Systematic Myostatin Expression Screening Platform for Identification and Evaluation of Myogenesis-Related Phytogenic in Pigs.

Skeletal muscle growth in livestock impacts meat quantity and quality. Concerns arise because certain feed additives, like beta-agonists, may affect food safety. Skeletal muscle is a specialized tissue consisting of nondividing and multinucleated muscle fibers. Myostatin (MSTN), a protein specific to skeletal muscle, is secreted and functions as a negative regulator of muscle mass by inhibiting the proliferation and differentiation of myoblasts. To enhance livestock muscle growth, phytogenic feed additives could be an alternative as they inhibit MSTN activity. The objective of this study was to establish a systematic screening platform using MSTN activity to evaluate phytogenics, providing scientific evidence of their assessment and potency. In this study, we established a screening platform to monitor myostatin promoter activity in rat L8 myoblasts. Extract of Glycyrrhiza uralensis (GUE), an oriental herbal medicine, was identified through this screening platform, and the active fractions of GUE were identified using a process-scale liquid column chromatography system. For in vivo study, GUE as a feed additive was investigated in growth-finishing pigs. The results showed that GUE significantly increased body weight, carcass weight, and lean content in pigs. Microbiota analysis indicated that GUE did not affect the composition of gut microbiota in pigs. In summary, this established rodent myoblast screening platform was used to identify a myogenesis-related phytogenic, GUE, and further demonstrated that the active fractions and compounds inhibited MSTN expression. These findings suggest a novel application for GUE in growth performance enhancement through modulation of MSTN expression. Moreover, this well-established screening platform holds significant potential for identifying and assessing a diverse range of phytogenics that contribute to the process of myogenesis.

Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization.

Traditional serological enzyme-linked immunosorbent assay (ELISA) is routinely used to monitor pathogens during quarantine in most animal facilities to prevent possible infection. However, the ELISA platform is a single-target assay, and screening all targeted pathogens is time-consuming and laborious. In this study, to increase sensitivity and to reduce diagnosis time for high-throughput processes, multiplex PCR and DNA biochip techniques were combined to develop a multi-pathogen diagnostic method for use instead of routine ELISA. Eight primer sets were designed for multiplex PCR to detect genes from seven targeted bacterial and viral pathogens. DNA-DNA hybridization was conducted on a biochip following the multiple PCR analysis. Using this method, a total of 24 clinical samples were tested, and the result showed that not only single infection but also co-infection by multi-pathogens can be detected. In conclusion, multiplex PCR coupled with a DNA biochip is an efficient method for detecting multi-pathogens in a reaction. This platform is a useful tool for quarantine services and disease prevention in animal facilities.

Genome-Wide Scan and Test of Candidate Genes in the Snail Biomphalaria glabrata Reveal New Locus Influencing Resistance to Schistosoma mansoni.

BACKGROUND: New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails. METHODOLOGY/PRINCIPAL FINDINGS: Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2 Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated. CONCLUSIONS/SIGNIFICANCE: The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation.

Hyperdiverse gene cluster in snail host conveys resistance to human schistosome parasites.

Schistosomiasis, a neglected global pandemic, may be curtailed by blocking transmission of the parasite via its intermediate hosts, aquatic snails. Elucidating the genetic basis of snail-schistosome interaction is a key to this strategy. Here we map a natural parasite-resistance polymorphism from a Caribbean population of the snail Biomphalaria glabrata. In independent experimental evolution lines, RAD genotyping shows that the same genomic region responds to selection for resistance to the parasite Schistosoma mansoni. A dominant allele in this region conveys an 8-fold decrease in the odds of infection. Fine-mapping and RNA-Seq characterization reveal a <1Mb region, the Guadeloupe Resistance Complex (GRC), with 15 coding genes. Seven genes are single-pass transmembrane proteins with putative immunological roles, most of which show strikingly high nonsynonymous divergence (5-10%) among alleles. High linkage disequilibrium among three intermediate-frequency (>25%) haplotypes across the GRC, a significantly non-neutral pattern, suggests that balancing selection maintains diversity at the GRC. Thus, the GRC resembles immune gene complexes seen in other taxa and is likely involved in parasite recognition. The GRC is a potential target for controlling transmission of schistosomiasis, including via genetic manipulation of snails.

Molecular screening of Chinese medicinal plants for progestogenic and anti-progestogenic activity.

Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species - Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita - were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 (mu)g/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40-100 (mu)g/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy.

Growth inhibition and antioxidative status induced by selenium-enriched broccoli extract and selenocompounds in DNA mismatch repair-deficient human colon cancer cells.

The effects of enzymatic-digested Se-enriched broccoli extracts (SeB) and selenocompounds on growth and antioxidative status in human colon cancer cells was investigated in this study. HCT116 and HCT116+Chr.3 cells were treated with selenocompounds (sodium selenite, sodium selenate, Se-Met, MeSeCys) or SeB [high-Se (H-SeB) or low-Se (L-SeB)]. The cytotoxicity induced by selenocompounds in HCT116 cells was not associated with cellular H2O2 level, while the differential cytotoxicity observed by sodium selenite between HCT116 and HCT116+Chr.3 cell lines was related to cellular H2O2 production with the change in antioxidative enzyme activity, and the restoration of chromosome 3. H-SeB was found to reduce the cellular H2O2 content in HCT116+Chr.3 cells. The results in this study indicate that regardless of Se content, the cytotoxicity in HCT116 cells of both SeB forms appeared to be H2O2-independent, whereas the cytotoxicity in HCT116+Chr.3 of either SeB form appeared to be H2O2-dependent with an increase in antioxidative ability for H-SeB.

Antibacterial characteristics and activity of water-soluble chitosan derivatives prepared by the Maillard reaction.

The antibacterial activity of water-soluble chitosan derivatives prepared by Maillard reactions against Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Shigella dysenteriae, and Salmonella typhimurium was examined. Relatively high antibacterial activity against various microorganisms was noted for the chitosan-glucosamine derivative as compared to the acid-soluble chitosan. In addition, it was found that the susceptibility of the test organisms to the water-soluble chitosan derivative was higher in deionized water than in saline solution. Metal ions were also found to reduce the antibacterial activity of the water-soluble chitosan derivative on S. aureus. The marked increase in glucose level, protein content and lactate dehydrogenase (LDH) activity was observed in the cell supernatant of S. aureus exposed to the water-soluble chitosan derivative in deionized water. The results suggest that the water-soluble chitosan produced by Maillard reaction may be a promising commercial substitute for acid-soluble chitosan.

Antioxidant properties and antioxidant compounds of various extracts from the edible basidiomycete Grifola frondosa (Maitake).

Grifola frondosa is an edible mushroom currently available in Taiwan. Ethanolic, cold-water and hot-water extracts were prepared and their antioxidant properties were investigated. At 1 mg/mL, G. frondosa T1 and T2 cold-water extracts showed high reducing powers of 1.02 and 0.50, respectively. Chelating abilities on ferrous ions of G. frondosa T1 and T2 were higher for cold-water extracts than for ethanolic and hot-water extracts. For the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, G. frondosa T1 and T2 extracts were effective in the following order: ethanolic > hot-water > cold-water. The G. frondosa hot-water extract showed high scavenging ability on superoxide anions. Total phenols, flavonoids, ascorbic acid and α-tocopherol are the major antioxidant components found in the various G. frondosa extracts. Based on EC(50) values (<20 mg/mL) obtained, the various extracts from G. frondosa investigated in this study display potent antioxidative properties.

Characterization and expression of chicken selenoprotein W.

As an essential trace element, selenium (Se) deficiency results in White Muscle Disease in livestock and Keshan disease in humans. The main objectives of this study were to clone and characterize the chicken selenoprotein W (SeW) gene and investigate SeW mRNA expression in chicken tissues. The deduced amino acid (AA) sequence of chicken SeW contains 85 AAs with UAG as the stop codon. Like all SeW genes identified in different species, chicken SeW contains one well-conserved selenocysteine (Sec) at the 13th position encoded by the UGA codon. The proposed glutathione (GSH)-binding site at the Cys(37) of SeW is not conserved in the chicken, but Cys(9) and Sec(13), with possible GSH binding, are conserved in SeWs identified from all species. There are 23-59% and 50-61% homology in cDNA and deduced AA sequences of SeW, respectively, between the chicken and other species. The predicted secondary structure of chicken SeW mRNA indicates that the selenocysteine insertion sequence element is type II with invariant adenosines within the apical bulge. The SeW mRNA expression is high in skeletal muscle followed by brain, but extremely low in other tissues from chickens fed a commercial maize-based diet. The SeW gene is ubiquitously expressed in heart, skeletal muscle, brain, testis, spleen, kidney, lung, liver, stomach and pancreas in chickens fed a commercial diet supplemented with sodium selenite. These results indicate that dietary selenium supplementation regulates SeW gene expression in the chicken and skeletal muscle is the most responsive tissue when dietary Se content is low.

