the open bursts of zCFTR are not easily resolved. Third, although abolishing ATP hydrolysis by replacing the catalytic glutamate with glutamine (i.e., E1372Q) drastically prolongs the open bursts defined by the macroscopic relaxation analysis in zCFTR, the Po within a “locked-open” burst of E1372Q-zCFTR is only ~ 0.35 (vs. Po > 0.94 in E1371Q-hCFTR). Collectively, our data not only provide a reasonable explanation for the unexpected closed-state structure of phosphorylated E1372Q-zCFTR with a canonical ATP-bound dimer of the nucleotide binding domains (NBDs), but also implicate significant structural and functional differences between these two evolutionarily distant orthologs. [ABSTRACT FROM AUTHOR]

Repairing damaged articular cartilage is particularly challenging because of the limited ability of cartilage to perform self-repair. Intra-articular injections of N-acetylglucosamine (GlcNAc) comprise a method of repairing full-thickness articular cartilage defects in the rabbit knee joint model. To date, the effects of administration of GlcNAc and hyaluronic acid (HA) have been investigated only in the context of osteoarthritis treatment. Therefore, we evaluated the therapeutic effects of using cell-free porous poly lactic-co-glycolic acid (PLGA) graft implants and intra-articular injections of GlcNAc or HA in a rabbit model of osteochondral regeneration to investigate whether they have the potential for inducing osteochondral regeneration when used alone or simultaneously. Twenty-four rabbits were randomized into one of four groups: the scaffold-only group (PLGA), the scaffold with intra-articular injections of GlcNAc (PLGA+G) group, twice per week for four weeks; the scaffold with intra-articular injections of HA group (PLGA+HA) group, once per week for three weeks; and the scaffold with intra-articular injections of GlcNAc and HA (PLGA+G+HA) group, once per week for three weeks. Knees were evaluated at 4 and 12 weeks after surgery. At the end of testing, only the PLGA+G+HA group exhibited significant bone reconstruction, chondrocyte clustering, and good interactions with adjacent surfaces at 4 weeks. Additionally, the PLGA+G+HA group demonstrated essentially original hyaline cartilage structures that appeared to have sound chondrocyte orientation, considerable glycosaminoglycan levels, and reconstruction of the bone structure at 12 weeks. Moreover, the PLGA+G+HA group showed organized osteochondral integration and significantly higher bone volume per tissue volume and trabecular thickness. However, there were no significant differences between the PLGA+G and PLGA+HA groups except for gap formation on subchondral bone in the PLGA+G group. This study demonstrated that PLGA implantation combined with intra-articular injections of GlcNAc and HA allowed for cartilage and bone regeneration and significantly promoted osteochondral regeneration in rabbits without supplementation of exogenous growth factors. And the combination of this two supplements with PLGA scaffold could also prolong injection interval and better performance than either of them alone for the reconstruction of osteochondral tissue in the knee joints of rabbits. [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]

Type 1 polysaccharide storage myopathy (PSSM1) is a glycogen storage disorder of known cause whereas the basis for type 2 PSSM (PSSM2) is unknown. The same diet and exercise regime prescribed for PSSM1 is recommended for PSSM2; however, the benefit of these recommendations for PSSM2 is undocumented. The objectives of this study were to determine traits of PSSM2 Warmblood horses (WB), determine the changes in exercise responses that occur with a recommended low-starch/fat-supplemented diet and exercise regime, and determine if glycogen concentrations correspond to the severity of signs. Owners of PSSM2 WB (2008–2016), completed a retrospective questionnaire regarding their horse. Glycogen concentrations were analyzed in skeletal muscle of PSSM2 WB (n = 36) obtained prior to recommendations and in control WB with no evident myopathy (n = 23). Chi-square, Fisher’s exact, McNemar’s tests with Bonferroni correction and Mann Whitney testing were utilized. Abnormal exercise responses reported by owners, began at approximately 6 years of age and included a decline in performance, a reluctance to collect and reluctance to go forward in over 50% of horses. With the recommended diet and exercise regime, 80% of PSSM2 WB owners reported an overall improvement with significant decreases in the proportion of horses showing a decline in performance and rhabdomyolysis. However, 53% of PSSM2 WB were still not advancing as expected with reluctance to go forward and collect persisting in approximately one third of horses. Median muscle glycogen concentrations did not differ between PSSM2 WB and WB with no evident myopathy. PSSM2 WB with the highest glycogen concentrations were significantly more likely to show a decline in performance than those with lower glycogen concentrations. In conclusion, diet and exercise recommendations ideal for PSSM1 improve but do not eliminate the decline in performance and reluctance to go forward under saddle characteristic of PSSM2. [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]

