Deciphering Estrus Expression in Gilts: The Role of Alternative Polyadenylation and LincRNAs in Reproductive Transcriptomics.

The fertility rate and litter size of female pigs are critically affected by the expression of estrus. The objective of this study was to elucidate the regulatory mechanisms of estrus expression by analyzing the differential expression of genes and long intergenic non-coding RNAs (lincRNA), as well as the utilization of alternative polyadenylation (APA) sites, in the vulva and vagina during the estrus and diestrus stages of Large White and indigenous Chinese Mi gilts. Our study revealed that the number of differentially expressed genes (DEG) in the vulva was less than that in the vagina, and the DEGs in the vulva were enriched in pathways such as "neural" pathways and steroid hormone responses, including the "Calcium signaling pathway" and "Oxytocin signaling pathway". The DEGs in the vagina were enriched in the "Metabolic pathways" and "VEGF signaling pathway". Furthermore, 27 and 21 differentially expressed lincRNAs (DEL), whose target genes were enriched in the "Endocrine resistance" pathway, were identified in the vulva and vagina, respectively. Additionally, we observed that 63 and 618 transcripts of the 3'-untranslated region (3'-UTR) were lengthened during estrus in the vulva and vagina, respectively. Interestingly, the genes undergoing APA events in the vulva exhibited species-specific enrichment in neural or steroid-related pathways, whereas those in the vagina were enriched in apoptosis or autophagy-related pathways. Further bioinformatic analysis of these lengthened 3'-UTRs revealed the presence of multiple miRNAs binding sites and cytoplasmic polyadenylation element (CPE) regulatory aspects. In particular, we identified more than 10 CPEs in the validated lengthened 3'-UTRs of the NFIX, PCNX4, CEP162 and ABHD2 genes using RT-qPCR. These findings demonstrated the involvement of APA and lincRNAs in the regulation of estrus expression in female pigs, providing new insights into the molecular mechanisms underlying estrus expression in pigs.

Characterization and analysis of transcriptomes of multiple tissues from estrus and diestrus in pigs.

A comprehensive understanding of the complex regulatory mechanisms governing estrus and ovulation across multiple tissues in mammals is imperative to improve the reproductive performance of livestock and mitigate ovulation-related disorders in humans. To comprehensively elucidate the regulatory landscape, we analyzed the transcriptome of protein-coding genes and long intergenic non-coding RNAs (lincRNAs) in 58 samples (including the hypothalamus, pituitary, ovary, vagina, and vulva) derived from European Large White gilts and Chinese Mi gilts during estrus and diestrus. We constructed an intricate regulatory network encompassing 358 hub genes across the five examined tissues. Furthermore, our investigation identified 85 differentially expressed lincRNAs that are predicted to target 230 genes associated with critical functions including behavior, receptors, and apoptosis. Importantly, we found that vital components of estrus and ovulation events involve "Apoptosis" pathway in the hypothalamus, "Autophagy" in the ovary, as well as "Hypoxia" and "Angiogenesis" in the vagina and vulva. We have identified several differentially expressed transcription factors (TFs), such as SPI1 and HES2, which regulate these pathways. SPI1 may suppress transcription in the autophagy pathway, promoting apoptosis and inhibiting the proliferation of ovarian granulosa cells. Our study provides the most comprehensive transcriptional profiling information related to estrus and ovulation events.

Comprehensive Genome and Transcriptome Analysis Identifies SLCO3A1 Associated with Aggressive Behavior in Pigs.

