Succinate induces skeletal muscle fiber remodeling via SUNCR1 signaling.

The conversion of skeletal muscle fiber from fast twitch to slow-twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber-type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss-of-function models generated by phospholipase Cß antagonists, SUNCR1 global knockout, or SUNCR1 gastrocnemius-specific knockdown, we found that the effects of succinate on skeletal muscle fiber-type remodeling are mediated by SUNCR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUNCR1 signaling pathway. These findings suggest the potential beneficial use of succinate-based compounds in both athletic and sedentary populations.

miR-361-3p regulates FSH by targeting FSHB in a porcine anterior pituitary cell model.

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis.

BACKGROUND: Milk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis. RESULTS: A total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot. CONCLUSIONS: These results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

Exploration of microRNAs in porcine milk exosomes.

BACKGROUND: Breast milk contains complex nutrients and facilitates the maturation of various biological systems in infants. Exosomes, membranous vesicles of endocytic origin found in different body fluids such as milk, can mediate intercellular communication. We hypothesized that microRNAs (miRNAs), a class of non-coding small RNAs of 18-25 nt which are known to be packaged in exosomes of human, bovine and porcine milk, may play important roles in the development of piglets. RESULTS: In this study, exosomes of approximately 100 nm in diameter were isolated from porcine milk through serial centrifugation and ultracentrifugation procedures. Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component. Solexa sequencing showed a total of 491 miRNAs, including 176 known miRNAs and 315 novel mature miRNAs (representing 366 pre-miRNAs), which were distributed among 30 clusters and 35 families, and two predicted novel miRNAs were verified targeting 3'UTR of IGF-1R by luciferase assay. Interestingly, we observed that three miRNAs (ssc-let-7e, ssc-miR-27a, and ssc-miR-30a) could be generated from miRNA-offset RNAs (moRNAs). The top 10 miRNAs accounted for 74.5% (67,154 counts) of total counts, which were predicted to target 2,333 genes by RNAhybrid software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using DAVID bioinformatics resources indicated that the identified miRNAs targeted genes enriched in transcription, immunity and metabolism processes, and 14 of the top 20 miRNAs possibly participate in regulation of the IgA immune network. CONCLUSIONS: Our findings suggest that porcine milk exosomes contain a large number of miRNAs, which potentially play an important role in information transfer from sow milk to piglets. The predicted miRNAs of porcine milk exosomes in this study provide a basis for future biochemical and biophysical function studies.

Differential gene expression pattern in hypothalamus of chickens during fasting-induced metabolic reprogramming: functions of glucose and lipid metabolism in the feed intake of chickens.

Fasting-induced hypothalamic metabolic reprogramming is involved in regulating energy homeostasis and appetite in mammals, but this phenomenon remains unclear in poultry. In this study, the expression patterns of a panel of genes related to neuropeptides, glucose, and lipid metabolism enzymes in the hypothalamus of chickens during fasting and refeeding were characterized by microarray analysis and quantitative PCR. Results showed that 48 h of fasting upregulated (P < 0.05) the mRNA expressions of orexigenic neuropeptide Y and agouti-related protein but downregulated (P < 0.05) that of anorexigenic neuropeptide pro-opiomelanocortin; growth hormone-releasing hormone; islet amyloid polypeptide; thyroid-stimulating hormone, ß; and glycoprotein hormones, α polypeptide. After 48 h of fasting, the mRNA expression of fatty acid ß-oxidation [peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A, and forkhead box O1], energy sensor protein [sirtuin 1 (SIRT1) and forkhead box O1], and glycolysis inhibitor (pyruvate dehydrogenase kinase, isozyme 4) were enhanced, but that of fatty acid synthesis and transport associated genes (acetyl-CoA carboxylase α, fatty acid synthase, apolipoprotein A-I, endothelial lipase, and fatty acid binding protein 7) were suppressed. Liver and muscle also demonstrated similar expression patterns of genes related to glucose and lipid metabolism with hypothalamus, except for that of acetyl-CoA carboxylase α, acyl-CoA synthetase long-chain family member 4, and apolipoprotein A-I. The results of intracerebroventricular (ICV) injection experiments confirmed that α-lipoic acid (ALA, pyruvate dehydrogenase kinase, isozyme 4 inhibitor, 0.10 µmol) and NADH (SIRT1 inhibitor, 0.80 µmol) significantly suppressed the appetite of chickens, whereas 2-deoxy-d-glucose (glycolytic inhibitor, 0.12 to 1.20 µmol) and NAD(+) (SIRT1 activator, 0.08 to 0.80 µmol) increased feed intake in chickens. The orexigenic effect of NAD(+) was also blocked by cotreatment with NADH. However, ICV injection of either GW7647 (PPARα agonist) or GW6471 (PPARα antagonist) showed no effects on feed intake. Results suggested that hypothalamic glycolysis (inhibited by ALA and promoted by 2-deoxy-d-glucose) and SIRT1 (inhibited by NADH and promoted by NAD(+)), not PPARα, were probably involved in feed intake regulation in chickens.

