CRISPR-Mediated Library Screening of Gene-Knockout Cell Lines for Investigating Antiviral Innate Immunity.

The application of CRISPR-mediated library screening has fundamentally transformed functional genomics by revealing the complexity of virus-host interactions. This protocol describes the use of CRISPR-mediated library screening to identify key functional genes regulating the innate immune response to PEDV infection. We detail a step-by-step process, starting from the design and construction of a customized CRISPR knockout library targeting genes involved in innate immunity to the effective delivery of these constructs into cells using lentiviral vectors. Subsequently, we outline the process of identifying functional genes postviral attack, including the use of next-generation sequencing (NGS), to analyze and identify knockout cells that exhibit altered responses to infection. This integrated approach provides researchers in immunology and virology with a resource and a robust framework for uncovering the genetic basis of host-pathogen interactions and the arsenal of the innate immune system against viral invasions.

KIAA0753 enhances osteoblast differentiation suppressed by diabetes.

Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.

ATP synthase subunit ATP5B interacts with TGEV Nsp2 and acts as a negative regulator of TGEV replication.

Coronavirus nonstructural protein 2 (Nsp2) is regarded as a virulence determinant and plays a critical role in virus replication, and innate immunity. Screening and identifying host cell proteins that interact with viral proteins is an effective way to reveal the functions of viral proteins. In this study, the host proteins that interacted with transmissible gastroenteritis virus (TGEV) Nsp2 were identified using immunoprecipitation combined with LC-MS/MS. 77 host cell proteins were identified as putative Nsp2 interaction host cell proteins and a protein-protein interaction (PPI) was constructed. The identified proteins were found to be associated with various subcellular locations and functional categories through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. It is hypothesized that the host cell proteins interacting with TGEV Nsp2 are mainly involved in the formation of the cytoplasmic translation initiation complex, mRNA binding, ribosomes, and proteasomes. Among these, the ATP5B, a core subunit of the mitochondrial ATP synthase was further studied. The Coimmunoprecipitation (Co-IP) and indirect immunofluorescence (IFA) results confirmed that TGEV Nsp2 interacted with ATP5B. Furthermore, the downregulation of ATP5B expression was found to promote TGEV replication, suggesting that ATP5B might function as a negative regulator of TGEV replication. Collectively, our results offer additional insights into the functions of Nsp2 and provide a novel antiviral target against TGEV.

Mitochondrial dysfunction and increased reactive oxygen species production in MECP2 mutant astrocytes and their impact on neurons.

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here, using human embryonic stem cell (hESC) lines, we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs), particularly pronounced in glia versus neurons. Employing a multiomics approach, we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally, RTT ASTs exhibited increased cytosolic amino acids under basal conditions, which were depleted upon increased energy demands. Notably, mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons, suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health.

Role of the GalNAc-galectin pathway in the healing of premature rupture of membranes.

BACKGROUND: Premature rupture of the membranes (PROM) is a key cause of preterm birth and represents a major cause of neonatal mortality and morbidity. Natural products N-acetyl-d-galactosamine (GalNAc), which are basic building blocks of important polysaccharides in biological cells or tissues, such as chitin, glycoproteins, and glycolipids, may improve possible effects of wound healing. METHODS: An in vitro inflammation and oxidative stress model was constructed using tumor necrosis-α (TNF-α) and lipopolysaccharide (LPS) action on WISH cells. Human amniotic epithelial cells (hAECs) were primarily cultured by digestion to construct a wound model. The effects of GalNAc on anti-inflammatory and anti-oxidative stress, migration and proliferation, epithelial-mesenchymal transition (EMT), glycosaminoglycan (GAG)/hyaluronic acid (HA) production, and protein kinase B (Akt) pathway in hAECs and WISH cells were analyzed using the DCFH-DA fluorescent probe, ELISA, CCK-8, scratch, transwell migration, and western blot to determine the mechanism by which GalNAc promotes amniotic wound healing. RESULTS: GalNAc decreased IL-6 expression in TNF-α-stimulated WISH cells and ROS expression in LPS-stimulated WISH cells (P < 0.05). GalNAc promoted the expression of Gal-1 and Gal-3 with anti-inflammatory and anti-oxidative stress effects. GalNAc promoted the migration of hAECs (50% vs. 80%) and WISH cells through the Akt signaling pathway, EMT reached the point of promoting fetal membrane healing, and GalNAc did not affect the activity of hAECs and WISH cells (P > 0.05). GalNAc upregulated the expression of sGAG in WISH cells (P < 0.05) but did not affect HA levels (P > 0.05). CONCLUSIONS: GalNAc might be a potential target for the prevention and treatment of PROM through the galectin pathway, including (i) inflammation; (ii) epithelial-mesenchymal transition; (iii) proliferation and migration; and (iv) regression, remodeling, and healing.