Copy number variation (CNV) represents a significant reservoir of genetic diversity within the genome and exhibits a strong association with economically valuable traits in livestock. The manifestation of aggressive behavior in pigs has detrimental effects on production efficiency, immune competency, and meat quality. Nevertheless, the impact of CNV on the aggressive behavior of pigs remains elusive. In this investigation, we employed an integrated analysis of genome and transcriptome data to investigate the interplay between CNV, gene expression changes, and indicators of aggressive behavior in weaned pigs. Specifically, a subset of pigs comprising the most aggressive pigs (MAP, n = 12) and the least aggressive pigs (LAP, n = 11) was purposefully selected from a herd of 500 weaned pigs following a mixing procedure based on their composite aggressive score (CAS). Subsequently, we thoroughly analyzed copy number variation regions (CNVRs) across the entire genome using next-generation sequencing techniques, ultimately revealing the presence of 6869 CNVRs. Using genome-wide association study (GWAS) analysis and evaluating variance-stabilizing transformation (VST) values, we successfully identified distinct CNVRs that distinguished the MAP and LAP counterparts. Among the prioritized CNVRs, CNVR-4962 (designated as the top-ranked p-value and VST value, No. 1) was located within the Solute Carrier Organic Anion Transporter Family Member 3A1 (SLCO3A1) gene. The results of our analyses indicated a significantly higher (p < 0.05) copy number of SLCO3A1 in the MAP compared to the LAP. Furthermore, this increased copy number exhibited a positive correlation with the CAS of the pigs (p < 0.05). Furthermore, we integrated genomic data with transcriptomic data from the temporal lobe to facilitate the examination of expression quantitative trait loci (eQTL). Importantly, these observations were consistent with the mRNA expression pattern of SLCO3A1 in the temporal lobe of both MAP and LAP (p < 0.05). Consequently, our findings strongly suggest that CNVs affecting SLCO3A1 may influence gene expression through a dosage effect. These results highlight the potential of SLCO3A1 as a candidate gene associated with aggressive traits in pig breeding programs.

Stevioside Improves Antioxidant Capacity and Intestinal Barrier Function while Attenuating Inflammation and Apoptosis by Regulating the NF-κB/MAPK Pathways in Diquat-Induced Oxidative Stress of IPEC-J2 Cells.

As a natural sweetener, stevioside is extracted from Stevia rebaudiana Bertoni and possesses potent antioxidant activity. However, little information is known about its protective role in maintaining the intestinal epithelial cells health under oxidative stress. The aim of this study was to investigate the protective effects and underlying mechanisms of stevioside on alleviating inflammation, apoptosis, and improving antioxidant capacity in intestinal porcine epithelial cells (IPEC-J2) under oxidative stress by diquat. The results demonstrated that the pretreatment with stevioside (250 µM) for 6 h increased cell viability and proliferation and prevented apoptosis induced by diquat at 1000 µM for 6 h in IPEC-J2 cells, compared with the diquat alone-treated cells. Importantly, stevioside pretreatment significantly reduced ROS and MDA production as well as upregulated T-SOD, CAT, and GSH-Px activity. Moreover, it also decreased cell permeability and improved intestinal barrier functions by significantly upregulating the tight junction protein abundances of claudin-1, occludin, and ZO-1. At the same time, stevioside significantly down-regulated the secretion and gene expression of IL-6, IL-8, and TNF-α and decreased the phosphorylation levels of NF-κB, IκB, and ERK1/2 compared with the diquat alone group. Taken together, this study demonstrated that stevioside alleviated diquat-stimulated cytotoxicity, inflammation, and apoptosis in IPEC-J2 cells, protecting cellular barrier integrity and mitigating oxidative stress by interfering with the NF-κB and MAPK signaling pathways.

PACAP inhibition alleviates neuropathic pain by modulating Nav1.7 through the MAPK/ERK signaling pathway in a rat model of chronic constriction injury.

BACKGROUND: Trigeminal neuralgia is a common chronic maxillofacial neuropathic pain disorder, and voltage-gated sodium channels (VSGCs) are likely involved in its pathology. Prior studies report that pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide highly expressed in the trigeminal ganglion, may contribute to dorsal root ganglion neuron excitability by modulating the Nav1.7. OBJECTIVE: We investigated whether PACAP can regulate Nav1.7 through the mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway in the trigeminal ganglion after chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. STUDY DESIGN: Sprague-Dawley rats underwent ION-CCI, followed by intrathecal injection of PACAP 6-38 (PAC1 receptor antagonist) and PD98059 (MEK/ERK antagonist). Quantitative real-time PCR and western blot were used to quantify ATF3, PACAP, ERK, p-ERK, and Nav1.7 expression. RESULTS: The mechanical pain threshold decreased from day 3 to day 21 after ION-CCI and reached the lowest testing value by day 14; however, it increased after PACAP 6-38 and PD98059 injections. Additionally, ION-CCI surgery increased ATF3, PACAP, and p-ERK expression in the rat trigeminal ganglion and decreased Nav1.7 and PAC1 receptor expression; however, there was no difference in ERK expression. PACAP 6-38 injection significantly decreased PACAP, p-ERK, and Nav1.7 expression and increased the PAC1 receptor expression, with no change in ERK expression. Moreover, PD98059 injection decreased PACAP, p-ERK, and Nav1.7 expression and increased the expression of PAC1 receptor. CONCLUSION: After ION-CCI, PACAP in the rat trigeminal ganglion can modulate Nav1.7 through the MEK/ERK pathway via the PAC1 receptor. Further, PACAP inhibition alleviates allodynia in ION-CCI rats.