Roles of α-linolenic acid on IGF-I secretion and GH/IGF system gene expression in porcine primary hepatocytes.

The main purposes of this study were to investigate the effects of α-linolenic acid (ALA) on the insulin-like growth factor (IGF) system of porcine primary hepatocytes with or without growth hormone (GH) or insulin and the potential role of peroxisome proliferator-activated receptor α and -γ (PPARα/γ) pathway. We found that 1 µM ALA increased IGF-I secretion from hepatocytes at 48 and 72 h. Expression of hepatocytes IGF-I, IGF-II, GH receptor (GHR), insulin receptor (IR), IGF-binding protein 3 (IGFBP3), and IGFBP4 mRNAs was up-regulated by ALA treatment. GH (15 nM) alone or co-treated with ALA increased hepatocytes IGF-I secretion and the expression of GHR and IGFBP1 mRNAs, but down-regulated IGFBP5 mRNA compared with appropriate control across ALA. GH also enhanced the ALA-induced increase in the transcript levels of IGF-II and GHR, but tended to attenuate that of IGFBP4. Insulin (1 µM) alone or co-treated with ALA improved IGF-I secretion and the expression of IGFBP3 mRNA, but decreased IGFBP1 mRNA versus appropriate control across ALA. Insulin also up-regulated the expression of GHR, IR, IGFBP3, and IGFBP4 mRNAs, and tended to prevent the transcript levels of IGF-I and IGFBP4 improved by ALA. Both PPARγ agonist rosiglitazone and its antagonist GW9662 could elevated the IGF-I secretion in dose-dependent manner but they had no interaction with ALA. However, GW7647, a PPARα agonist, increased IGF-I secretion dose-dependently, but the antagonist GW6471 was without effect. Moreover, GW6471 prevented the IGF-I promoting effect of ALA. This suggests that the IGF-I promoting effect of ALA may be mediated by the PPARα pathway.

Identification and comparison of microRNAs from skeletal muscle and adipose tissues from two porcine breeds.

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that negatively regulate gene expression at the post-transcriptional level. Although an increasing number of porcine miRNAs recently have been identified, research has yet to identify the full repertoire of miRNAs in pig skeletal and adipose tissues and their differences between breeds. We extracted small RNA from skeletal muscle and adipose tissues of Landrace and Lantang pigs, and the expression of a total of 184 known porcine miRNAs (113 from Solexa sequencing and 171 from miRNA chip hybridization) as well as 521 novel miRNA candidates was detected. Moreover, 20 miRNAs were selected randomly from the 184 miRNAs and analysed by quantitative real-time PCR to confirm the aforementioned results. In the skeletal muscle tissues, 21 miRNAs were up-regulated in Lantang and another 33 were highly expressed in Landrace pigs. In the adipose tissues, 25 miRNAs were down-regulated in Lantang and another 23 were lowly expressed in Landrace pigs. miRNA divergence between tissues was also detected in this study. Ten miRNAs were highly expressed in the skeletal muscle tissue in comparison with adipose tissue, and another 10 miRNAs exhibited the opposite expression profile. To investigate the regulatory mechanism of the miRNAs in muscle and adipose tissues, the 10 miRNAs with the most divergent expression profiles were functionally categorized using the Kyoto Encyclopedia of Genes and Genomes database. Most of the miRNAs strongly corresponded to myogenesis and adipogenesis processes. In addition, 84 of the 521 miRNA candidates were potentially porcine-specific miRNAs. This study adds new valuable information to comparative miRNA profiles of skeletal muscle and adipose tissues in porcine species. The great diversity of miRNA composition and expression levels both between breeds and between tissues suggests that a complex regulatory network exists in porcine subcutaneous fat development.

The effect of dietary Panax ginseng polysaccharide extract on the immune responses in white shrimp, Litopenaeus vannamei.

