Multi-omics study and ncRNA regulation of anti-BmNPV in silkworms, Bombyx mori: an update.

Bombyx mori silkworm is an important economic insect which has a significant contribution to the improvement of the economy. Bombyx mori nucleopolyhedrovirus (BmNPV) is a vitally significant purulent virus that impedes the sustainable and stable development of the silkworm industry, resulting in substantial economic losses. In recent years, with the development of biotechnology, transcriptomics, proteomics, metabolomics, and the related techniques have been used to select BmNPV-resistant genes, proteins, and metabolites. The regulatory networks between viruses and hosts have been gradually clarified with the discovery of ncRNAs, such as miRNA, lncRNA, and circRNA in cells. Thus, this paper aims to highlight the results of current multi-omics and ncRNA studies on BmNPV resistance in the silkworm, providing some references for resistant strategies in the silkworm to BmNPV.

Distinct roles of carbohydrate-binding modules in multidomain ß-1,3-1,4-glucanase on polysaccharide degradation.

Lam16A is a novel GH16 ß-1,3-1,4-lichenase isolated from the genus Caldicellulosiruptor which can utilize untreated carbohydrate components of plant cell walls. Its catalytic module has been characterized that the six carbohydrate-binding modules (CBMs) were queued in the C-terminus, but their roles were still unclear. Here, full-length and CBM-truncated mutants of Lam16A were purified and characterized through heterologous expression in Escherichia coli. The profiles of these proteins, including the enzyme activity, degrading efficiency, substrate-binding affinity, and thermostability, were explored. Full-length Lam16A with six CBMs showed excellent thermostability and the highest activity against barley ß-glucan and laminarin with optimum pH of 6.5. The CBMs stimulated degrading ability of the catalytic module, especially against ß-1,3(4)-glucan-based polysaccharides. The released products from ß-1,3-1,4-glucan by Lam16A or its truncated mutants revealed an endo-type glycoside hydrolase. Lam16As exhibited strong binding affinities to the insoluble polysaccharides, especially Lam16A-1CBM. The degradation of yeast cell walls by Lam16A enzyme solution relative to the control reduced the absorbance values at OD800 by ~ 85% ± 1.2, enabling the release of up to ~ 0.057 ± 0.0039 µg/mL of the cytoplasmic protein into the supernatant, lowering the viability of the cells by ~ 70.3% ± 6.9, thus causing significant damage in the cell wall structure. Taken together, CBMs could influence the substrate specificity, thermal stability, and binding affinity of ß-1,3-1,4-glucanase. These results demonstrate the great potential of these enzymes to promote the bioavailability of ß-1,3-glucan oligosaccharides for health benefits. KEY POINTS: • Carbohydrate-binding modules strongly influenced the enzyme activity and binding affinity, and further impacted glycoside hydrolase activity. • Lam16A enzymes have sufficient ability to hydrolyze ß-1,3-1,4-glucan-based polysaccharides. • Lam16As provide a powerful tool to promote the bioavailability of ß-1,3-glucan oligosaccharides.

Identification and Validation of Common Reference Genes for Normalization of Esophageal Squamous Cell Carcinoma Gene Expression Profiles.

Esophageal squamous cell carcinoma (ESCC) is one of the subtypes of esophageal cancer with Chinese characteristics, and its five-year survival rate is less than 20%. Early diagnosis is beneficial to improving the survival rate of ESCC significantly. Quantitative Real-Time Polymerase Chain Reaction is a high-throughput technique that can quantify tumor-related genes for early diagnosis. Its accuracy largely depends on the stability of the reference gene. There is no systematic scientific basis to demonstrate which reference gene expression is stable in ESCC and no consensus on the selection of internal reference. Therefore, this research used four software programs (The comparative delta-Ct method, GeNorm, NormFinder, and BestKeeper) to evaluate the expression stability of eight candidate reference genes commonly used in other tumor tissues and generated a comprehensive analysis by RefFinder. Randomly selected transcriptome sequencing analysis confirmed the SPP1 gene is closely related to ESCC. It was found that the expression trend of SPP1 obtained by RPS18 and PPIA as internal reference genes were the same as that of sequencing. The results show that RPS18 and PPIA are stable reference genes, and PPIA + RPS18 are a suitable reference gene combination. This is a reference gene report that combines transcriptome sequencing analysis and only focuses on ESCC, which makes the quantification more precise, systematic, and standardized, and promotes gene regulation research and the early diagnosis of ESCC in the future.

Biofuel and chemical production from carbon one industry flux gas by acetogenic bacteria.

