Effects of different precursors on the structure of lignin-based biochar and its ability to adsorb benzopyrene from sesame oil.

Agricultural by-products of sesame are promising bioresources in food processing. This study extracted lignin from the by-products of sesame oil production, namely, the capsules and straw of black and white sesame. Using acid, alkali, and ethanol methods, 12 distinct lignins were obtained to prepare biochar, aiming to investigate both the structural characteristics of lignin-based biochar (LBB) and its ability to remove benzo[a]pyrene (BaP) from sesame oil. The results showed that white sesame straw was the most suitable raw material for preparing biochar. In terms of the preparation method, acid-extracted lignin biochar was more effective in removing BaP than alkaline or ethanol methods. Notably, WS-1LB (white sesame straw acid-extracted lignin biochar) exhibited the highest BaP adsorption efficiency (91.44 %) and the maximum specific surface area (1065.8187 m2/g), characterized by porous structures. The pseudo 2nd and Freundlich models were found to be the best fit for the adsorption kinetics and isotherms of BaP on LBB, respectively, suggesting that a multilayer adsorption process was dominant. The high adsorption of LBB mainly resulted from pore filling. This study provides an economical and highly efficient biochar adsorbent for the removal of BaP in oil.

Comparison of methods for activating sesame stalk lignin biochar for removing benzo[a]pyrene from sesame oil.

In this study, three activators and two activation methods were employed to activate sesame lignin-based biochar. The biochar samples were comprehensively characterized, their abilities to adsorb benzo[a]pyrene (BaP) from sesame oil were assessed, and the mechanism was analyzed. The results showed that the biochar obtained by one-step activation was more effective in removing BaP from sesame oil than the biochar produced by two-step activation. Among them, the biochar generated by one-step activation with ZnCl2 as the activator had the largest specific surface area (1068.8776 m3/g), and the richest mesoporous structure (0.7891 m3/g); it removed 90.53 % of BaP from sesame oil. BaP was mainly adsorbed by the mesopores of biochar. Mechanistically, pore-filling, π-π conjugations, hydrogen bonding, and n-π interactions were involved. The adsorption was spontaneous and heat-absorbing. In conclusion, the preparation of sesame lignin biochar using one-step activation with ZnCl2 as the activator was found to be the best for removing BaP from sesame oil. This biochar may be an economical adsorbent for the industrial removal of BaP from sesame oil.

Mechanism of gemcitabine combined with lobaplatin in interventional treatment of locally advanced cervical cancer.

In order to investigate the mechanism of gemcitabine combined with lobaplatin in the interventional treatment of locally advanced cervical cancer (LACC), 90 patients with LACC were divided into control group (oxaliplatin + gemcitabine) and experimental group (lobaplatin + gemcitabine) according to different perfusion drugs and embolization drugs, 45 cases in each group. They were treated with arterial chemotherapy and arterial embolization. Postoperative recurrence, metastasis, and survival, as well as changes in serum vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) levels before and after treatment were observed in both groups. The results showed that the recurrence rate of cervical cancer at 0.5, 1, 2, 3, 4, and 5 years after operation in the experimental group was significantly lower than that in the control group, P  < 0.05; there was no significant difference in the postoperative cervical cancer metastasis rate, P  > 0.05. Before treatment, the serum VEGF in the experimental group and the control group were (642.76 ±â€…216.67) ng/L and (626.30 ±â€…275.43) ng/L, respectively, and MMP-9 were (580.61 ±â€…194.12) ng/L and (575.28 ±â€…202.55) ng/L, respectively. After treatment, the serum VEGF levels in the experimental group and the control group were (429.24 ±â€…132.69) ng/L and (554.63 ±â€…178.11) ng/L, respectively, and MMP-9 levels were (357.60 ±â€…123.11) ng/L and (461.83 ±â€…144.45) ng/L, respectively. There was no significant difference in the serum VEGF and MMP-9 levels between the two groups before treatment ( P  > 0.05); after treatment, the serum VEGF and MMP-9 levels in the experimental group were significantly lower than those in the control group, P  < 0.05. Therefore, gemcitabine combined with lobaplatin interventional therapy can improve the cure rate of LACC by reducing VEGF and MMP-9 levels in the serum of patients.

