Identification and characterization of extrachromosomal circular DNA in Wei and Large White pigs by high-throughput sequencing.

Wei pig (WP) and Large White pig (LP) are fatty and lean breeds, respectively. Extrachromosomal circular DNA (eccDNA) plays an important role in regulating signaling pathway processes of cell. However, there are few reports regarding the eccDNA and ecDNA profiles in WP and LP. The present work aimed to investigate the eccDNA and ecDNA profiles between WP and LP. Three WPs and three LPs (100 ± 1.3 kg) were selected for analysis of eccDNA and ecDNA in the ear samples. Results showed that there were 39,686,953,656-58,411,217,258 and 53,824,168,657-58,311,810,737 clean data for WP and LP, respectively. Sequencing yielded 15,587-25,479 and 71,123-79,605 eccDNAs from the ear samples of WP and LP, respectively. There were 15,111 and 22,594 eccDNA-derived genes in the WP and LP, respectively, and 13,807 eccDNA-derived genes were common in the ear samples of both pigs. Sequencing yielded 13-19 and 27-43 ecDNAs in the ears of WP and LP, respectively. There were 1,005 and 1,777 ecDNA-derived genes in WP and LP, respectively, and 351 ecDNA-derived genes were common in the ear samples of both pigs. The most significant KEGG pathways of eccDNA-derived genes were axon guidance, focal adhesion, metabolic pathways, MAPK signaling pathway, Hedgehog signaling pathway, microRNAs in cancer, tight junction, phospholipase D signaling pathway, endocytosis, and sphingolipid signaling pathway. Furthermore, the most significant KEGG pathways of ecDNA-derived genes were olfactory transduction, B cell receptor signaling pathway, and chemical carcinogenesis. The eccDNA00044301 was lower abundance, while the ecDNA00000060 was higher abundance in WP compared with that in LP. Summary, we found that eccDNAs and ecDNAs are common in WP and LP and occur in sizes large enough to carry one or several partial or complete genes. These findings have expanded the knowledge repertoire of circular DNA in pig and will provide a reference for the use of pigs as a medical model and help discovery of new genetic markers to select high-quality breeds.

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin.

Background: Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-ß superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole. Methods: Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-ß superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA. Results: Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin. Conclusion: High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Adding distiller's grains and molasses on fermentation quality of rice straw silages / Adição de melaço na qualidade de fermentação de silagem de palha de arroz

ABSTRACT: Ensilage is a simple and low-cost strategy to enable long term preservation and environmentally friendly utilization of agricultural by-products, such as straws and distiller's grains (DG) for ruminants. Effect of mixing different proportions of DG and rice straw (i.e. 0, 10, 20 or 30% of DG) with or without 5% molasses addition on fermentation and chemical variables of silages was evaluated. The study was conducted as a randomized blocks design in a 4 × 2 factorial arrangement, with three replications, using laboratory silos of 1L capacity (n=24). Despite a significant interaction (P<0.01) between DG and molasses addition was observed for most variables, in general the increased addition of DG linearly decreased the pH value, acetic acid (AA), butyric acid (BA) and ammonia N concentration (P<0.01), and increased the lactic acid (LA) concentration (P<0.01). Exception was the propionic acid concentration which linearly decreased without molasses addition and linearly increased with molasses addition at increased proportion of DG (P<0.01). In both silages with or without molasses the addition of DG increased the dry matter, water soluble carbohydrates and crude protein (P<0.01), and decreased the NDF content (P<0.01). Based on the perspective of maximum utilization of rice straw, the mixture of 10% of DG associated to 5% molasses at ensilage process is recommended.

RESUMO: Ensilagem é uma estratégia simples e de baixo custo que habilita a preservação de sub-produtos agrícolas por longo tempo e com mínimo impacto ambiental, tal como a preservação de palha de arroz e resíduos da destilação de grãos (DG) para uso na alimentação de ruminantes. O objetivo deste estudo foi avaliar o efeito de incluir diferentes proporções de DG e palha de arroz (i.e. 0, 10, 20 or 30% of DG) com ou sem inclusão de 5% de melaço sobre variáveis da fermentação e composição química do material ensilado. O estudo foi conduzido em blocos casualizados em um esquema fatorial 4 × 2, com três repetições, utilizando mini-silos de 1L de capacidade (n=24). Embora a interação entre DG e melaço foi significativa (P<0,01) para a maior parte das variáveis, em geral a adição de DG diminuiu linearmente o pH e as concentrações de ácido acético, ácido butírico e amônia (P<0,01), e aumentou linearmente a concentração de ácido láctico (P<0,01). Exceção foi a concentração de ácido propiônico que diminuiu linearmente sem a adição de melaço enquanto aumentou linearmente com a adição de melaço, à incrementados níveis de inclusão de DG (P<0,01). Em ambos casos, com ou sem adição de melaço, a adição de DG aumentou linearmente o teor de matéria seca, de carboidratos solúveis em água e de proteína bruta, e diminuiu o teor de fibra em detergente neutro do material ensilado the NDF content (P<0,01). Baseado na perspectiva de máxima utilização de palha da arroz, recomenda-se a mistura de 10% de DG associado com 5% de melaço no processo de ensilagem.

Efficient expression and characterization of a cold-active endo-1, 4-β -glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.