Carbon one industry flux gas generated from fossil fuels, various industrial and domestic waste, as well as lignocellulosic biomass provides an innovative raw material to lead the sustainable development. Through the chemical and biological processing, the gas mixture composed of CO, CO2, and H2, also termed as syngas, is converted to biofuels and high-value chemicals. Here, the syngas fermentation process is elaborated to provide an overview. Sources of syngas are summarized and the influences of impurities on biological fermentation are exhibited. Acetogens and carboxydotrophs are the two main clusters of syngas utilizing microorganisms, their essential characters are presented, especially the energy metabolic scheme with CO, CO2, and H2. Synthetic biology techniques and microcompartment regulation are further discussed and proposed to create a high-efficiency cell factory. Moreover, the influencing factors in fermentation and products in carboxylic acids, alcohols, and others such like polyhydroxyalkanoate and poly-3-hydroxybutyrate are addressed. Biological fermentation from carbon one industry flux gas is a promising alternative, the latest scientific advances are expatiated hoping to inspire more creative transformation.

Induction of goat bone marrow mesenchymal stem cells into putative male germ cells using mRNA for STRA8, BOULE and DAZL.

Bone mesenchymal stem cells (BMSCs) have the capacity to differentiate into germ cells (GCs). This study was conducted to develop a non-integrated method of using RNA transfection to derive putative male GCs from goat BMSCs (gBMSCs) in vitro by overexpressing STRA8, BOULE and DAZL. The gBMSCs were induced by co-transfection these three mRNAs together (mi-SBD group) or sequential transfection according to their expression time order in vivo (mi-S + BD group). After transfection, a small population of gBMSCs transdifferentiated into early germ cell-like cells and had the potential to enter meiosis. These cells expressed primordial germ cell specific genes STELLA, C-KIT and MVH, as well as premeiotic genes DAZL, BOULE, STRA8, PIWIL2 and RNF17. Importantly, the expression level of meiotic marker synaptonemal complex protein 3 significantly increased in these transfected two groups compared with control cells by qRT-PCR, immunofluorescence and western blot analysis (P < 0.05). Moreover, the protein expression of MVH was significantly higher in mi-S + BD group than that in mi-SBD group (P < 0.05). In addition, compared with control group, the methylation rate of imprinted gene H19 decreased in these two transfected group (P < 0.05), and the rate was significantly lower in mi-S + BD group compared with mi-SBD group (P < 0.05). This study helps to understand the mechanisms of action of key genes in GCs differentiation and also provides a novel system for in vitro induction of male GCs from stem cells.

Energy restriction affect liver development in Hu sheep ram lambs through Hippo signaling pathway.

This study aimed to evaluate the effects of dietary energy restriction on postnatal liver development in Hu sheep ram lambs. A total of 16 ram lambs were randomly divided into two groups: 100% energy requirements diet and 55% energy requirements diet, which were fed for 75 d. Results showed that the final body and liver weights decreased with energy restriction (p <0.05). Energy restriction caused a significant decrease in the levels of circulating insulin-like growth factor 1 (IGF1) and an increase in growth hormone secretion (p <0.05), which can be explained by the decreased mRNA expression levels of the growth hormone receptor (GHR) and IGF1 (p <0.05). The proliferating cell nuclear antigen (PCNA), Ki-67 and apoptosis-related proteins (BAX and BCL2) were mainly located in the nucleus and the cytoplasm of hepatocytes, respectively. The transcription and protein levels of PCNA and BAX were significantly decreased and increased by energy restriction, respectively (p <0.05). The caspase9 and caspase3 mRNA and activity were increased in energy restriction group (p <0.05). Moreover, Hippo signaling pathway proteins [mammalian sterile 20-like protein kinase 1 (MST1), large tumor suppressor kinase 1 (LATS1), and yes-associated protein 1 (YAP1)] were mainly observed around the hepatic portal area, and the expression levels of their mRNA and proteins were significantly decreased in energy restriction group (p <0.05). In summary, energy restriction in ram lambs impairs liver development by increasing apoptosis, which may occur via the Hippo signaling pathway.

Effects of NRF1 on steroidogenesis and apoptosis in goat luteinized granulosa cells.

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

Effects of diet and arginine treatment during the luteal phase on ovarian NO/PGC-1α signaling in ewes.

The aim of this study was to determine whether arginine (Arg) supplementation of malnourished ewes affects the expression of key NO/PGC-1α signaling pathway genes in the ovary. On Day 6-15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ± 1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9) and l-arginine group (AG; n = 9), and administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. The results of our analyses showed that both short-term feed-restriction and/or supplementation with L-Arg-HCl affected the number of different size follicles observed in the ovary, and the relative day of estrus behavior initiation of ewes. Specifically, the relative day of estrus behavior initiation was significantly advanced in AG compared with that in RG ewes (P < 0.05). Both the number of ≤2 mm-ovarian follicles (P < 0.05) and the total number of ovarian follicles (P < 0.05) were significantly increased in the RG and AG compared with that in the CG ewes. RG ewes exhibited a higher proportion of ≤2 mm (P < 0.05), but a lower proportion of >5 mm follicles than did CG ewes (P < 0.05). The mean number of corpus lutea ≥5 mm was significantly increased in AG as compared to that in either CG or RG ewes. Furthermore, the expression of eNOS, nNOS, iNOS, PDE5A, PDE9A, PRKG2, and PPARGC1A varied significantly among the treatment groups (P < 0.05). GUCY1A3 mRNA levels were significantly increased in RG and AG as compared to those in CG ewes (P < 0.05), whereas conversely, GUCY1B3 mRNA levels were significantly decreased in CG and RG as compared to those in AG ewes (P < 0.05). P53 mRNA levels were found to vary significantly among the three experimental treatment groups (P < 0.05), and similarly, the relative expression levels of P53 were greater in AG and RG than in CG ewes (P < 0.05). The levels of eNOS protein were significantly higher in RG than in either CG or AG ewes (P < 0.05). The relative expression levels of PGC-1α were significantly higher in RG (P < 0.05) and significantly lower in AG ewes (P < 0.05) than in CG ewes. In conclusion, the results of the present study indicate that feed-restriction negatively affects follicular development, and that Arg-supplementation may modulate the expression of key NO/PGC-1α signaling pathway genes in the ovary and thereby accelerate ovulatory processes and the estrous rate. Elucidation of mechanisms underlying these effects of Arg on gene expression in the ewe ovary requires further investigation.