Kinase-mediated protein phosphorylation is a central mechanism for regulation of cellular responses and phenotypes. While considerable information is available regarding the evolutionary relationships within the kinase family, as well as the evolutionary conservation of phosphorylation sites, each aspect of this partnership is typically considered in isolation, despite their clear functional relationship. Here, to offer a more holistic perspective on the evolution of protein phosphorylation, the conservation of protein phosphorylation sites is considered in the context of the conservation of the corresponding modifying kinases. Specifically, conservation of defined kinase-phosphorylation site pairings (KPSPs), as well as of each of the component parts (the kinase and the phosphorylation site), were examined across a range of species. As expected, greater evolutionary distance between species was generally associated with lower probability of KPSP conservation, and only a small fraction of KPSPs were maintained across all species, with the vast majority of KPSP losses due to the absence of the phosphorylation site. This supports a model in which a relatively stable kinome promotes the emergence of functional substrates from an evolutionarily malleable phosphoproteome. [ABSTRACT FROM AUTHOR]

While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2% of canine neoplasia, and is characterized by both aggressive local growth/invasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3K/Akt/mTOR pathway signaling. The purpose of this study was to examine the in vitro effects of a novel dual PI3K/mTOR inhibitor, VDC-597, in three canine HSA cell lines (DEN-, CIN-, and SB-HSA). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, and vascular endothelial growth factor production in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin. These results suggest that inhibitors of the PI3K/mTOR pathway may act against multiple components of the neoplastic process, including proliferation/apoptosis, chemosensitivity, migration, and angiogenesis, and justify the evaluation of PI3K/mTOR inhibitors in canine, and potentially human, HSA. [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]

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning. [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]

Vandetanib, a multi-kinase inhibitor used for the treatment of various cancers, has been reported to induce several adverse cardiac effects. However, the underlying mechanisms of vandetanib-induced cardiotoxicity are unclear. This study aimed to investigate the mechanism of vandetanib-induced cardiotoxicity using intracellular electrophysiological recordings on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), rabbit Purkinje fibers, and HEK293 cells transiently expressing human ether-a-go-go-related gene (hERG; the rapidly activating delayed rectifier K+ channel, IKr), KCNQ1/KCNE1 (the slowly activating delayed rectifier K+ current, IKs), KCNJ2 (the inwardly rectifying K+ current, IK1) or SCN5A (the inward Na+ current, INa). Purkinje fiber assays and ion channel studies showed that vandetanib at concentrations of 1 and 3 μM inhibited the hERG currents and prolonged the action potential duration. Alanine scanning and in silico hERG docking studies demonstrated that Y652 and F656 in the hERG S6 domain play critical roles in vandetanib binding. In hiPSC-CMs, vandetanib markedly reduced the maximum rate of depolarization during the AP upstroke. Ion channel studies revealed that hiPSC-CMs were more sensitive to inhibition of the INa by vandetanib than in a heterogeneously expressed HEK293 cell model, consistent with the changes in the AP parameters of hiPSC-CMs. The subclasses of Class I antiarrhythmic drugs inhibited INa currents in a dose-dependent manner in hiPSC-CMs and SCN5A-encoded HEK293 cells. The inhibitory potency of vandetanib for INa was much higher in hiPSC-CMs (IC50: 2.72 μM) than in HEK293 cells (IC50: 36.63 μM). These data suggest that AP and INa assays using hiPSC-CMs are useful electrophysiological models for prediction of drug-induced cardiotoxicity. [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]

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]

Abelson tyrosine kinase (Abl) is a key regulator of actin-related morphogenetic processes including axon guidance, where it functions downstream of several guidance receptors. While the long C-terminal domain (CTD) of Abl is required for function, its role is poorly understood. Here, a battery of mutants of Drosophila Abl was created that systematically deleted large segments of the CTD from Abl or added them back to the N-terminus alone. The functionality of these Abl transgenes was assessed through rescue of axon guidance defects and adult lethality in Abl loss-of-function, as well as through gain-of-function effects in sensitized slit or frazzled backgrounds that perturb midline guidance in the Drosophila embryonic nerve cord. Two regions of the CTD play important and distinct roles, but additive effects for other regions were also detected. The first quarter of the CTD, including a conserved PxxP motif and its surrounding sequence, regulates Abl function while the third quarter localizes Abl to axons. These regions feature long stretches of intrinsically disordered sequence typically found in hub proteins and are associated with diverse protein-protein interactions. Thus, the CTD of Abl appears to use these disordered regions to establish a variety of different signaling complexes required during formation of axon tracts. [ABSTRACT FROM AUTHOR]

Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell. [ABSTRACT FROM AUTHOR]