Production of bioactive human alpha-defensin 5 in Pichia pastoris.

Human alpha-defensin 5 (HD5), a small cationic peptide, is expressed in Paneth cell granules of small intestinal crypts. HD5 exhibits high antimicrobial activity against a broad spectrum of pathogenic agents, including bacteria, fungi, and viruses. In this study, the constitutive expression of HD5 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris (P. pastoris). HD5 cDNA was amplified by polymerase chain reaction (PCR) using human lung cell cDNA as template. The 96-bp DNA fragment encoding mature HD5 peptide (amino acid 63-94) was subcloned into the yeast expression vector and transfected into P. pastoris X-33 expression host by electroporation. The recombinant HD5 (rHD5) was detected in the supernatant of transfected yeast by western blot analysis. The recombinant HD5 crude extract from transfected P. pastoris showed antimicrobial activities against Salmonella typhimurium, Staphylococcus aureus and pathogenic E. coli. However, rHD5 did not inhibit the growth of lactic acid bacteria such as Lactobacillus bulgaricus, Bifidobacterium bifidum, or B. longum. These results indicated that the rHD5 expressed in P. pastoris selectively inhibited the growth of specific bacteria.

Glutathione regulation in arsenic-induced porcine aortic endothelial cells.

The objective was to investigate the regulation of glutathione (GSH) turnover in porcine aortic endothelial cells (PAECs) treated with sodium arsenite (NaAsO(2)), arsenic trioxide (As(2)O(3)) or sodium arsenate (Na(2)HAsO(4)) up to 72 hr at 0, 1, 5, and 10 microM, respectively. Intracellular GSH and glutathione disulfide (GSSG) contents, as well as the activities and mRNA levels of glutamate-cysteine lyase (GCL; gamma-glutamylcysteine synthetase) and gamma-glutamyl transpeptidase (GGT), were examined. The trivalent arsenic compounds increased GSH and GSSG contents in PAECs. An increase in GCL activity was observed at 24hr whereas an increase in GCL mRNA level was observed at 72 hr. The increase in GGT activity was only observed at 72 hr. In addition, a tendency of increase in GGT mRNA level was observed. Na(2)HAsO(4) treatment did not affect GSH content and the turnover-related enzymes. A differential GSH modulation in PAECs by trivalent arsenic compounds was found. The regulatory mechanism responsible for the As(2)O(3)-induced GSH increase is related to the GSH-turnover enzymes, GCL and GGT, while that for the NaAsO(2)-induced GSH increase may not be related to expression of GSH-turnover enzymes.

Safety evaluation of water extracts of Toona sinensis Roemor leaf.

The purposes of this study were to evaluate the safety of water extracts of Toona sinensis Roemor leaf (TSL-1). The mutagenic properties of TSL-1 was investigated using the Ames test, and no mutagenicity was found toward all tester strains (Salmonella typhimurium TA98, TA100, TA102 and TA1535). In the acute oral toxicity study, a single limit dose of 5000 mgTSL-1/kg bw was given to male and female ICR mice, then observed for a 14-day period. In the subacute study, TSL-1 was administered as oral daily dose of 1000 mg/kg bw/day for 28 days. The results showed no acute lethal effect at a maximal tested dose of 5000 mg/kg bw TSL-1 in male and female mice. The subacute toxicity showed the oral administration of 1000 mg/kg bw for consecutive 28 days was safe in male mice. TSL-1 treated female mice showed decreases of food intake and kidney relative weight in acute oral toxicity test, and decreases of body weight gain, food intake and lung relative weight in subacute toxicity trial. However, no remarked toxic effects were found in the biochemical and histopathological parameters of TSL-1 treated female mice. These effects whether related to the major components or other ingredients in TSL-1 need to elucidate in the further studies.

Effect of maternal myostatin antibody on offspring growth performance and body composition in mice.