A Functional Single Nucleotide Polymorphism in the 3' Untranslated Region of the Porcine JARID2 Gene Is Associated with Aggressive Behavior of Weaned Pigs after Mixing.

In pig production, pigs often show more aggressive behavior after mixing, which adversely affects animal welfare and growth performance. The Jumonji and structural domain-rich AT interaction domain 2 (JARID2) gene plays an important role in neurodevelopment in mice and various psychiatric disorders in humans. The JARID2 gene may impact the aggressive behavior of pigs. By observing the behavior of 500 weaned pigs during the first 72 h after mixing, the ear tissue samples of the 12 most aggressive and 12 least aggressive pigs were selected for DNA resequencing based on the intensity of their aggressive behavior. Large group correlation analysis indicated that the rs3262221458 site located in the 3'-UTR region of the porcine JARID2 gene has a strong relationship with the aggressive behavior of weaned pigs. Pigs with the mutant TT genotype of rs3262221458 have more aggressive behavior than those pigs with the GG and GT genotypes. The dual luciferase assay indicated that the luciferase activity of the plasmids containing the G allele of rs326221458 was significantly less than that of plasmids containing the T allele of rs326221458 and control groups. The binding ability of miR-9828-3p to sequences containing the T allele was less than that of sequences containing the G allele. The overexpression of miR-9828-3p in porcine neuroglial cells (PNGCs) and PK15 cells significantly decreased the mRNA and protein levels of the JARID2 gene. In addition, miR-9828-3p inhibited the proliferation of PNGCs. After inhibiting miR-9828-3p, the mRNA and protein expression levels of JARID2 increased, and the proliferation of PNGCs showed an opposite trend to the cells that forced the expression of miR-9828-3p. In addition, interference with the JARID2 gene by siRNA can effectively inhibit the proliferation of PNGCs. In summary, we found that the rs326221458 locus regulates the expression of the JARID2 gene by affecting the binding of miR-9828-3p and the JARID2 gene, thereby affecting the aggressive behavior of weaned pigs after mixing.

Acidifiers Attenuate Diquat-Induced Oxidative Stress and Inflammatory Responses by Regulating NF-κB/MAPK/COX-2 Pathways in IPEC-J2 Cells.

In this study, we evaluated the protective effects and potential mechanisms of acidifiers on intestinal epithelial cells exposure to oxidative stress (OS). IPEC-J2 cells were first pretreated with 5 × 10-5 acidifiers for 4 h before being exposed to the optimal dose of diquat to induce oxidative stress. The results showed that acidifiers attenuated diquat-induced oxidative stress, which manifests as the improvement of antioxidant capacity and the reduction in reactive oxygen species (ROS) accumulation. The acidifier treatment decreased cell permeability and enhanced intestinal epithelial barrier function through enhancing the expression of claudin-1 and occludin in diquat-induced cells. Moreover, acidifier treatment attenuated diquat-induced inflammatory responses, which was confirmed by the decreased secretion and gene expression of pro-inflammatory (TNF-α, IL-8) and upregulated anti-inflammatory factors (IL-10). In addition, acidifiers significantly reduced the diquat-induced gene and protein expression levels of COX-2, NF-κB, I-κB-ß, ERK1/2, and JNK2, while they increased I-κB-α expression in IPEC-J2 cells. Furthermore, we discovered that acidifiers promoted epithelial cell proliferation (increased expression of PCNA and CCND1) and inhibited apoptosis (decreased expression of BAX, increased expression of BCL-2). Taken together, these results suggest that acidifiers are potent antioxidants that attenuate diquat-induced inflammation, apoptosis, and maintain cellular barrier integrity by regulating the NF-κB/MAPK/COX-2 signaling pathways.

The new role of nursing in digital inclusion: Reflections on smartphone use and willingness to increase digital skills among Chinese older adults.