Endurance exercise-induced histone methylation modification involved in skeletal muscle fiber type transition and mitochondrial biogenesis.

Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise.

Valsartan Rescues Suppressed Mitochondrial Metabolism during Insulin Resistance in C2C12 Myotubes.

Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models.

The PI3K/Akt-Nrf2 Signaling Pathway and Mitophagy Synergistically Mediate Hydroxytyrosol to Alleviate Intestinal Oxidative Damage.

Oxidative stress is a major pathogenic factor in many intestinal diseases, such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). The Nrf2 signaling pathway and mitophagy can reduce reactive oxygen species (ROS) and alleviate oxidative stress, but their relationship is unclear. Hydroxytyrosol (HT), a polyphenolic compound abundant in olive oil, has strong antioxidant activity and may help treat these diseases. We used pigs as a model to investigate HT's effect on intestinal oxidative damage and its mechanisms. Diquat (DQ) induced oxidative stress and impaired intestinal barrier function, which HT mitigated. Mechanistic studies in IPEC-J2 cells showed that HT protected against oxidative damage by activating the PI3K/Akt-Nrf2 signaling pathway and promoting mitophagy. Our study highlighted the synergistic relationship between Nrf2 and mitophagy in mediating HT's antioxidant effects. Inhibition studies confirmed that disrupting either pathway compromised HT's protective effects. Maintaining redox balance through Nrf2 and mitophagy is important for eliminating excess ROS. Nrf2 increases antioxidant enzymes to clear existing ROS, while mitophagy removes damaged mitochondria and reduces ROS generation. This study demonstrates that these pathways collaboratively modulate the antioxidant effects of HT, with neither being dispensable. Targeting Nrf2 and mitophagy could be a promising strategy for treating oxidative stress-related intestinal diseases, with HT as a potential treatment.

Mitochondrial pyruvate carrier 2 mitigates acute kidney injury via sustaining mitochondrial metabolism.

Cisplatin, a chemotherapeutic drug, can result in acute kidney injury (AKI). Currently, there are no effective prevention methods. An incomplete understanding of the pathogenesis of AKI is a major barrier to the development of effective therapies. Metabolism reprogramming shift to glycolysis was involved in AKI pathogenesis. Glycolysis results in the pyruvate production. The mitochondrial pyruvate carrier (MPC) conveys cytosol pyruvate into mitochondria, promoting the tricarboxylic acid cycle. In this current study, we found a reduction in MPC2 expression in mice and cultured HK2 cells with cisplatin-induced AKI. MPC2 overexpression attenuated cisplatin-mediated nephrotoxicity both in vitro and in vivo via restoring pyruvate metabolism and mitochondrial function. Knockdown of MPC2 reversed this effect. Furthermore, artemether, an MPC2 potential activator, could mitigate AKI via regulating MPC2-mediated pyruvate metabolism. Our findings revealed that MPC2-pyruvate metabolism axis was a promising strategy to alleviate AKI induced by cisplatin.

Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage.

Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.

The identification of key molecules and pathways in the crosstalk of calcium oxalate-treated TCMK-1 cells and macrophage via exosomes.

The interplay between crystals and epithelial cells forms the cornerstone of kidney stone development, communication between epithelial cells and macrophages emerging as a pivotal role in this process. We conducted next-generation sequencing on the secreted exosomes of TCMK-1 cells treated with calcium oxalate monohydrate (OX_EXO) or controls (NC_EXO), and on the macrophage cell line RAW264.7 stimulated with OX_EXO or NC_EXO, followed by validation of differentially expressed target proteins and miRNAs through Western blot and PCR. UPSET plots were employed to identify genes co-targeted by exosomal miRNAs. Various bioinformatic analyses were employed to predict potential mechanisms of the dysregulated genes. We integrated sequencing data from the GEO database, and validated findings using clinical patient urine and kidney tissues. We identified 665 differentially expressed exosomal miRNAs between OX_EXO and NC_EXO. Among the top 10 down-regulated miRNAs, the most targeted genes were AAK1 and NUFIP2, whereas PLCB1 was significantly targeted among the top 10 up-regulated miRNAs. In clinical specimens, we confirmed the differential expressions of five homologous miRNAs, as well as CNOT3, CNCNA1C, APEX1, and TMEM199. In conclusion, treatment of TCMK-1 cells with calcium oxalate significantly alerted the expression profile of exosomal miRNAs, subsequently influencing gene expression in macrophages, thereby modulating the processes of kidney stone formation.