Myostatin (GDF8) is a member of the transforming growth factor beta (TGF-beta) superfamily. The finding that animals with a knockout or mutation of the myostatin-encoding gene show increased muscle mass suggests that myostatin negatively regulates muscle growth. The study reported here was designed to investigate the effect of induction of maternal myostatin antibody on the growth performance and body composition of the mouse. Female mice were induced to produce myostatin antibody by immunization with synthetic myostatin peptide prior to mating with male mice. The body masses of offspring were measured weekly and the body compositions of offspring were determined at 8 weeks of age. The results showed that myostatin antibody was detected in both immunized female mice and their 8-week-old offspring. The growth performance of offspring from the myostatin antibody-induced (mstn Ab-induced) group was higher than that from the control group at 8 weeks of age. The body composition of both male and female offspring from the mstn Ab-induced group contained higher crude protein and lower crude fat than those from the control group (P<0.05). The litter number from the maternal mstn Ab-induced group was less than that from control mice, while embryo development was normal in both groups. However, the amount of developing follicle in ovaries of the mstn Ab-induced group was lower than that in the control group. It is concluded that induction of maternal mstn Ab enhances the growth performance of offspring and influences the offspring body composition by increasing the crude protein and reducing crude fat.

Mechanism for proliferation inhibition by various selenium compounds and selenium-enriched broccoli extract in rat glial cells.

The objective of this study was to investigate the differential effects of various selenium (Se) compounds and Se-enriched broccoli extracts on cell proliferation and the possible mechanism responsible for the Se-induced growth inhibition. C6 rat glial cells were incubated with graded concentrations up to 1000 nM of selenite, selenate, selenomethionine (SeM), Se-methyl-selenocysteine (SeMCys), high-Se broccoli (H-SeB) extract or low-Se broccoli (L-SeB) extract for 24 and 48 h. MTT results indicated that all Se sources and levels examined inhibited C6 cell proliferation at 48 h. The results from cell cycle progression and apoptosis analysis indicated that SeM, SeMCys, H-SeB or L-SeB treatments at the concentration of 1000 nM reduced the cell population in G(0)/G(1) phase, but induced G(2)/M phase arrest and increased apoptosis and secondary necrosis in C6 cells at 24 h. The populations of apoptotic cells and secondary necrotic cells were increased by all Se sources examined. The COMET assay indicated that there was no significant DNA single-strand break found for all Se treatments in C6 cells for 48 h. In addition, the Se-induced proliferation inhibition may involve a hydrogen peroxide (H(2)O(2))-dependent mechanism with elevated cellular glutathione peroxidase (cGPX) activity. Both H-SeB and L-SeB inhibited C6 cell proliferation but H-SeB was less inhibitory than L-SeB. The proliferation inhibition by H-SeB in C6 cells is apparently related to the increased H(2)O(2) with the elevated cGPX activity, but the inhibition by L-SeB was H(2)O(2)-independent without change in cGPX activity.

Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation.

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.

Modulation of the arsenic effects on cytotoxicity, viability, and cell cycle in porcine endothelial cells by selenium.

The differential effects of arsenic compounds and the effect of selenium on arsenic-induced changes in cytotoxicity, viability, and cell cycle of porcine aorta endothelial cells (PAECs) were investigated. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay indicated that arsenic trioxide (As(2)O(3)) and sodium arsenite (NaAsO(2)) showed similar cytotoxicity, whereas sodium arsenate (Na(2)HAsO(4)) did not show cytotoxicity in PAECs. As(2)O(3) and NaAsO(2) at 20 microM decreased PAEC viability, decreased G0/G1 phase, and increased apoptosis. An increased G2/M phase was observed in NaAsO(2)-treated PAECs, whereas an increase in secondary necrosis (late apoptosis) was observed in As(2)O(3)-treated PAECs. As(2)O(3)-induced apoptosis was associated with upregulation of p53 and caspase 3, whereas NaAsO(2)-induced apoptosis was associated with p53 upregulation. Sodium selenite (Na(2)SeO(3)) at 1 nM reduced 20 microM As(2)O(3)-induced cytotoxicity, but not apoptosis, at 24 h. Increased glutathione peroxidase (GPX) activity by Na(2)SeO(3) pretreatment in 20 microM As(2)O(3)-treated PAECs suggests that Na(2)SeO(3) modulates As(2)O(3)-induced cytoxicity by GPX modulation.