OBJECTIVE: This study explores the willingness of older adults to use smartphones and improve their digital skills and encourages nursing to actively participate in bridging the digital divide. METHODS: Subject analysis was used to conduct qualitative research, and 23 older adults were interviewed. RESULTS: We identified four themes: (1) the current situation of smartphone use; (2) the digital dilemma of smartphone use; (3) social support for digital skills; and (4) the willingness to learn digital skills. Older adults in China are willing to accept and use smartphones for simple operations, and peer learning may be an effective way to improve their digital skills. CONCLUSION: Community support is necessary to develop the digital skills of older adults with smartphones and reduce the digital divide to the greatest extent possible. Nursing may play a role in promoting digital inclusion for older adults.

Effects of dietary supplement with a Chinese herbal mixture on growth performance, antioxidant capacity, and gut microbiota in weaned pigs.

Weaning stress decreases the growth performance of piglets and is one of the main concerns of pig industries. Traditional Chinese herbal medicines have been used to reduce the adverse effects of weaning stress as both nutritional supplements and antibiotic substitutes. This study aimed to evaluate the effects of a Chinese herbal mixture (Kangtaile, which contained Paeonia lactiflora, licorice, dandelion, and tea polyphenols) on the growth performances, immune response, antioxidant capacity, and intestinal microbiota of weaned pigs. A total of 400 weaned pigs [Duroc × (Landrace × Yorkshire)] were randomly allocated into one of four treatments: the CON group, fed with basic diet; the HM1 group, fed with basal diet supplemented with 0.5 g herbal mixture/kg diet; the HM2 group, fed with basal diet supplemented with 1.0 g herbal mixture/kg diet; or the HM3 group, fed with basal diet supplemented with 1.5 g herbal mixture/kg diet. The results revealed that dietary supplementation with the herbal mixture for 28 days improved average daily gain and feed conversion ratio, while decreased the diarrhea rate of weaned pigs. Moreover, dietary supple-mentation with the herbal mixture improved the antioxidant capacity through increasing the activity of catalase (CAT) and the total antioxidant capacity (T-AOC) level, while decreasing the concentration of malondialdehyde (MDA) in the serum. Pigs supplemented with herbal mixture presented an increased serum immunoglobulin (Ig)M level on day 14 compared with control pigs. The herbal mixture altered the composition of intestinal microbiota by influencing the relative abundances of Firmicutes and Bacteroidetes at the phylum level. The relative abundances of the Firmicutes and Bacteroidetes were significantly related to the body weight gain of pigs. In conclusion, supplementation of herbal mixture to the diet improved growth performance, immunity, and antioxidant capacity and modified the composition of intestinal microbiota in weaning pigs. This study provided new insights into the nutritional regulation effects of the herbal mixtures on weaned pigs.

Genome-Wide Detection of Copy Number Variations and Evaluation of Candidate Copy Number Polymorphism Genes Associated With Complex Traits of Pigs.

Copy number variation (CNV) has been considered to be an important source of genetic variation for important phenotypic traits of livestock. In this study, we performed whole-genome CNV detection on Suhuai (SH) (n = 23), Chinese Min Zhu (MZ) (n = 11), and Large White (LW) (n = 12) pigs based on next-generation sequencing data. The copy number variation regions (CNVRs) were annotated and analyzed, and 10,885, 10,836, and 10,917 CNVRs were detected in LW, MZ, and SH pigs, respectively. Some CNVRs have been randomly selected for verification of the variation type by real-time PCR. We found that SH and LW pigs are closely related, while MZ pigs are distantly related to the SH and LW pigs by CNVR-based genetic structure, PCA, VST, and QTL analyses. A total of 14 known genes annotated in CNVRs were unique for LW pigs. Among them, the cyclin T2 (CCNT2) is involved in cell proliferation and the cell cycle. The FA Complementation Group M (FANCM) is involved in defective DNA repair and reproductive cell development. Ten known genes annotated in 47 CNVRs were unique for MZ pigs. The genes included glycerol-3-phosphate acyltransferase 3 (GPAT3) is involved in fat synthesis and is essential to forming the glycerol triphosphate. Glutathione S-transferase mu 4 (GSTM4) gene plays an important role in detoxification. Eleven known genes annotated in 23 CNVRs were unique for SH pigs. Neuroligin 4 X-linked (NLGN4X) and Neuroligin 4 Y-linked (NLGN4Y) are involved with nerve disorders and nerve signal transmission. IgLON family member 5 (IGLON5) is related to autoimmunity and neural activities. The unique characteristics of LW, MZ, and SH pigs are related to these genes with CNV polymorphisms. These findings provide important information for the identification of candidate genes in the molecular breeding of pigs.