NAT10-mediated ac4C acetylation of TFRC promotes sepsis-induced pulmonary injury through regulating ferroptosis.

BACKGROUND: Sepsis-induced pulmonary injury (SPI) is a common complication of sepsis with a high rate of mortality. N4-acetylcytidine (ac4C) is mediated by the ac4C "writer", N-acetyltransferase (NAT)10, to regulate the stabilization of mRNA. This study aimed to investigate the role of NAT10 in SPI and the underlying mechanism. METHODS: Twenty-three acute respiratory distress syndrome (ARDS) patients and 27 non-ARDS volunteers were recruited. A sepsis rat model was established. Reverse transcription-quantitative polymerase chain reaction was used to detect the expression of NAT10 and transferrin receptor (TFRC). Cell viability was detected by cell counting kit-8. The levels of Fe2+, glutathione, and malondialdehyde were assessed by commercial kits. Lipid reactive oxygen species production was measured by flow cytometric analysis. Western blot was used to detect ferroptosis-related protein levels. Haematoxylin & eosin staining was performed to observe the pulmonary pathological symptoms. RESULTS: The results showed that NAT10 was increased in ARDS patients and lipopolysaccharide-treated human lung microvascular endothelial cell line-5a (HULEC-5a) cells. NAT10 inhibition increased cell viability and decreased ferroptosis in HULEC-5a cells. TFRC was a downstream regulatory target of NAT10-mediated ac4C acetylation. Overexpression of TFRC decreased cell viability and promoted ferroptosis. In in vivo study, NAT10 inhibition alleviated SPI. CONCLUSION: NAT10-mediated ac4C acetylation of TFRC aggravated SPI through promoting ferroptosis.

Protective effect of ghrelin in oxidative stress-induced age-related macular degeneration in vitro and in vivo.

Oxidative damage to human retinal pigment epithelial (RPE) cells is the main cause of age-related macular degeneration (AMD), in our previous work, we showed that ghrelin has an antioxidative effect on human lens epithelium (HLE) cells, however, the studies of using ghrelin in treating the degenerative diseases of the retina have rarely been reported. In this article, we assessed the effect of ghrelin on preventing oxidative stress induced by hydrogen peroxide (H2O2) in ARPE-19 cells and its mechanism. We observed that pretreatment with ghrelin protected ARPE-19 cells from H2O2-induced cell oxidative injuries and apoptosis responses. Furthermore, an oxidative stress-induced mouse model of AMD was established via injection of sodium iodate (NaIO3) to tail veins, and treatment with ghrelin preserved retinal function, and protected photoreceptors.

P4HB regulates the TGFß/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells.

BACKGROUND: Diabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it's role in HG-induced TECs remains to be elucidated. METHODS: The HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFß/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB. RESULTS: HG induced an increase in the expression of P4HB and TGFß, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFß/SMAD3-related proteins in HK-2 cells. CONCLUSION: P4HB regulated the TGFß/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.

Melatonin inhibits bovine viral diarrhea virus replication by ER stress-mediated NF-κB signal pathway and autophagy in MDBK cells.

Bovine viral diarrhea-mucosal disease (BVD-MD) is a contagious disease in cattle, caused by the bovine viral diarrhea virus (BVDV). This virus continues to spread globally, exerting pressure on both public health and the economy. Despite its impact, there are currently no effective drugs for treating BVDV. This study utilized Madin-Darby bovine kidney (MDBK) cells as a model to investigate the antiviral effects of melatonin against Bovine Viral Diarrhea Virus (BVDV) and its connection with endoplasmic reticulum (ER) stress. Our results show that melatonin can suppress BVDV proliferation in MDBK cells by modulating the endoplasmic reticulum (ER) stress-mediated NF-κB pathway and autophagy. Specifically, melatonin alleviated ER stress, inhibited the activation of IκBα and p65, regulated autophagy, and reduced the expression levels of pro-inflammatory cytokines. Further, when we treated BVDV-infected cells with the ER stress inducer thapsigargin, it led to significant activation of the NF-κB pathway and autophagy. Conversely, treating the cells with the ER stress inhibitor 4-phenylbutyric acid reversed these effects. These findings suggest that melatonin exerts its antiviral effects primarily through the PERK-eIF2α-ATF4 of ER stress-mediated NF-κB pathway and autophagy. Overall, our study underscores the potential of melatonin as an effective protective and therapeutic option against BVDV, offering insights into its anti-infective mechanisms.

