MicroRNA-216a-5p Alleviates Acute Kidney Injury of Mice via Suppressing FAS Ligand Expression.

INTRODUCTION: The aim of this present work was to investigate the mechanism of the microRNA (miR)-216a-5p/FASL axis in mice with acute kidney injury (AKI). METHODS: Mice kidney ischemia/reperfusion (I/R) injury was used as AKI models in this study. I/R mice were injected with miR-216a-5p- and FASL-related constructs to investigate potential mechanisms of kidney protection. Kidney function, inflammation, oxidative stress, and kidney cell apoptosis were assessed after 24 h of reperfusion. In vitro, the hypoxia-reoxygenation (H/R) model was used with kidney tubular epithelial cells (TECs) to mimic kidney I/R injury. H/R-treated TECs were transfected with miR-216a-5p- and FASL-related constructs to detect cell viability, inflammation, and oxidative stress. MiR-216a-5p and FASL expression levels in mouse kidney tissues and in H/R-treated TECs were detected. RESULTS: MiR-216a-5p was downregulated and FASL was upregulated in kidney tissues of I/R mice and H/R-treated TECs. Upregulating miR-216a-5p attenuated kidney cell apoptosis and the damage of kidney function, and reduced inflammatory factor levels and oxidative stress response in kidney tissues of I/R mice. Upregulating miR-216a-5p advanced cell viability and reduced inflammatory factor levels and oxidative stress response in H/R-treated TECs. Downregulation of FASL effectively reversed the influences of downregulation of miR-216a-5p on kidney injury in mice and kidney TEC survival. CONCLUSION: Our study reveals that miR-216a-5p reduces I/R-induced pathological kidney damage in AKI via suppressing FASL.

Dexmedetomidine attenuates ferroptosis by Keap1-Nrf2/HO-1 pathway in LPS-induced acute kidney injury.

Previous research has demonstrated that Dexmedetomidine (DEX), an α2 adrenergic agonist commonly used for its sedative and analgesic properties, can attenuate lipopolysaccharide (LPS)-induced acute kidney injury (AKI). This study explores the possibility that DEX's protective effects in LPS-induced AKI are mediated through the inhibition of ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, and the activation of the antioxidant response through the Keap1/Nrf2/HO-1 signaling pathway. We induced AKI in 42 mice using LPS and divided them into six groups: saline control, LPS, LPS + DEX, LPS + Ferrostatin-1 (LPS + Fer-1; a ferroptosis inhibitor), LPS + DEX with α2-receptor antagonist Altipamizole (LPS + DEX + ATI), and LPS + DEX with Nrf2 inhibitor ML385 (LPS + DEX + ML385). After 24 h, we analyzed blood and kidney tissues. LPS exposure resulted in AKI, with increased serum creatinine, BUN, and cystatin C, and tubular damage, which DEX and Fer-1 ameliorated. However, Altipamizole and ML385 negated these improvements. The LPS group exhibited elevated oxidative stress markers and mitochondrial damage, reduced by DEX and Fer-1, but not when α2-adrenergic or Nrf2 pathways were blocked. Nrf2 and HO-1 expression declined in the LPS group, rebounded with LPS + DEX and LPS + Fer-1, and fell again with inhibitors; inversely, Keap1 expression varied. Our results demonstrate that DEX may protect against LPS-induced AKI, at least partially by regulating ferroptosis and the α2-adrenergic receptor/Keap1/Nrf2/HO-1 pathway, suggesting a potential therapeutic role for DEX in AKI management by modulating cell death and antioxidant defenses.

Genetic analysis and QTL mapping for silique density in rapeseed (Brassica napus L.).

KEY MESSAGE: Genetic models, QTLs and candidate gene for silique density on main inflorescence of rapeseed were identified. Silique density is one of the critical factors to determine seed yield and plant architecture in rapeseed (Brassica napus L.); however, the genetic control of this trait is largely unknown. In this study, the genetic model for silique density on main inflorescence (SDMI) of rapeseed was estimated according to the phenotypic data of P1 (an inbreed line with high SDMI), P2 (an inbreed line with low SDMI), F1, F2, BC1P1 and BC1P2 populations, revealing that SDMI is probably controlled by multi-minor genes with or without major gene. The QTLs for SDMI and its component characters including silique number on main inflorescence (SNMI) and main inflorescence length (MIL) were consequently mapped from a DH population derived from P1 and P2 by using a genetic linkage map constructed by restriction site-associated DNA sequencing (RAD seq) technology. A total of eight, 14 and three QTLs were identified for SDMI, SNMI and MIL under three environments, respectively, with an overlap among SDMI and SNMI in 55.7-75.4 cm on linkage group C06 which corresponding to 11.6-27.3 Mb on chromosome C06. Genomic resequencing was further conducted between a high- and a low-SDMI pool constructed from the DH population, and QTL-seq analysis identified a 0.15 Mb interval (25.98-26.13 Mb) from the C06-QTL region aforementioned. Transcriptome sequencing and qRT-PCR identified one possible candidate gene (BnARGOS) from the 0.15 Mb interval. This study will provide novel insights into the genetic basis of SD in rapeseed.