Functional variants in the promoter region of sulfotransferase 1C1 gene associated with estrogen degradation in gilts.

Chinese indigenous Mi gilts have clearer estrus expression than European Large White gilts, and sulfotransferase 1C1 (SULT1C1) gene was differentially expressed between them. To investigate the differential expression mechanism of porcine SULT1C1 gene, we cloned its promoter region and predicted its activity. Six deletion expression vectors (P1, P2, P3, P4, P5, and P6) for the promoter of SULT1C1 gene were constructed. Vector P3 (-1084/+261) had the highest expression activity, whereas vector P4 (-642/+261) showed a reduced in promoter activity, which suggests that the core promoter region of SULT1C1 gene is located between -1084 bp and -642 bp. Two single nucleotide polymorphisms (SNPs), c. - 994 G > A (rs345070974) and c. - 946 G > A (rs337902009) were found in Mi and Large White gilts between -1100 and -661 bp, and the expression vectors with four haplotypes (GG, AA, GA, and AG) of two SNPs were constructed. The relative luciferase activity of vector with haplotype GG was the greatest among four vectors. These indicate that c. - 994 G > A and c. - 946 G > A are key mutations for promoter activity of SULT1C1 gene. Porcine SULT1C1 promoter with -994 G allele and -946 G allele significantly improved the gene expression level. It could be involved in different estrus expression between Large White and Mi gilts.

Drinking Water Supplemented with Acidifiers Improves the Growth Performance of Weaned Pigs and Potentially Regulates Antioxidant Capacity, Immunity, and Gastrointestinal Microbiota Diversity.

This study evaluated the potential effects of adding acidifiers to the drinking water on the growth performance, complete blood count, antioxidant indicators, and diversity of gastrointestinal microbiota for weaned pigs. A total of 400 weaned pigs were randomly divided into four treatments. Pigs were fed the same basal diet and given either water (no acidifier was added, control) or water plus blends of different formulas of acidifiers (acidifier A1, A2, or A3) for 35 days. On d 18 and 35 of the experimental period, 64 pigs (four pigs per pen) were randomly selected to collect blood for a CBC test (n = 128) and an antioxidant indicators test (n = 128); 24 pigs (six pigs per group) were randomly selected to collect fresh feces (n = 48) from the rectum for 16S rRNA gene sequencing. Compared to the control, supplementing the drinking water with acidifiers improved the growth performance and survival rate of weaned pigs. Acidifier groups also increased serum catalase (CAT) and total antioxidant capacity (T-AOC) activities, while also displaying a decreased malondialdehyde (MDA) concentration compared to the control. The relative abundance of Firmicutes in the acidifier A1 group was greater than that in the control group (p < 0.05) on d 35; the relative abundance of Lactobacillus in the acidifier A1 group was greater than that in the control group (p < 0.05) on d 18 and 35. The microbial species Subdoligranulum or Ruminococcaceae_UCG-005 had significantly positive correlations with ADG and ADFI or with serum antioxidant indicators, respectively. These findings suggest that supplementing the drinking water with an acidifier has a potential as an antioxidant, which was reflected in the improvement of growth performance, immunity, antioxidant capacity, and intestinal flora.

Comprehensive Transcriptome Analysis of Follicles from Two Stages of the Estrus Cycle of Two Breeds Reveals the Roles of Long Intergenic Non-Coding RNAs in Gilts.

Visible and long-lasting estrus expression of gilts and sows effectively sends a mating signal. To reveal the roles of Long Intergenic Non-coding RNAs (lincRNAs) in estrus expression, RNA-seq was used to investigate the lincRNAs expression of follicular tissues from Large White gilts at diestrus (LD) and estrus (LE), and Chinese Mi gilts at diestrus (MD) and estrus (ME). Seventy-three differentially expressed lincRNAs (DELs) were found in all comparisons (LE vs. ME, LD vs. LE, and MD vs. ME comparisons). Eleven lincRNAs were differentially expressed in both LD vs. LE and MD vs. ME comparisons. Fifteen DELs were mapped onto the pig corpus luteum number Quantitative Trait Loci (QTL) fragments. A protein-protein interaction (PPI) network that involved estrus expression using 20 DEGs was then constructed. Interestingly, three predicted target DEGs (PTGs) (CYP19A1 of MSTRG.10910, CDK1 of MSTRG.10910 and MSTRG.23984, SCARB1 of MSTRG.1559) were observed in the PPI network. A competitive endogenous RNA (ceRNA) network including three lincRNAs, five miRNAs, and five genes was constructed. Our study provides new insight into the lincRNAs associated with estrus expression and follicular development in gilts.