MicroRNA-223 alleviates inflammatory response in renal ischemia-reperfusion injury by targeting NLRP3.

We investigated the potential correlation between miR-223 and NAcHT, LRR, and PYd domain-containing protein 3 (NLRP3) in the context of renal ischemia-reperfusion injury (RIRI), which is a leading cause of acute renal failure with significant mortality rates. Additionally, miR-223 has been implicated in renal inflammation, further highlighting its relevance to this study. C57BL/6 male mice were used as RIRI models. After successful modeling, pathological examinations and serum creatinine and miR-223 levels were tested. Pro-inflammatory cytokine (IL-1ß, IL-6, IL-8, NLPR3, TLR4) expression was detected in mice by western blot (kidney tissue) and enzyme-linked immunosorbent assay (serum). HK-2 cells were used for in vitro experiments. A hypoxia/reoxygenation (H/R) model was used, and miR-223 and pro-inflammatory cytokine levels were detected using PCR and western blot assays, respectively. A dual-luciferase reporter assay was conducted to confirm the binding of miR-223 to NLPR3. Next, NLRP3 was knocked down to determine whether the anti-inflammatory function of miR-223 is dependent on NLRP3. MiR-223 expression was lower in RIRI mice than in the sham operation group. The level of miR-223 negatively correlated with serum creatinine levels and the severity of tubule injury. Increased proinflammatory cytokine levels in RIRI mice were observed. In vitro, miR-223 alleviated the inflammatory response in H/R treated cells by inhibiting proinflammatory cytokines. Dual-luciferase reporter and western blot assays confirmed the binding of miR-223 to NLRP3. NLRP3 knockdown reversed the anti-inflammatory effects of miR-223 in HK-2 cells. MiR-223 plays an anti-inflammatory role in RIRI by targeting NLRP3 to repress pro-inflammatory factors.

Cylindromatosis lysine 63 deubiquitinase (CYLD) suppress TLR3-mediated CCL5 expression in human renal proximal tubular epithelial cells.

BACKGROUND: One of the causes of tubulointerstitial nephritis is viral infection, with innate immune responses affecting its pathogenesis. Toll-like receptor 3 (TLR3) recognizes viral infections and acts antivirally by activating signaling to produce inflammatory cytokines/chemokines, including C-C motif chemokine ligand 5 (CCL5) and interferon-ß (IFN-ß). Although cylindromatosis lysine 63 deubiquitinase (CYLD) is known to be associated with tubulointerstitial nephritis and renal function, its role in the antiviral innate immune response in tubular epithelial cells remains unknown. In this study, we investigated the association between CYLD and TLR3-mediated CCL5 production in cultured human renal proximal tubular epithelial cells (hRPTECs). METHODS AND RESULTS: Polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand, was used to stimulate hRPTECs. mRNA expression was measured using reverse transcription-quantitative polymerase chain reaction. Protein expression was assayed using western blotting or an enzyme-linked immunosorbent assay. Knockdown of IFN-ß, nuclear factor-kappa B (NF-κB) p65, and CYLD was performed by transfecting cells with specific small interfering RNAs. The intracellular localization of CYLD in hRPTECs was analyzed using immunofluorescence. Poly IC induced CCL5 expression in a time- and concentration-dependent manner, and knockdown of either IFN-ß or p65 reduced poly IC-induced CCL5 expression. CYLD knockdown increased the poly IC-induced CCL5, phosphorylated IκB kinase α/ß (IKK complex), and phosphorylated p65 expression. The CYLD protein was localized in the cytoplasm, and poly IC did not alter its expression. CONCLUSION: CYLD may prevent excessive inflammation due to an antiviral innate immune response by suppressing IKK complex and NF-κB activation downstream of TLR3 in hRPTECs.