Regulatory Effects of the Kiss1 Gene in the Testis on Puberty and Reproduction in Hezuo and Landrance Boars.

Kisspeptin, a neuropeptide encoded by the Kiss1 gene, combines with its receptor Kiss1R to regulate the onset of puberty and male fertility by the hypothalamic-pituitary-gonadal axis. However, little is known regarding the expression signatures and molecular functions of Kiss1 in the testis. H&E staining revealed that well-arranged spermatogonia, spermatocytes, round and elongated spermatids, and spermatozoa, were observed in 4-, 6-, and 8-month-old testes compared to 1- and 3-month-old testes of Hezuo pigs; however, these were not observed in Landrance until 6 months. The diameter, perimeter, and cross-sectional area of seminiferous tubules and the perimeter and area of the tubular lumen increased gradually with age in both pigs. Still, Hezuo pigs grew faster than Landrance. The cloning results suggested that the Hezuo pigs' Kiss1 CDS region is 417 bp in length, encodes 138 amino acids, and is highly conserved in the kisspeptin-10 region. qRT-PCR and Western blot indicated that the expression trends of Kiss1 mRNA and protein were essentially identical, with higher expression levels at post-pubertal stages. Immunohistochemistry demonstrated that the Kiss1 protein was mainly located in Leydig cells and post-pubertal spermatogenic cells, ranging from round spermatids to spermatozoa. These studies suggest that Kiss1 is an essential regulator in the onset of puberty and spermatogenesis of boars.

Molecular characterization, expression patterns and cellular localization of BCAS2 gene in male Hezuo pig.

Background: Breast carcinoma amplified sequence 2 (BCAS2) participates in pre-mRNA splicing and DNA damage response, which is implicated in spermatogenesis and meiosis initiation in mouse. Nevertheless, the physiological roles of BCAS2 in the testes of large mammals especially boars remain largely unknown. Methods: In this study, testes were collected from Hezuo pig at three development stages including 30 days old (30 d), 120 days old (120 d), and 240 days old (240 d). BCAS2 CDS region was firstly cloned using RT-PCR method, and its molecular characteristics were identified using relevant bioinformatics software. Additionally, the expression patterns and cellular localization of BCAS2 were analyzed by quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry and immunofluorescence. Results: The cloning and sequence analysis indicated that the Hezuo pig BCAS2 CDS fragment encompassed 678 bp open reading frame (ORF) capable of encoding 225 amino acid residues, and possessed high identities with some other mammals. The results of qRT-PCR and Western blot displayed that BCAS2 levels both mRNA and protein were age-dependent increased (p < 0.01). Additionally, immunohistochemistry and immunofluorescence results revealed that BCAS2 protein was mainly observed in nucleus of gonocytes at 30 d testes as well as nucleus of spermatogonia and Sertoli cells at 120 and 240 d testes. Accordingly, we conclude that BCAS2 is critical for testicular development and spermatogenesis of Hezuo pig, perhaps by regulating proliferation or differentiation of gonocytes, pre-mRNA splicing of spermatogonia and functional maintenance of Sertoli cells, but specific mechanism still requires be further investigated.

Dysregulated Arginine Metabolism Is Linked to Retinal Degeneration in Cep250 Knockout Mice.