Identification and Functional Assignment of Genes Implicated in Sperm Maturation of Tibetan Sheep.

While traveling through the epididymis, immature sheep spermatozoa undergo a sequence of processes that ultimately give them the capacity to swim and fertilize an egg. Different gene expression patterns may be found in the epididymal caput, corpus, and cauda, conferring variant or unique biological roles during epididymis development and sperm maturation. To search for candidate genes associated with ovine sperm maturation and assess their possible modulating mechanisms, we characterized gene expression in each epididymal segment derived from pre- and post-pubertal Tibetan sheep by RNA sequencing. Compared with pre-puberty, 7730 (3724 upregulated and 4006 downregulated), 7516 (3909 upregulated and 3607 downregulated), and 7586 (4115 elevated and 3471 downregulated) genes were found to be differentially expressed in the post-pubertal caput, corpus, and cauda epididymis, respectively, and real-time quantitative PCR verified the validity of the gathered expression patterns. Based on their functional annotations, most differential genes were assigned to the biological processes and pathways associated with cellular proliferation, differentiation, immune response, or metabolic activities. As for the post-pubertal epididymis, 2801, 197, and 186 genes were specifically expressed in the caput, corpus, and cauda, respectively. Functional annotation revealed that they were mainly enriched to various distinct biological processes associated with reproduction (including the caput binding of sperm to the zona pellucida; fertilization in the caput and corpus; and meiosis in the caput and cauda) and development (such as cell differentiation and developmental maturation in the caput; cell proliferation and metabolism in the corpus; and regulation of tube size and cell division/cell cycle in the cauda). Additionally, we focused on the identification of genes implicated in immunity and sperm maturation, and subsequent functional enrichment analysis revealed that immune-related genes mainly participated in the biological processes or pathways associated with the immune barrier (such as JAM3 and ITGA4/6/9) and immunosuppression (such as TGFB2, TGFBR1, TGFBR2, and SMAD3), thus protecting auto-immunogenic spermatozoa. Additionally, sperm maturation was mostly controlled by genes linked with cellular processes, including cell growth, proliferation, division, migration, morphogenesis, and junction. Altogether, these results suggest that most genes were differentially expressed in developmental epididymal regions to contribute to microenvironment development and sperm maturation. These findings help us better understand the epididymal biology, including sperm maturation pathways and functional differences between the epididymal regions in Tibetan sheep and other sheep breeds.

Dexmedetomidine Alleviates Ischemia/Reperfusion-Associated Acute Kidney Injury by Enhancing Autophagic Activity via the α2-AR/AMPK/mTOR Pathway.

BACKGROUND: Dexmedetomidine (DEX) reportedly protects against ischemia-reperfusion (I/R) injury and associated damage to the kidneys, but the underlying mechanisms have yet to be established. METHODS: Unilateral nephrectomy was performed in Wistar rats, and the remaining kidney was clamped for 1 h prior to reperfusion to establish an experimental model system. These animals were then randomized into Sham, DEX + Sham, DEX + I/R, ATI (Altepamizole, α2-adrenergic receptor inhibitor) + DEX + I/R, and 3-MA (3-methyladenine, autophagy inhibitor) + DEX + I/R groups. Serum renal function biomarkers, acute kidney injury (AKI) histopathological scores, serum inflammatory factors, redox biomarkers, markers of autophagic flux, and autophagosome numbers were assessed. Levels of proteins related to the autophagic pathway, including mTOR and AMPK, were also analyzed. RESULTS: Serum creatinine and urea nitrogen levels in the I/R group were significantly elevated over those in sham control rats, as were AKI scores, serum inflammatory cytokine concentrations (IL-6, IL-1ß, and TNF-α), and serum levels of the oxidative stress biomarker malondialdehyde (MDA). All of these parameters were significantly reduced in the DEX + I/R group relative to I/R model rats. I/R group rats also exhibited significant decreases in renal levels of autophagic flux-related biomarkers and autophagosome numbers relative to sham controls, while DEX administration partially restored normal autophagic flux in these rats. Acute I/R also suppress the expression of AMPK in the kidney while increasing mTOR expression, and DEX reversed these effects. The beneficial impact of DEX on I/R-associated AKI was ablated by ATI or 3-MA administration. CONCLUSIONS: These analyses provide strong evidence for the ability of DEX to protect against I/R-associated AKI via the α2-AR/AMPK/mTOR pathway-mediated enhancement of autophagic activity.