METTL14-mediated lncRNA-FAS-AS1 promotes osteoarthritis progression by up-regulating ADAM8.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative disease. We explored the role and regulatory mechanisms of lncRNA-FAS-AS1 in OA progression. METHODS: We exposed human immortalized chondrocytes to IL-1ß for 24 h to induce an OA cell model. The target molecule levels were assessed using western blot and quantitative real-time PCR (RT-qPCR). Cell viability and apoptosis were measured using CCK-8 and flow cytometry. The m6A modification of FAS-AS1 was determined using MeRIP. We examined the binding relationships between FAS-AS1, Fragile X mental retardation 1 (FMR1), and A disintegrin and metalloproteinase 8 (ADAM8) using RIP and RNA pull-down. The OA animal model was established by separating the medial collateral ligament and medial meniscus. Safranin-O staining and Mankin's scale were employed to evaluate pathological changes within the cartilage. RESULTS: FAS-AS1, METTL14, and ADAM8 were upregulated, and the JAK/STAT3 signaling pathway was activated in OA mice and IL-1ß-induced chondrocytes. FAS-AS1 knockdown inhibited extracellular matrix degradation in IL-1ß-induced chondrocytes; however, ADAM8 overexpression reversed this effect. FAS-AS1 maintained the stability of ADAM8 mRNA by recruiting FMR1. METTL14 knockdown repressed FAS-AS1 expression in an m6A-dependent manner. FAS-AS1 overexpression reversed the inhibitory effects of METTL14 knockdown on JAK/STAT3 signaling and cartilage damage in the OA model both in vitro and in vivo. CONCLUSION: METTL14-mediated FAS-AS1 promotes OA progression through the FMR1/ADAM8/JAK/STAT3 axis.

Activin A plays an essential role in migration and proliferation of hepatic stellate cells via Smad3 and calcium signaling.

Activin A and hepatic stellate cells (HSCs) are involved in tissue repair and fibrosis in liver injury. This study investigated the impact of activin A on HSC activation and migration. A microfluidic D4-chip was used for examining the cell migration of mouse hepatic stellate cell line MHSteC. The analysis of differentially expressed genes revealed that activin ßA (Inhba), activin receptor type 1A (Acvr1a) and type 2A (Acvr2a) mRNAs were more significantly expressed in human HSCs than in the hepatocytes. Moreover, activin A promoted MHSteC proliferation and induced MHSteC migration. Furthermore, the MHSteCs treated with activin A exhibited increased levels of migration-related proteins, N-cadherin, Vimentin, α-SMA, MMP2 and MMP9, but a decreased level of E-cadherin. Additionally, activin A treatment significantly increased the p-Smad3 levels and p-Smad3/Smad3 ratio in the MHSteCs, and the Smad3 inhibitor SIS3 attenuated activin A-induced MHSteC proliferation and migration. Simultaneously, activin A increased the calcium levels in the MHSteCs, and the migratory effects of activin A on MHSteCs were weakened by the intracellular calcium ion-chelating agent BAPTA-AM. These data indicate that activin A can promote MHSteC activation and migration through the canonical Smad3 signaling and calcium signaling.

EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction in lipopolysaccharide-induced tubular epithelial cells.

Sepsis represents an organ dysfunction resulting from the host's maladjusted response to infection, and can give rise to acute kidney injury (AKI), which significantly increase the morbidity and mortality of septic patients. This study strived for identifying a novel therapeutic strategy for patients with sepsis-induced AKI (SI-AKI). Rat tubular epithelial NRK-52E cells were subjected to lipopolysaccharide (LPS) exposure for induction of in-vitro SI-AKI. The expressions of E1A binding protein p300 (EP300) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in NRK-52E cells were assessed by western blot and qRT-PCR, and their interaction was explored by chromatin immunoprecipitation performed with antibody for H3K27 acetylation (H3K27ac). The effect of them on SI-AKI-associated mitochondrial dysfunction of tubular epithelial cells was investigated using transfection, MTT assay, TUNEL staining, 2',7'-Dichlorodihydrofluorescein diacetate probe assay, Mitosox assay, and JC-1 staining. MTHFD2 and EP300 were upregulated by LPS exposure in NRK-52E cells. LPS increased the acetylation of H3 histone in the MTHFD2 promoter region, and EP300 suppressed the effect of LPS. EP300 ablation inhibited the expression of MTHFD2. MTHFD2 overexpression antagonized LPS-induced viability reduction, apoptosis promotion, reactive oxygen species overproduction, and mitochondrial membrane potential collapse of NRK-52E cells. By contrast, MTHFD2 knockdown and EP300 ablation brought about opposite consequences. Furthermore, MTHFD2 overexpress and EP300 ablation counteracted each other's effect in LPS-exposed NRK-52E cells. EP300-mediated H3 acetylation elevates MTHFD2 expression to reduce mitochondrial dysfunction of tubular epithelial cells in SI-AKI.