Purpose: Degeneration of retinal photoreceptors is frequently observed in diverse ciliopathy disorders, and photoreceptor cilium gates the molecular trafficking between the inner and the outer segment (OS). This study aims to generate a homozygous global Cep250 knockout (KO) mouse and study the resulting phenotype. Methods: We used Cep250 KO mice and untargeted metabolomics to uncover potential mechanisms underlying retinal degeneration. Long-term follow-up studies using optical coherence tomography (OCT) and electroretinography (ERG) were performed. Results: OCT and ERG results demonstrated gradual thinning of the outer nuclear layer (ONL) and progressive attenuation of the scotopic ERG responses in Cep250-/- mice. More TUNEL signal was observed in the ONL of these mice. Immunostaining of selected OS proteins revealed mislocalization of these proteins in the ONL of Cep250-/- mice. Interestingly, untargeted metabolomics analysis revealed arginine-related metabolic pathways were altered and enriched in Cep250-/- mice. Mis-localization of a key protein in the arginine metabolism pathway, arginase 1 (ARG1), in the ONL of KO mice further supports this model. Moreover, adeno-associated virus (AAV)-based retinal knockdown of Arg1 led to similar architectural and functional alterations in wild-type retinas. Conclusions: Altogether, these results suggest that dysregulated arginine metabolism contributes to retinal degeneration in Cep250-/- mice. Our findings provide novel insights that increase understanding of retinal degeneration in ciliopathy disorders.

Overexpression of SIRT1 alleviates oxidative damage and barrier dysfunction in CPB2 toxin-infected IPEC-J2 cells.

Clostridium perfringens (C. perfringens) beta2 (CPB2) toxin may induce necrotizing enteritis (NE) in pigs. Sirtuin1 (SIRT1) is involved in inflammatory intestinal diseases and affects intestinal barrier function. However, the effects of SIRT1 on piglet intestinal disease caused by CPB2 toxin are unclear. This study revealed the role of pig SIRT1 in CPB2 toxin-exposed intestinal porcine epithelial cells (IPEC-J2). Herein, we manifested that SIRT1 was dramatically decreased in IPEC-J2 cells infected with CPB2 toxin. Subsequently, we silenced and overexpressed SIRT1 using siRNA and a overexpression vector in CPB2 toxin-treated IPEC-J2 cells. The results indicated that overexpression of SIRT1 suppressed reactive oxygen species (ROS) generates, the expression tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and Bax, nuclear factor-kappa B (NF-κB p65), phospho (p)-NF-kB p65 and lactate dehydrogenase (LDH) activity and apoptosis in CPB2 toxin-treated IPEC-J2 cells, and increased IL-10, mitochondrial membrane potential (ΔΨm), Bcl-2, Claudin1 and Occludin levels and cell viability. These results indicated that SIRT1 protects IPEC-J2 cells against CPB2 toxin-induced oxidative damage and tight junction (TJ) disruption, which provides a theoretical basis for further study of the molecular regulatory mechanism of SIRT1 in C. perfringens-infected NE in piglets.

lnc001776 Affects CPB2 Toxin-Induced Excessive Injury of Porcine Intestinal Epithelial Cells via Activating JNK/NF-kB Pathway through ssc-let-7i-5p/IL-6 Axis.

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.

METTL3-Mediated LncRNA EN_42575 m6A Modification Alleviates CPB2 Toxin-Induced Damage in IPEC-J2 Cells.

Long non-coding RNAs (lncRNAs) modified by n6-methyladenosine (m6A) have been implicated in the development and progression of several diseases. However, the mechanism responsible for the role of m6A-modified lncRNAs in Clostridium perfringens type C piglet diarrhea has remained largely unknown. We previously developed an in vitro model of CPB2 toxin-induced piglet diarrhea in IPEC-J2 cells. In addition, we previously performed RNA immunoprecipitation sequencing (MeRIP-seq), which demonstrated lncRNA EN_42575 as one of the most regulated m6A-modified lncRNAs in CPB2 toxin-exposed IPEC-J2 cells. In this study, we used MeRIP-qPCR, FISH, EdU, and RNA pull-down assays to determine the function of lncRNA EN_42575 in CPB2 toxin-exposed IPEC-J2 cells. LncRNA EN_42575 was significantly downregulated at different time points in CPB2 toxin-treated cells. Functionally, lncRNA EN_42575 overexpression reduced cytotoxicity, promoted cell proliferation, and inhibited apoptosis and oxidative damage, whereas the knockdown of lncRNA EN_42575 reversed these results. Furthermore, the dual-luciferase analysis revealed that METTL3 regulated lncRNA EN_42575 expression in an m6A-dependent manner. In conclusion, METTL3-mediated lncRNA EN_42575 exerted a regulatory effect on IPEC-J2 cells exposed to CPB2 toxins. These findings offer novel perspectives to further investigate the function of m6A-modified lncRNAs in piglet diarrhea.