Influencing Factors and Improvement Path of Synergistic Value Creation Efficiency of Emerging Enterprises.

The cooperation between emerging enterprises and different enterprises can stimulate innovation enthusiasm and realize synergistic value creation. At present, there is no regular pattern and rules for synergistic value creation among emerging enterprises, which cannot achieve effective synergistic value creation. Based on the complex network relationship and dynamic model of game evolution among multiple entities of value creation within emerging enterprises, the factors that affect the efficiency of synergistic value creation in the aspect of input, benefit assignment, and interaction mechanism of synergistic value creation are analyzed and then the path of the promotion of synergistic value creation among the entities of new enterprises is studied. The results show that the core enterprises play a leading role in the cooperation input among emerging enterprises, and if the balance of interests among cooperative enterprises is guaranteed, the revenue effect is the best, and the network cooperation density is the strongest. The game evolution shows that, in order to improve the efficiency of synergistic value creation, it is necessary to improve the expected coefficient and number of synergies between enterprises and establish a mechanism for equitable distribution of synergies by strengthening information exchange among enterprises in emerging industries, so as to build an atmosphere of synergies, the complementarity of assets and synergies.

A Single-Nucleotide Polymorphism in the Promoter of Porcine ARHGAP24 Gene Regulates Aggressive Behavior of Weaned Pigs After Mixing by Affecting the Binding of Transcription Factor p53.

Pigs are important biomedical model animals for the study of human neurological diseases. Similar to human aggressive behavior in children and adolescents, weaned pigs also show more aggressive behavior after mixing, which has negative effects on animal welfare and growth performance. The identification of functional single-nucleotide polymorphisms (SNPs) related to the aggressive behavior of pigs would provide valuable molecular markers of the aggressive behavioral trait for genetic improvement program. The Rho GTPase-activating protein 24 (ARHGAP24) gene plays an important role in regulating the process of axon guidance, which may impact the aggressive behavior of pigs. By resequencing the entire coding region, partially adjacent introns and the 5' and 3' flanking regions, six and four SNPs were identified in the 5' flanking region and 5' untranslated region (UTR) of the porcine ARHGAP24 gene, respectively. Association analyses revealed that nine SNPs were significantly associated with aggressive behavioral traits (p = < 1.00 × 10-4-4.51 × 10-2), and their haplotypes were significantly associated with aggressive behavior (p = < 1.00 × 10-4-2.99 × 10-2). The core promoter region of the ARHGAP24 gene has been identified between -670 and -1,113 bp. Furthermore, the luciferase activity of allele A of rs335052970 was significantly less than that of allele G, suggesting that the transcriptional activity of the ARHGAP24 gene was inhibited by allele A of rs335052970. It was identified that the transcription factor p53 bound to the transcription factor binding sites (TFBSs) containing allele A of rs335052970. In porcine primary neural cells, p53 binds to the target promoter region of the ARHGAP24 gene, reduces its promoter transcriptional activity, and then reduces its messenger RNA (mRNA) and protein expression. The results demonstrated that the ARHGAP24 gene had significant genetic effects on aggressive behavioral traits of pigs. Therefore, rs335052970 in the ARHGAP24 gene can be used as a molecular marker to select the less aggressive pigs.

Identification of functional single nucleotide polymorphisms in the porcine SLC6A4 gene associated with aggressive behavior in weaned pigs after mixing.