The immunostimulatory effects of orally administered Panax ginseng root or its polysaccharides (GSP) in white shrimp, Litopenaeus vannamei, were investigated in this study. Shrimp were fed a diet containing 0.4 g kg⁻¹ GSP over a period of 84 days, during which the activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), acid phosphatase (ACP), and alkaline phosphatase (AKP), as well as malondialdehyde (MDA) content, and expressions of cytosolic superoxide dismutase (cyt-SOD), CAT, GSH-Px, and peroxiredoxin (Prx) genes were determined in various tissues of the shrimp. Results showed that the shrimp fed the GSP diet had significantly increased ACP and AKP activities in the gills. The GSP-fed shrimp also displayed significantly increased T-SOD and GSH-Px activities in the gills and hepatopancreas of the shrimp; meanwhile there was enhanced CAT activity in the gills, but decreased MDA content in the gills, hepatopancreas and muscle. The mRNA expressions of cyt-SOD, CAT, GSH-Px and Prx were significantly elevated in the gills and hepatopancreas of the shrimp fed the GSP diet for 84 days, compared with that of the control. Therefore, GSP can be used as an immunostimulant for shrimp through dietary administration to increase immune enzyme activity and modify expression of immune genes in shrimp.

Comparative Analysis of MicroRNA Expression Profiles Between Skeletal Muscle- and Adipose-Derived Exosomes in Pig.

Skeletal muscle and adipose tissues are both involved in regulation of metabolism. In the skeletal muscle-adipose tissue crosstalk, exosomes may play an important role but the main components of exosomes are not clear. In this study, we found skeletal muscle-derived exosomes can inhibit adipogenesis of porcine preadipocytes. We identified microRNA expression profiles of muscle exosomes and adipose exosomes by high-throughput sequencing. There were 104 (both novel and known microRNAs) microRNAs differentially expressed (DE miRNAs) between M-EXO (muscle-derived exosomes) and A-EXO (adipose-derived exosomes) groups. A total of 2,137 target genes of DE miRNAs for M-EXO and 2,004 target genes of DE miRNAs for A-EXO were detected. Bioinformatic analyses revealed that some DE miRNAs of M-EXO (especially miR-221-5p) were mainly enriched in lipid-related metabolism processes. The findings may serve as a fundamental resource for understanding the detailed functions of exosomes between the skeletal muscle-adipose crosstalk and the potential relationship between skeletal muscle atrophy and obesity.

Description of two new species of the genus Heterochelamon Türkay & Dai, 1997 (Crustacea: Decapoda: Brachyura: Potamidae), from southern China.

This study describes two new species of freshwater crab of the genus Heterochelamon Türkay & Dai, 1997 from southern China, H. huidongense from Guangdong Province and H. jinxiuense from Guangxi Zhuang Autonomous Region. The two new species can be differentiated from congeners by characters derived from the shape of the epibranchial tooth, external orbital angle, cheliped proportions and structure of the male first gonopod. The present study brings the number of Heterochelamon species to seven. We used the mitochondrial 16S rRNA gene for a molecular analysis and the results are consistent with the morphological features that support the recognition of two new taxa.

A new species of freshwater crab of the genus Qianguimon Huang, 2018 (Decapoda: Brachyura: Potamidae) from Guangxi, Southern China.

A new species of freshwater crab of the genus Qianguimon Huang, 2018, is described from Guangxi Zhuang Autonomous Region, southern China. It can be distinguished from congeners by the following characters: male first gonopods bent inward at about 45° at base of terminal segment, carapace regions distinct and rugged and the female vulva opening inwards and downwards. In addition, molecular evidence derived from the 16S rRNA gene supported the species described in this study as a new species of Qianguimon.

A new species of freshwater crab of the genus Mediapotamon Türkay & Dai, 1997 (Crustacea, Decapoda, Brachyura, Potamidae) from Guizhou, China.

A new species of Mediapotamon Türkay & Dai, 1997 from a karst system in southwest China is described. The new species can be separated from congeners by the combination of a sharp and distinct epibranchial tooth, the anterolateral region lined with few scattered granules, the terminal segment of the male first gonopod distinctly bent with a constant diameter, and the position of the female vulvae. Mitochondrial 16S rDNA genetic data was used to investigate the systematic position of the new species, which is supported as a new taxon.

Description of A New Species of Freshwater Crab of the Genus Qianguimon Huang, 2018 (Crustacea: Decapoda: Brachyura: Potamidae) from Yulin, Guangxi, Southern China.