S100A9 plays a key role in Clostridium perfringens beta2 toxin-induced inflammatory damage in porcine IPEC-J2 intestinal epithelial cells.

BACKGROUND: As an important regulator of autoimmune responses and inflammation, S100A9 may serve as a therapeutic target in inflammatory diseases. However, the role of S100A9 in Clostridium perfringens type C infectious diarrhea is poorly studied. The aim of our study was to screen downstream target genes regulated by S100A9 in Clostridium perfringens beta2 (CPB2) toxin-induced IPEC-J2 cell injury. We constructed IPEC-J2 cells with S100A9 knockdown and a CPB2-induced cell injury model, screened downstream genes regulated by S100A9 using RNA-Seq technique, and performed functional enrichment analysis. The function of S100A9 was verified using molecular biology techniques. RESULTS: We identified 316 differentially expressed genes (DEGs), of which 221 were upregulated and 95 were downregulated. Functional enrichment analysis revealed that the DEGs were significantly enriched in cilium movement, negative regulation of cell differentiation, immune response, protein digestion and absorption, and complement and coagulation cascades. The key genes of immune response were TNF, CCL1, CCR7, CSF2, and CXCL9. When CPB2 toxin-induced IPEC-J2 cells overexpressed S100A9, Bax expression increased, Bcl-2 expression and mitochondrial membrane potential decreased, and SOD activity was inhibited. CONCLUSION: In conclusion, S100A9 was involved in CPB2-induced inflammatory response in IPEC-J2 cells by regulating the expression of downstream target genes, namely, TNF, CCL1, CCR7, CSF2, and CXCL9; promoting apoptosis; and aggravating oxidative cell damage. This study laid the foundation for further study on the regulatory mechanism underlying piglet diarrhea.

Information fields of written texts protect cells from oxidative damage and accelerate repair.

OBJECTIVE: Written words demonstrated specific effects on biochemical measures and cell growth in cultured mammalian cells in previous study. We examined the protection and reparative effects of Chinese texts with positive meaning and the word "Buddha" on oxidative-damaged cells, with the goal of exploring the therapeutic effect of such characters. METHODS: The human embryonic kidney cell line HEK293T was utilized, with the oxidative- damaged cell model produced by hydrogen peroxide (H2O2). Double-blind experiments were set up to ensure all operators involved in the experiments did not know the contents of the texts in envelopes. Petri-dishes (9 plates) containing oxidative-damaged cells were selected randomly for three controls, three treatments by texts with positive meaning, and another three treatment by the word "Buddha". Cells were sub-sampled at 3 h and 24 h to examine growth and mitochondrial functions. All data are expressed relative to the controls. RESULTS: Compared with the control group, mitochondrial functions were enhanced as evidenced by increased adenosine triphosphate (ATP) levels, accompanied by the significant improvement in cell growth rate for the groups treated by positive texts and the word "Buddha". Reactive oxygen species (ROS) levels were decreased significantly in positive texts treatment, which suggested that the antioxidant capacity of cells was also improved. CONCLUSIONS: This study revealed that certain texts can potentially offer protection and accelerate the cellular repair for oxidative-damaged cells.

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.