METTL3 Regulates the Inflammatory Response in CPB2 Toxin-Exposed IPEC-J2 Cells through the TLR2/NF-κB Signaling Pathway.

Clostridium perfringens beta2 (CPB2) toxin is one of the main pathogenic toxins produced by Clostridium perfringens, which causes intestinal diseases in animals and humans. The N6-methyladenosine (m6A) modification is the most common reversible modification in eukaryotic disease processes. Methyltransferase-like 3 (METTL3) regulates immunity and inflammatory responses induced by the bacterial infections in animals. However, METTL3's involvement in CPB2-treated intestinal porcine epithelial cell line-J2 (IPEC-J2) remains unclear. In the current study, we used methylated RNA immunoprecipitation-quantitative polymerase chain reaction, Western blotting and immunofluorescence assay to determine the role of METTL3 in CPB2-exposed IPEC-J2 cells. The findings revealed that m6A and METTL3 levels were increased in CPB2 treated IPEC-J2 cells. Functionally, METTL3 overexpression promoted the release of inflammatory factors, increased cytotoxicity, decreased cell viability and disrupted tight junctions between cells, while the knockdown of METTL3 reversed these results. Furthermore, METTL3 was involved in the inflammatory response of IPEC-J2 cells by activating the TLR2/NF-κB signaling pathway through regulating TLR2 m6A levels. In conclusion, METTL3 overexpression triggered the TLR2/NF-κB signaling pathway and promoted CPB2-induced inflammatory responses in IPEC-J2 cells. These findings may provide a new strategy for the prevention and treatment of diarrhea caused by Clostridium perfringens.

Analysis of Influencing Factors of Apathy in Patients with Parkinson's Disease.

BACKGROUND: Apathy is a common non-motor symptom of Parkinson's disease (PD). The influencing factors of apathy are currently controversial. This study aimed to describe the clinical characteristics of PD-associated apathy and to analyze the associated risk factors. METHODS: Two hundred patients diagnosed with PD were selected. Included patients were divided into an apathetic group and a non-apathetic group. Demographic and clinical data, motor symptoms, non-motor symptoms and medication use of the two groups were assessed. RESULTS: The incidence of apathy was 69%. Demographic and clinical data, motor symptoms, non-motor symptoms and medications use were statistically significant. CONCLUSIONS: PD patients with more severe motor symptoms, cognitive impairment, depression, anxiety, RBD, excessive daytime sleep, fatigue, low education level, long disease course, poor quality of life and lower DA dosage are more prone to apathy. Cognitive function, quality of life, educational level, DA and LEDD are independent risk factors for apathy.

Proteome Informatics in Tibetan Sheep (Ovis aries) Testes Suggest the Crucial Proteins Related to Development and Functionality.

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein-protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

FTO Regulates Apoptosis in CPB2-Treated IPEC-J2 Cells by Targeting Caspase 3 Apoptotic Protein.

N6-methyladenosine (m6A) modification can accommodate mRNA processing, stability, and translation in mammals, and fat mass and obesity associated protein (FTO) is a vital demethylase in the m6A modification pathway. Clostridium perfringens type C (C. perfringens type C) causes diarrhea in piglets and has a serious impact on the pig industry. However, our understanding of the effect of m6A in the process of C. perfringens type C infectious piglet diarrhea (CPTCIPD) is limited. Here, an in vitro model of CPTCIPD was constructed by treating the intestinal porcine epithelial cell line-J2 (IPEC-J2) with Clostridium perfringens beta2 (CPB2) toxin, and the role of FTO was analyzed using quantitative real-time polymerase chain reaction, Western blotting, and flow cytometry. The results revealed that the overall RNA m6A contents at the tissue and cell levels were significantly up-regulated after C. perfringens infection (p < 0.05). FTO expression was significantly reduced in CPB2-treated IPEC-J2 cells. Functionally, FTO knockdown in the treated cells inhibited their proliferation and promoted apoptosis and the inflammation phenotype, whereas FTO overexpression had the opposite effects. Inhibiting FTO prolonged the half-life and up-regulated the expression of Caspase 3, leading to apoptosis. Therefore, this work explored the regulation of FTO in IPEC-J2 cells after CPB2 treatment and enhanced our understanding of the effect of the m6A modification in CPTCIPD.