A new species of freshwater crab of the genus Qianguimon Huang, 2018, Q. rongxianense sp. nov., is described from Rong County, Yulin City, Guangxi Zhuang Autonomous region, southern China. This new species resembles its congeners and some species in the genus Yarepotamon Dai & Türkay, 1997, but can be distinguished from these by its combination of the carapace, third maxilliped, male gonopod, female vulvae characters and size. Molecular data derived from the mitochondrial 16S rDNA supports the establishment of the new species, but does not provide further evidence as to its generic placement.

Two new species and the molecular phylogeography of the freshwater crab genus Bottapotamon (Crustacea: Decapoda: Brachyura: Potamidae).

Bottapotamon chenzhouense sp. n. and B. luxiense sp. n. are described from Hunan Province and Jiangxi Province, respectively. These species both have diagnostic features of the genus Bottapotamon and discernible characteristics as new species. B. chenzhouense sp. n. can be distinguished from co-geners by features such as the G1, which has a fold covering the surface of the entire subterminal article with a distal region. B. luxiense sp. n. has an elliptical carapace, and a sturdy and blunt terminal article of G1. The molecular phylogeny and biogeography of the genus Bottapotamon (Decapoda: Brachyura: Potamidae) were studied, using mitochondrial cytochrome oxidase I (mtDNA COI), 16S rRNA and nuclear histone H3 gene fragments. The results support the assignment of the two new species to the genus Bottapotamon. In addition, the divergence time of the genus Bottapotamon was estimated to be 3.49-1.08 Ma, which coincided with various vicariant and dispersal events that occurred in the geological area where the genus Bottapotamon is commonly distributed. Mountains appear to have played an important role in the distribution of this genus. The Wuyi Mountains gradually formed offshore and inland of southeastern China by the compression of the Pacific plate and the Indian plate in the Neogene-Quaternary, and the Luoxiao Mountains formed continuously in the continued forming in the north-south direction because of neotectonic movement, have resulted in the geographical distribution pattern of the genus Bottapotamon, which was also established gradually.

Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells.

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 µM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans.

CDX2 increases SLC7A7 expression and proliferation of pig intestinal epithelial cells.

Nutrient absorption mediated by nutrient transporters expressed in the intestinal epithelium supplies substrates to support intestinal processes, including epithelial cell proliferation. We evaluated the role of Caudal type homeobox 2 (CDX2), an intestine-specific transcription factor, in the proliferation of pig intestinal epithelial cells (IPEC-1) and searched for novel intestinal nutrient transporter genes activated by CDX2. Our cloned pig CDX2 cDNA contains a "homeobox" DNA binding motif, suggesting it is a transcriptional activator. CDX2 overexpression in IPEC-1 cells increased cell proliferation, the percentage of cells in S/G2 phase, and the abundance of transcripts of the cell cycle-related genes Cyclin A2; Cyclin B; Cyclin D2; proliferating cell nuclear antigen; and cell cycle cyclin-dependent kinases 1, 2 and 4, as well as the predicted CDX2 target genes SLC1A1, SLC5A1 and SLC7A7. In addition, luciferase reporter and chromatin immunoprecipitation assays revealed that CDX2 binds directly to the SLC7A7 promoter. This is the first report of CDX2 function in pig intestinal epithelial cells and identifies SLC7A7 as a novel CDX2 target gene. Our findings show that nutrient transporters are activated during CDX2-induced proliferation of normal intestinal epithelial cells.

[Analysis of cost-effectiveness of molluscacide combined with herbicide by spraying].

OBJECTIVE: To explore an economical and efficient molluscicidal method suitable for large area of nursery stock field. METHODS: Two nursery stock fields with Oncomelania hupensis were selected as experimental sites, and an experimental group and a control group were set. In the experimental group, the molluscacide and herbicide were alternately used (a purification molluscicidal method) during the period of May to October, 2011. In the control group, grass shoveling and soil burying combined with molluscacide were used in the same period. The snail control effects of the two groups were observed and the costs of the two methods were analyzed. RESULTS: No living snails were found in both experimental and control groups three consecutive years after the snail control intervention above mentioned. The costs of snail control intervention in the experimental group and control group were 0.90 and 1.80 Yuan/m2, respectively. CONCLUSION: The effect of the purification molluscicidal method in nursery stock field is satisfying, and the cost is lower.