LncRNA ALDB-898 modulates intestinal epithelial cell damage caused by Clostridium perfringens type C in piglet by regulating ssc-miR-122-5p/OCLN signaling.

Diarrhea of piglets caused by Clostridium perfringens type C (C. perfringens type C) infection is a global problem afflicting piglet production. Long noncoding RNA (LncRNA) and microRNA (miRNA) have emerged as critical regulators of this pathological process, but the underlying molecular mechanisms remain unclear. In this study, we first observed the expression changes of ALDBSSCG0000000898 (ALDB-898) and ssc-miR-122-5p in infected ileum tissue of piglets with C. perfringens type C, and then used C. perfringens beta2 toxin (CPB2) to induce intestinal porcine epithelial cells (IPEC-J2) to construct an injury model. Cytometry kit 8 (CCK-8), lactate dehydrogenase (LDH), real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, flow cytometry and fluorescein isothiocyanate-dextran 4 (FITC-Dextran 4) flux assays were performed to study the effect of ALDB-898 and ssc-miR-122-5p in apoptosis, inflammation and intestinal barrier damage and inflammatory in IPEC-J2 cells induced by CPB2. In addition, dual-luciferase reporter gene analysis was performed to confirm the relationship between ssc-miR-122-5p and ALDB-898 or ssc-miR-122-5p and occludin (OCLN), respectively. There were lower expression levels of ALDB-898 and OCLN and higher expression levels of ssc-miR-122-5p in diarrhea piglets caused by Clostridium perfringens type C. ALDB-898 and OCLN were significantly decreased and ssc-miR-122-5p was increased in IPEC-J2 after exposure to the CPB2 in a dose- and time-dependent manner. ALDB-898 overexpression mitigated CPB2-induced cell injury by promoting viability, restraining apoptosis, cytotoxicity, and inflammatory response, as well as weakening the destruction of the intestinal barrier. Further mechanisms disclosed that ALDB-898 functioned as a competing endogenous RNA (ceRNA) via binding to ssc-miR-122-5p, and OCLN was a target of ssc-miR-122-5p. Importantly, the ssc-miR-122-5p mimic led to abolishing the protective function of ALDB-898 on CPB2-induced IPEC-J2 cell damage, and the addition of OCLN reversed the negative impact of ssc-miR-122-5p, thereby restoring the protection of ALDB-898. Our data showed that ALDB-898 could enhance the expression of OCLN through competitive binding ssc-miR-122-5p to suppress CPB2-induced damage. The ALDB-898/ssc-miR-122-5p/OCLN signaling may be a candidate therapeutic pathway for diarrhea of piglets.

Effects of different music on HEK293T cell growth and mitochondrial functions.

BACKGROUND: Mitochondria are considered a portal to receive, process and integrate external energy and information to maintain cellular homeostasis. Previous studies demonstrated that mitochondrial function and antioxidant capacity of in-vitro cultured mammalian cells is modified by different energetic stimuli including electromagnetic energy, acoustic energy, external Qi and subtle energy emitted by written texts in relatively short time frames. Thus, individual cells can act as a rapid and sensitive biological sensor and act as a platform to evaluate and understand the subtle effect of different biophysical stimuli. Regarding acoustic energy, several different types of music have been reported to produce beneficial effects on human health. OBJECTIVE: To compare eastern and western music styles on cell function at the biochemical level to understand the underlying mechanisms involved. Methods In this study, we compared the effects of Chinese five-element music with two types of western music (heavy-metal and classical) on mitochondrial function, oxidative capacity and growth using HEK293T cells (human embryonic kidney cells). RESULTS: Unlike rock and classical music, exposing cells to five-element music produced several beneficial physiological effects, including statistically significant increases in the production of adenosine triphosphate (ATP) by 17%, glutathione (GSH) by 21% and cell growth rates (14%), as well as a significant reduction in the reactive oxygen species (ROS) by 13%. For the group treated with classical music, there was only a trend toward increased GSH (8%), although the increased growth rates (14%) did reach significance. In sharp contrast, the cells treated with heavy-metal music responded with an opposite and significant 16% increase in ROS and a significant 11% reduction in cell viability. CONCLUSIONS: This study revealed the dramatically different and even opposite effects of different styles of music on specific biochemical measures in cultured human cells. These results help explain the underlying biochemical mechanisms of the effects of the different types of music.