TMT labeled comparative proteomic analysis reveals spleen active immune responses during Clostridium perfringens type C infected piglet diarrhea.

Background: Clostridium perfringens (C. perfringens) type C is the principal pathogenic clostridia of swine, frequently causing hemorrhagic diarrhea, even necrotic enteritis in piglets, leading to severe economic loss for swine industr ies worldwide. However, there are no specific and effective prevention measures. Therefore, clarifying the molecular mechanisms of hosts against pathogenesis infection is very important to reduce the incidence of C. perfringens type C infected piglet diarrhea disease. Methods: We performed an TMT labeling-based quantitative spleen proteomic analysis of the control group (SC), tolerance group (SR) and susceptible group (SS) to identify the differentially expressed proteins (DEPs), and screened potential molecular markers of piglet spleen tissues in response to C. perfringens type C infection. Results: In this study, a total of 115, 176 and 83 DEPs were identified in SR vs SC, SS vs SC, and SR vs SC, respectively, which may play the important regulatory roles in the process of piglet spleens in response toC. perfringens type C-infected diarrhea diseases. GO enrichment analysis revealed that the DEPs were mostly significantly enriched in acute inflammatory response, defense response, antimicrobial response, transporter activity, cellular metabolic process and so on, and KEGG pathway enrichment analysis showed that the significantly enriched immune related pathways of the PPAR signaling pathway, IL-17 signaling pathway, antigen processing and presentation, which hints at the immune defense process of piglet spleen against C. perfringens infection. This study helps to elucidate the protein expressional pattern of piglet spleen against C. perfringens type C-infected diarrhea disease, which can contribute to the prevention and control for pig diarrhea disease and the further development of diarrhea resistant pig breeding.

ssc-microRNA-132 targets DACH1 to exert anti-inflammatory and anti-apoptotic effects in Clostridium perfringens beta2 toxin-treated porcine intestinal epithelial cells.

Clostridium perfringens (C. perfringens) type C (CPC) is one of the chief pathogens that causes diarrhea in piglets, and C. perfringens beta2 (CPB2) toxin is the main virulence factor of CPC. Our previous research demonstrated that ssc-microR-132 was differentially expressed in ileal tissues of CPC-mediated diarrheic piglets and healthy piglets, which implied a potential role of ssc-microR-132 in this process. Here, we found that ssc-microR-132 was notably down-regulated in CPB2-exposed intestinal porcine epithelial cells (IPEC-J2), which was consistent with the ileal tissue expression. Moreover, ssc-microR-132 upregulation alleviated CPB2-induced inflammatory damage and apoptosis in IPEC-J2, whereas ssc-microR-132 knockdown presented the opposite effects. Furthermore, the dual-luciferase reporter assay indicated that ssc-microR-132 directly targeted Dachshund homolog 1 (DACH1). Moreover, DACH1 overexpression intensified CPB2-induced inflammatory injury and apoptosis in IPEC-J2. Remarkably, the introduction of DACH1 weakened the anti-inflammatory and anti-apoptotic effects of ssc-microR-132 in CPB2-exposed IPEC-J2. Overall, the results reveal that ssc-microR-132 targeted DACH1 to alleviate CPB2-mediated inflammation and apoptosis in IPEC-J2.

N6-Methyladenosine Methylation Analysis of Long Noncoding RNAs and mRNAs in IPEC-J2 Cells Treated With Clostridium perfringens beta2 Toxin.

Background: The n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear. Methods: Here, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells. Results: We identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells. Conclusion: In summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as "hyper-down" genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Integrating miRNA and mRNA Profiling to Assess the Potential miRNA-mRNA Modules Linked With Testicular Immune Homeostasis in Sheep.