Variation in genes of the serotonergic system influences aggressive behavior by affecting serotonin levels in the central and cortical nervous system. SLC6A4 (serotonin transporter) is a master regulator of 5-HT signaling and involved in the regulation of aggressive behavior in humans and rodents. To identify potential functional single nucleotide polymorphisms (SNPs) for the porcine SLC6A4 gene associated with aggressive behavior, a total of 500 pigs (268 barrows and 232 gilts) were selected and mixed in 51 pens. Their behavior was recorded and observed for 72 h after mixing. Based on a composite aggressive score (CAS), the most aggressive and the least aggressive pigs within each pen were selected separately (a total of 204 pigs). Ear tissue was sampled to extract genomic DNA. Eight SNPs in the 5'-flanking region, coding region, and 3'-untranslated region (3'-UTR) of SLC6A4 were genotyped, of which 6 SNPs had significant differences (P < 0.05) in allele frequency between the most aggressive and least aggressive pigs. Luciferase activity was greater in plasmids of genotype GG than plasmids of genotype CC of rs345058216 (P < 0.01). Computational analysis nominated MAZ as putative transcription factor (TF) with higher probability to bind the SLC6A4 promoter at the SNP (rs345058216) site. Also, we demonstrated that MAZ overexpression modulates SLC6A4 promoter activity in allele-specific manner with an in vitro assay. In addition, we demonstrated that SLC6A4 was a direct target of miR-671-5p. The dual luciferase reporter gene assay and cell transfection were performed to examine the role of miR-671-5p in regulating SLC6A4 expression. The luciferase assays revealed that the SNP rs332335871 affects regulation of miR-671-5p in SLC6A4 expression. After overexpression of miR-671-5p in porcine primary neural cells, the SLC6A4 mRNA levels can be significantly reduced. In conclusion, we here found that miR-671-5p and MAZ mediated porcine SLC6A4 expression level, which provides the possible molecular mechanism of aggressive behavior.

The current study identified the functional single nucleotide polymorphisms for the porcine SLC6A4 (serotonin transporter) gene associated with aggressive behavior of pigs after mixing. We investigated the underlying molecular regulation mechanism of those functional variations. SNP rs345058216 (c.-1694C > G) affects binding of the transcription factor MAZ (MYC associated zinc finger protein) to the core promoter region of the SLC6A4 gene, whereas SNP rs332335871 (c.*1586G > A) affects the binding of miR-671-5p to the 3ʹ-UTR of SLC6A4 gene. These results suggest that rs345058216 and rs332335871 could be used as candidate molecular markers for aggressive behavior of pigs after mixing.

Functionally Antagonistic Transcription Factors IRF1 and IRF2 Regulate the Transcription of the Dopamine Receptor D2 Gene Associated with Aggressive Behavior of Weaned Pigs.

Aggressive behavior has negative effects on animal welfare and growth performance in pigs. The dopamine receptor D2 (DRD2) has a critical neuromodulator role in the dopamine signal pathway within the brain to control behavior. A functional single-nucleotide polymorphism (SNP), rs1110730503, in the promoter region of the porcine DRD2 gene was identified, which affects aggressive behavior in pigs. A chromatin immunoprecipitation (ChIP) assay was used to identify the interactions between interferon regulatory factor 1 (IRF1) and IRF2 with the DRD2 gene. The overexpression or knockdown of these two transcription factors in porcine kidney-15 (PK15) and porcine neuronal cells (PNCs) indicate that the binding of IRF1 to DRD2 promotes the transcription of the DRD2 gene, but the binding of IRF2 to the DRD2 gene inhibits its transcription. Furthermore, IRF1 and IRF2 are functionally antagonistic to each other. The downregulation of DRD2 or upregulation of IRF2 increased the apoptosis rate of porcine neuroglial cells. Taken together, we found that transcriptional factors IRF1 and IRF2 have vital roles in regulating the transcription of the DRD2 gene, and rs1110730503 (-915A/T) is a functional SNP that influences IRF2 binding to the promoter of the DRD2 gene. These findings will provide further insight towards controlling aggressive behavior in pigs.

MiR-31 targets HSD17B14 and FSHR, and miR-20b targets HSD17B14 to affect apoptosis and steroid hormone metabolism of porcine ovarian granulosa cells.

Porcine 17-hydroxysteroid dehydrogenase type 14 (HSD17B14) and FSH reporter (FSHR) genes play important roles in the metabolism of steroid hormones and the apoptosis of ovarian granulosa cells (GCs). Our bioinformatics analyses and the dual luciferase reporter assays indicated that porcine miR-20b and miR-31 target the 3'-UTR region of HSD17B14 gene, and miR-31 also targets the 3'-UTR region of FSHR gene. Overexpression of porcine HSD17B14 gene promoted the conversion from estradiol (E2) to estrone (E1) and increased the apoptosis of porcine GCs. Overexpression of miR-20b down-regulated the mRNA and protein expression level of HSD17B14 gene, decreased the concentration of progesterone (P4) and E1, increased E2, as well as reduced apoptosis of GCs. Moreover, overexpression of miR-31 also down-regulated the protein expression level of HSD17B14 gene, decreased the concentration of P4 and E1, and increased E2. However, miR-31 promoted apoptosis of GCs by targeting to the 3'-UTR of porcine FSHR gene. Taken together, we found that both porcine miR-20b and miR-31 target HSD17B14 gene, but miR-31 also targets FSHR gene to regulate the metabolism of steroid hormones and the apoptosis of porcine ovarian GCs. These findings expand the epigenetic regulatory mechanism of porcine miR-31 and miR-20b in ovarian GCs.