Effect of PGC-1α overexpression or silencing on mitochondrial apoptosis of goat luteinized granulosa cells.

During goat follicular development, abnormal expression of peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1α) in granulosa cells (GCs) may contribute to follicular atresia with unknown regulatory mechanisms. In this study, we investigate the effect of ectopic expression or interference of PGC-1α on cell apoptosis of goat first passage granulosa cells (FGCs) in vitro. The results indicate that PGC-1α silencing by short hairpin RNA (shRNA) in goat FGCs significantly reduced mitochondrial DNA (mtDNA) copy number (P < 0.05), changed mitochondria ultrastructure, and induced cell apoptosis (P < 0.05). The transcription and translation levels of the apoptosis-related genes BCL-2-associated X protein (BAX), caspase 3, and caspase 9 were significantly up-regulated (P < 0.05, respectively). Moreover, the ratio of BAX/B-cell lymphoma 2 (BCL-2) was reduced (P < 0.05), and the release of cytochrome c (cyt c) and lactate dehydrogenase (LDH) was significantly enhanced (P < 0.05, respectively) in PGC-1α interference goat FGCs. Furthermore, the expression of anti-oxidative related genes superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase (CAT) was down-regulated (P < 0.05, respectively) and the activity of glutathione/glutathione disulfide (GSH/GSSG) was inhibited (P < 0.05). While enforced expression of PGC-1α increased the levels of genes involved in the regulation of mitochondrial function and biogenesis, and enhanced the anti-oxidative and anti-apoptosis capacity. Taken together, our results reveal that lack of PGC-1α may lead to mitochondrial dysfunction and disrupt the cellular redox balance, thus resulting in goat GCs apoptosis through the mitochondria-dependent apoptotic pathway.

Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells.

Myostatin, a member of the transforming growth factor-ß family, acts as a negative regulator of skeletal muscle mass. In this study, myostatin-targeted caprine fibroblasts were obtained and subjected to SCNT to determine whether myostatin-knockout goats could be created. Fibroblasts from a 2-mo-old goat were transfected with a myostatin-targeted vector to prepare transgenic donor cells for nuclear transfer. After serum-starvation (for synchronization of the cell cycle), the percentage of transgenic fibroblasts in the G(0)/G(1) phase increased (66.2% vs. 82.9%; P < 0.05) compared with that in the control group, whereas the apoptosis rate and mitochondrial membrane potential were unaffected (P > 0.05). There were no significant differences between in vivo- and in vitro-matured oocytes as recipient cytoplasts for rates of fusion (86.5% vs. 78.4%), pregnancy (21.6% vs. 16.7%), or kidding (2.7% vs. 0%). One female kid from an in vivo-matured oocyte was born, but died a few hours later. Microsatellite analysis and polymerase chain reaction identification confirmed that this kid was genetically identical to the donor cells. Based on Western blot analysis, myostatin of the cloned kid was not expressed compared with that of nontransgenic kids. In conclusion, SCNT using myostatin-targeted 2-mo-old goat fibroblasts as donors has potential as a method for producing myostatin-targeted goats.

[Progress and application prospect of pig induced pluripotent stem cells].

Pig has always been the focus of establishing a big ungulate animal ES cell lines because of its convenient source, genetic similarity with humans, and their importance in animal husbandry, but little development is achieved. Induced pluripotent stem cells technology creates a new method of reprogramming somatic cells to pluripotent state. As the pig iPS cells is established and perfected, pig ES cells will be established in the coming years. The pig iPS cells will give a hint on other livestock ES cells. On the other hand, pig iPS cells can be used to improve the efficiency of transgenic cloning pigs to conduct effective breeding and conservation of breeds. It is particularly important that the pig iPS cells can provide new model for human medical research, a new donor cells for human tissue and organ engineering, and have extensive and far-reaching impact on the biomedical field. Here, we briefly review the major progress of iPS cells, and emphasize current state of pig iPS cells and its application prospect in biomedicine and animal husbandry in order to provide a useful reference for researchers working in this area.