Beyond its well-known role in spermatogenesis and androgen production, mammalian testes are increasingly recognized as an immune-privileged organ for protecting autoantigenic germ cells, especially meiotic and postmeiotic germ cells, from systemic immune responses. Despite its importance, the molecular mechanisms underlying this regulation in mammals, including sheep, are far from known. In this study, we searched for the genes associated with testicular immune privilege and assessed their possible modulating mechanisms by analyzing systematic profiling of mRNAs and miRNAs on testicular tissues derived from prepubertal and postpubertal Tibetan sheep acquired by RNA sequencing. We identified 1,118 differentially expressed (DE) mRNAs associated with immunity (245 increased mRNAs and 873 decreased mRNAs) and 715 DE miRNAs (561 increased miRNAs and 154 decreased miRNAs) in postpubertal testes compared with prepuberty. qPCR validations for 20 DE mRNAs and 16 miRNAs showed that the RNA-seq results are reliable. By using Western blot, the postpubertal testes exhibited decreased protein abundance of CD19 and TGFBR2 (two proteins encoded by DE mRNAs) when compared with prepuberty, consistent with mRNA levels. The subsequent immunofluorescent staining showed that the positive signals for the CD19 protein were observed mainly in Sertoli cells and the basement membrane of pre- and postpubertal testes, as well as the prepubertal testicular vascular endothelium. The TGFBR2 protein was found mostly in interstitial cells and germ cells of pre- and postpubertal testes. Functional enrichment analysis indicated that DE mRNAs were mainly enriched in biological processes or pathways strongly associated with the blood-testis barrier (BTB) function. Many decreased mRNAs with low expression abundance were significantly enriched in pathways related to immune response. Also, multiple key miRNA-target negative correlation regulatory networks were subsequently established. Furthermore, we verified the target associations between either oar-miR-29b or oar-miR-1185-3p and ITGB1 by dual-luciferase reporter assay. Finally, a putative schematic model of the miRNA-mRNA-pathway network mediated by immune homeostasis-related genes was proposed to show their potential regulatory roles in sheep testicular privilege. Taken together, we conclude that many immune-related genes identified in this study are negatively regulated by potential miRNAs to participate in the homeostatic regulation of testicular immune privilege of sheep by sustaining BTB function and inhibiting immune responses under normal physiological conditions. This work offers the first global view of the expression profiles of miRNAs/mRNAs involved in sheep testicular immune privilege and how the genes potentially contribute to immune-homeostatic maintenance.

Effects of miR-204 on apoptosis and inflammatory response of Clostridium perfringens beta2 toxin induced IPEC-J2 cells via targeting BCL2L2.

Clostridium perfringens beta2 (CPB2) toxin can cause intestinal damage and inflammatory responses in a variety of animals, which seriously endanger the healthy development of animal husbandry. Increasing evidence has demonstrated that microRNAs (miRNAs) can play an important regulatory role in the process of pathogenic infection. In our previous study, we found that miR-204 was highly expressed in the ileum tissues of the susceptible group diarrhea piglets after infection with Clostridium perfringens (C. perfringens) type C. In this study, we found that miR-204 was also up-regulated in different time points after CPB2 toxin treatment. Overexpression of miR-204 promoted apoptosis and inflammatory response of intestinal porcine epithelial cells (IPEC-J2), whereas the opposite results were displayed after transfected with miR-204 inhibitor. Furthermore, the luciferase reporter assays confirmed that BCL2L2 was a direct target gene of miR-204. Interestingly, we found that overexpression BCL2L2 attenuated the apoptosis and inflammatory response of CPB2 toxin induced IPEC-J2 cells. In conclusion, these results find that miR-204 promotes the apoptosis and intensify inflammatory response of CPB2 toxin induced IPEC-J2 cells via targeting BCL2L2. These data provide a valuable reference for the piglets resistance diarrhea at the molecular level.

Unraveling Stage-Dependent Expression Patterns of Circular RNAs and Their Related ceRNA Modulation in Ovine Postnatal Testis Development.