General Anesthesia Increased the Risk of Atrial Fibrillation and Acute Kidney Injury in Transcatheter Aortic Valve Replacement.

PURPOSE: To examine key impacts of anesthesia on new-onset atrial fibrillation (AF) and acute kidney injury (AKI) in transcatheter aortic valve replacement (TAVR). METHODS: All consecutive patients who underwent transfemoral, transapical, and transaortic TAVR in Fuwai Hospital from 2012 to 2018 were retrospectively analyzed and dichotomized into 2 groups: TAVR under conscious sedation (CS) and under general anesthesia (GA). The primary endpoint was a composite of all-cause mortality, stroke, AF, permanent pacemaker implantation, myocardial infarction, heart failure, high-grade atrioventricular block, and AKI at 1 year. Binary logistic regression and adjusted multilevel logistic regression were performed to analyze the predictors of AF and AKI. RESULTS: A total of 107 patients were under CS and 66 patients under GA. No significant difference was observed in the composite endpoint (51.5% vs. 41.2%, GA vs. CS, P = .182) and ≥ mild paravalvular leakage (36.4% vs. 31.4%, GA vs. CS, P = .589) at 1 year. However, the GA group had a significantly higher rate of intensive care unit (ICU) admission (84.8% vs. 6.5%, P < .001), AKI (28.8% vs. 14.0%, P = .018), new-onset AF (15.2% vs. 5.5% at 1 year, P = .036). Multivariable analysis revealed GA to be the significant predictor of new-onset AF (odds ratio 3.237, 95% confidence interval 1.059 to 9.894, P = .039) and AKI (odds ratio 2.517, 95% confidence interval 1.013 to 6.250, P = .047). CONCLUSION: GA was associated with higher rates of ICU admission, postoperative AKI, and new-onset AF. The results may provide new evidence that CS challenges universal GA.

Transapical transcatheter aortic valve implantation using the J-Valve system: A 1-year follow-up study.

OBJECTIVE: Transcatheter aortic valve implantation has become a routine procedure to treat screened inoperable or high-risk patients. In this study, we present the first outcome of echocardiographic midterm using a new second-generation transcatheter aortic valve implantation system, the J-Valve system (Jie Cheng Medical Technologies, Suzhou, China), in patients with aortic stenosis or aortic regurgitation. METHODS: From July 2014 to June 2015, 21 patients with isolated aortic valve disease at high risk for open surgery received transapical transcatheter aortic valve implantation using the J-Valve system. The primary end point was a combined efficacy end point after 1 year, which included all-cause mortality after more than 30 days and failure of current therapy for aortic stenosis or aortic regurgitation requiring hospitalization for symptoms of valve-related cardiac decompensation or prosthetic heart valve dysfunction. Secondary end points were cardiovascular mortality, major stroke, and life-threatening, disabling, or major bleeding after 6 and 12 months. RESULTS: The mean age of the study cohort was 75.52 ± 5.22 years, the European System for Cardiac Operative Risk Evaluation II score was 11.33% ± 1.28%, and the mean logistic European System for Cardiac Operative Risk Evaluation I score was 31.13% ± 9.68%. Transcatheter aortic valve implantation with the J-Valve system was successfully performed in 19 of the 21 patients (90.5%). For patients with aortic stenosis, 12-month follow-up echocardiography demonstrated an increase in mean effective valve area from 0.62 ± 0.17 cm2 to 1.52 ± 0.35 cm2 and a decrease in transvalvular mean gradient from 61 ± 15 mm Hg to 18 ± 9 mm Hg. All surviving patients (n = 18) reported improvements in at least 1 of the New York Heart Association classes. The combined ratio of successful implantation and absence of adverse events in our cohort (n = 21) was 76.19%. CONCLUSIONS: The J-Valve system exhibits a convincing midterm performance and is a safe and feasible procedure with low perioperative and postoperative complications.