Vascular characteristics and expression of hypoxia genes in Tibetan pigs' hearts.

BACKGROUND: Tibetan pigs have exhibited unique characteristics from low-altitudes pigs and adapted well to the Qinghai-Tibet Plateau. OBJECTIVES: The current study was undertaken to investigate the hypoxic adaptation of heart in Tibetan pigs. METHODS: The hearts of Tibetan pigs and Landrace pigs raised at high or low altitudes were compared using 3D casting technology, scanning electron microscopy and real-time quantitative PCR (qRT-PCR). RESULTS: We found that the ratio of the major axis to the minor axis and the density of the heart were significantly higher in Tibetan pigs than in Landrace pigs (p < 0.05). Tibetan pigs had larger diameters and higher densities of arterioles than Landrace pigs (p < 0.05), and these features have a similar variation with the expression of vascular endothelial growth factor (VEGF). The cardiac expression levels of hypoxia-inducible factor-1α (HIF-1α) and endothelial nitric oxide synthase (eNOS) were significantly higher in pigs reared at high altitudes than in those reared at low altitudes (p < 0.05). In contrast, Egl nine homolog 1 (EGLN1) had the opposite trend with respect to HIF-1α and eNOS and was related to red blood cell (RBC) counts. Notably, the expressions of erythropoietin (EPO) and endothelial PAS domain-containing protein 1 (EPAS1) were significantly higher in Landrace pigs kept at high altitudes than in the others (p < 0.05) and were associated with haemoglobin. CONCLUSIONS: These findings show that the regulation of the heart function of Tibetan pigs in a hypoxic environment is manifested at various levels to ensure the circulation of blood under extreme environmental conditions.

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.

Sodium Selenite Enhances Antibiotics Sensitivity of Pseudomonas aeruginosa and Deceases Its Pathogenicity by Inducing Oxidative Stress and Inhibiting Quorum Sensing System.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is commonly found in clinical settings and immuno-compromised patients. It is difficult to be eradicated due to its strong antibiotic resistance, and novel inactivation strategies have yet to be developed. Selenium is an essential microelement for humans and has been widely used in dietary supplement and chemoprevention therapy. In this study, the physiological and biochemical effects of sodium selenite on P. aeruginosa PAO1 were investigated. The results showed that 0~5 mM sodium selenite did not impact the growth of PAO1, but increased the lethality rate of PAO1 with antibiotics or H2O2 treatment and the antibiotics susceptibility both in planktonic and biofilm states. In addition, sodium selenite significantly reduced the expression of quorum sensing genes and inhibited various virulence factors of this bacterium, including pyocyanin production, bacterial motilities, and the type III secretion system. Further investigation found that the content of ROS in cells was significantly increased and the expression levels of most genes involved in oxidative stress were up-regulated, which indicated that sodium selenite induced oxidative stress. The RNA-seq result confirmed the phenotypes of virulence attenuation and the expression of quorum sensing and antioxidant-related genes. The assays of Chinese cabbage and Drosophila melanogaster infection models showed that the combination of sodium selenite and antibiotics significantly alleviated the infection of PAO1. In summary, the results revealed that sodium selenite induced oxidative stress and inhibited the quorum sensing system of P. aeruginosa, which in turn enhanced the antibiotic susceptibility and decreased the pathogenicity of this bacterium. These findings suggest that sodium selenite may be used as an effective strategy for adjunct treatment of the infections caused by P. aeruginosa.

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.

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.