Circular RNAs (circRNAs) have been shown to function in the reproductive systems including testis. However, their expression, as well as function in testicular development of sheep remain undefined. Herein, we performed RNA sequencing to reveal circRNA temporal expression patterns in testes of Tibetan sheep from different stages of maturation (3M, 3-month-old; 1Y, 1-year-old; 3Y, 3-year-old). A total of 3,982, 414, and 4,060 differentially expressed (DE) circRNAs were uncovered from 3M vs 1Y, 1Y vs 3Y, and 3M vs 3Y, respectively. Functional enrichment assessment indicated that the source genes of DE circRNAs were primarily engaged in spermatogenesis and testicular immune privilege including blood-testis barrier (BTB). We subsequently constructed the core circRNA-miRNA-mRNA interaction network for genes related to testicular function, such as spermatogenesis, germ cell development, BTB, and cell cycle/meiosis. Furthermore, we validated the target associations between either circ_024949, circ_026259 or IGF1, and oar-miR-29b in this network, and revealed their similar expression signatures in developmental testes that they were extensively expressed in germ cells, Leydig cells, and Sertoli cells, thus suggesting their broad functions in the functional maintenance of Leydig cells and Sertoli cells, as well as the development and maturation of male germ cells. Meanwhile, circ_026259 was shown to promote IGF1 expression through inhibition of oar-miR-29b in sheep Sertoli cells. This work gives the first global view for the expression and regulation of circRNAs in sheep testis, which will be helpful for providing new insights into the molecular mechanism of ovine testis function.

Epigenetic upregulation of ssc-miR-124a following treatment with Clostridium perfringens beta2-toxin attenuates both apoptosis and inflammation in intestinal porcine epithelial cells.

Clostridium perfringens (C. perfringens) is a globally recognized zoonotic pathogen. It has been reported that the beta2-toxin produced by C. perfringens can cause a variety of gastrointestinal diseases and even systemic inflammation. MicroRNA-124a (miR-124a) has been reported to play important roles in the host response to pathogenic infection. Although C. perfringens beta2-toxin induced injury in intestinal porcine epithelial (IPEC-J2) cells has been established, the underlying molecular mechanism is not completely unraveled. Here we show that a significant upregulation of ssc-miR-124a in IPEC-J2 cells after beta2-toxin stimulation was associated with the MiR-124A-1 and MiR-124A-2 gene promoter demethylation status. Importantly, overexpression of ssc-miR-124a significantly increased cell proliferation and decreased apoptosis and cytotoxicity in beta2-toxin treated IPEC-J2 cells. Transfection of IPEC-J2 cells with ssc-miR-124a mimic suppressed beta2-toxin induced inflammation. On the contrary, ssc-miR-124a inhibitor promoted aggravation of cell apoptosis and excessive damage. Furthermore, rho-associated coiled-coil-containing protein kinase 1 (ROCK1) was identified as the direct target gene of ssc-miR-124a in IPEC-J2 cells and its siRNA transfection reversed the promotion of apoptosis and aggravation of cellular damage induced by ssc-miR-124a inhibitor. Overall, we speculated that the miR-124A-1/2 gene was epigenetically regulated in IPEC-J2 cells after beta2-toxin treatment. Upregulation of ssc-miR-124a may restrain ROCK1, and attenuate apoptosis and inflammation induced by beta2-toxin that prevent IPEC-J2 cells from severe damages. We discover a new molecular mechanism by which IPEC-J2 cells counteract beta2-toxin-induced damage through the ssc-miR-124a/ROCK1 axis partially.

Substantia nigra neuromelanin magnetic resonance imaging in patients with different subtypes of Parkinson disease.

Neuromelanin (NM) is a dark pigment that mainly exists in neurons of the substantia nigra pars compacta (SNc). In Parkinson disease (PD) patients, NM concentration decreases gradually with degeneration and necrosis of dopamine neurons, suggesting potential use as a PD biomarker. We aimed to evaluate associations between NM concentration in in vivo SN and PD progression and different motor subtypes using NM magnetic resonance imaging (NM-MRI). Fifty-four patients with idiopathic PD were enrolled. Patients were divided into groups by subtypes with different clinical symptoms: tremor dominant (TD) group and postural instability and gait difficulty (PIGD) group. Fifteen healthy age-matched volunteers were enrolled as controls. All subjects underwent clinical assessment and NM-MRI examination. PD patients showed significantly decreased contrast-to-noise ratio (CNR) values in medial and lateral SN (P < 0.05) compared to controls. CNR values in lateral SN region decreased linearly with PD progression (P = 0.001). PIGD patients showed significant decreases in CNR mean values in lateral SN compared to TD patients (P = 0.004). Diagnostic accuracy of using lateral substantia nigra (SN) in TD and PIGD groups was 79% (sensitivity 76.5%, specificity 78.6%). NM concentration in PD patients decreases gradually during disease progression and differs significantly between PD subtypes. NM may be a reliable biomarker for PD severity and subtype identification.

MicroRNA-21-5p targets PDCD4 to modulate apoptosis and inflammatory response to Clostridium perfringens beta2 toxin infection in IPEC-J2 cells.

Clostridium perfringens (C. perfringens), a toxin-producing enteric pathogen, causes a variety of intestinal infections in humans and animals. C. perfringens beta2 (CPB2) toxin has been considered to be a strong virulence factor for C. perfringens infectious enteric diseases (CPED). Altered levels and functions of microRNA-21-5p (miR-21-5p) have been associated with apoptosis and inflammation response in pathological processes. However, little is known about its functional mechanism in CPED. Here, we found that miR-21-5p expressed in multiple tissues of pig, had a highest level in jejunum, and significantly upregulated in intestinal porcine epithelial cells (IPEC-J2) exposed to CPB2 toxin. Noteworthily, transfection of CPB2-treated IPEC-J2 cells with miR-21-5p mimic increased cell viability and Bcl2 expression, as well as reduced cytotoxicity, apoptosis rates and Bax level. Moreover, overexpression of miR-21-5p significantly suppressed the levels of interleukin (IL)-6, IL-8, TNF-α, IL-1ß and nuclear factor-kappa B (NF-κB p65) activity induced by CPB2 toxin, whereas that of the IL-10 was increased in IPEC-J2 cells. On the contrary, transfection of miR-21-5p inhibitor promoted CPB2-induced cell apoptosis and inflammation. Furthermore, we validated that programmed cell death 4 (PDCD4) was strikingly downregulated in CPB2-treated IPEC-J2 cells. PDCD4 exhibited opposing effects to those of miR-21-5p mimic on IPEC-J2 cells, and restoration of PDCD4 expression counteracted the suppressive effect of miR-21-5p on CPB2-induced apoptosis and inflammatory response. Collectively, our findings demonstrated that miR-21-5p was involved in regulating the immune response triggered by CPB2 toxin and contributed to protective effects in CPB2-induced CPED cell model by targeting PDCD4.

ssc-miR-185 targets cell division cycle 42 and promotes the proliferation of intestinal porcine epithelial cell.

OBJECTIVE: microRNAs (miRNAs) can play a role in a variety of physiological and pathological processes, and their role is achieved by regulating the expression of target genes. Our previous high-throughput sequencing found that ssc-miR-185 plays an important regulatory role in piglet diarrhea, but its specific target genes and functions in intestinal porcine epithelial cell (IPEC-J2) are still unclear. We intended to verify the target relationship between porcine miR-185 and cell division cycle 42 (CDC42) gene in IPEC-J2 and to explore the effect of miR-185 on the proliferation of IPEC-J2 cells. METHODS: The TargetScan, miRDB, and miRanda software were used to predict the target genes of porcine miR-185, and CDC42 was selected as a candidate target gene. The CDC423' UTR-wild type (WT) and CDC42-3'UTR-mutant type (MUT) segments were successfully cloned into pmirGLO luciferase vector, and the luciferase activity was detected after co-transfection with miR-185 mimics and pmirGLO-CDC42-3'UTR. The expression level of CDC42 was analyzed using quantitative polymerase chain reaction and Western blot. The proliferation of IPEC-J2 was detected using cell counting kit-8 (CCK-8), methylthiazolyldiphenyltetrazolium bromide (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays. RESULTS: Double enzyme digestion and sequencing confirmed that CDC42-3'UTR-WT and CDC42-3'UTR-MUT were successfully cloned into pmirGLO luciferase reporter vector, and the luciferase activity was significantly reduced after co-transfection with miR-185 mimics and CDC42-3'UTR-WT. Further we found that the mRNA and protein expression level of CDC42 were down-regulated after transfection with miR-185 mimics, while the opposite trend was observed after transfection with miR-185 inhibitor (p<0.01). In addition, the CCK-8, MTT, and EdU results demonstrated that miR-185 promotes IPEC-J2 cells proliferation by targeting CDC42. CONCLUSION: These findings indicate that porcine miR-185 can directly target CDC42 and promote the proliferation of IPEC-J2 cells. However, the detailed regulatory mechanism of miR-185/CDC42 axis in piglets' resistance to diarrhea is yet to be elucidated in further investigation.