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Background: Sepsis causes inflammation in response to infection, often leading to acute lung injury (ALI). Yazhicao (Commelina communis L., YZC) is widely distributed in the global tropics and has good anti-respiratory inflammatory activity; however, the protection of YZC against septic-ALI has not been established., Purpose: The role of YZC in septic-ALI will be investigated in this study., Methods and Results: In this study, YZC was shown to inhibit excessive inflammation and alleviate septic-ALI. Network pharmacology predicts that Quercetin, Acacetin and Diosmetin have the potential to serve as the pharmacological substance basis of YZC in alleviating septic-ALI. The metabolomics results indicated that YZC could improve the metabolic disorders caused by septic-ALI, which were mostly concerned with energy metabolism and amino acid metabolism, with Trimethylamine (TMA)/Trimethylamine N-oxide (TMAO) being potential small molecule metabolic markers for the clinical diagnosis and treatment of septic-ALI. YZC inhibits the initiation and progression of septic-ALI by controlling the TMA/TMAO metabolites. Our results also suggest that YZC protects the intestinal barrier from damage. Furthermore, our research indicated that YZC reduces TMAO synthesis by inhibiting TMA production through remodeling the intestine microbiota. We investigated the mechanism of YZC-mediated protection against septic-ALI and showed that YZC reduced the expression of proteins associated with NLRP3 inflammatory vesicles in the lung by inhibiting the expression of NF-κB., Conclusion: These results show that YZC inhibits the NF-κB/NLRP3 signaling pathway by regulating metabolic and intestinal flora disorders in septic-ALI mice to reduce TMAO synthesis. This study presents a theoretical groundwork for the advancement of novel medications and clinical use of YZC to enhance septic-ALI and furnishes a theoretical rationale for regulating intestinal microbiota as a therapeutic instrument to treat sepsis and septic-ALI., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier GmbH.)

Pathological retinal angiogenesis is not only the hallmark of retinopathies, but also a major cause of blindness. Guanylate binding protein 2 (GBP2) has been reported to be associated with retinal diseases such as diabetic retinopathy and hypoxic retinopathy. However, GBP2-mediated pathological retinal angiogenesis remains largely unknown. The present study aimed to investigate the role of GBP2 in pathological retinal angiogenesis and its underlying molecular mechanism. In this study, we established oxygen-induced retinopathy (OIR) mice model for in vivo study and hypoxia-induced angiogenesis in ARPE-19 cells for in vitro study. We demonstrated that GBP2 expression was markedly downregulated in the retina of mice with OIR and ARPE-19 cells treated with hypoxia, which was associated with pathological retinal angiogenesis. The regulatory mechanism of GBP2 in ARPE-19 cells was studied by GBP2 silencing and overexpression. The regulatory mechanism of GBP2 in the retina was investigated by overexpressing GBP2 in the retina of OIR mice. Mechanistically, GBP2 downregulated the expression and secretion of vascular endothelial growth factor (VEGFA) in ARPE-19 cells and retina of OIR mice. Interestingly, overexpression of GBP2 significantly inhibited neovascularization in OIR mice, conditioned medium of GBP2 overexpressing ARPE-19 cells inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, we confirmed that GBP2 downregulated VEGFA expression and angiogenesis by inhibiting the AKT/mTOR signaling pathway. Taken together, we concluded that GBP2 inhibited pathological retinal angiogenesis via the AKT/mTOR/VEGFA axis, thereby suggesting that GBP2 may be a therapeutic target for pathological retinal angiogenesis., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests regarding the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Context: Qi-dan-dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood., Objective: This study reveals the mechanism by which QDD ameliorates DKD., Materials and Methods: The compounds in QDD were identified by high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD-low (QDD-L), and 2% QDD-high (QDD-H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose-treated HK-2 and NRK-52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated., Results: A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial-mesenchymal transition (EMT) via the p38MAPK and AKT-mammalian target of rapamycin (mTOR) pathways., Discussion and Conclusion: QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD., (© 2024 Wiley Periodicals LLC.)

Salidroside, a principal bioactive component of Rhodiola crenulata, is neuroprotective across a wide time window in stroke models. We investigated whether salidroside induced neurogenesis after cerebral ischemia and aimed to identify its primary molecular targets. Rats, subjected to transient 2 h of middle cerebral artery occlusion (MCAO), received intraperitoneal vehicle or salidroside ± intracerebroventricular HSC70 inhibitor VER155008 or TrkB inhibitor ANA-12 for up to 7 days. MRI, behavioural tests, immunofluorescent staining and western blotting measured effects of salidroside. Reverse virtual docking and enzymatic assays assessed interaction of salidroside with purified recombinant HSC70. Salidroside dose-dependently decreased cerebral infarct volumes and neurological deficits, with maximal effects by 50 mg/kg/day. This dose also improved performance in beam balance and Morris water maze tests. Salidroside significantly increased BrdU + /nestin + , BrdU + /DCX + , BrdU + /NeuN + , BrdU - /NeuN + and BDNF + cells in the peri-infarct cortex, with less effect in striatum and no significant effect in the subventricular zone. Salidroside was predicted to bind with HSC70. Salidroside dose-dependently increased HSC70 ATPase and HSC70-dependent luciferase activities, but it did not activate HSP70. HSC70 immunoreactivity concentrated in the peri-infarct cortex and was unchanged by salidroside. However, VER155008 prevented salidroside-dependent increases of neurogenesis, BrdU - /NeuN + cells and BDNF + cells in peri-infarct cortex. Salidroside also increased BDNF protein and p-TrkB/TrkB ratio in ischemic brain, changes prevented by VER155008 and ANA-12, respectively. Additionally, ANA-12 blocked salidroside-dependent neurogenesis and increased BrdU - /NeuN + cells in the peri-infarct cortex. Salidroside directly activates HSC70, thereby stimulating neurogenesis and neuroprotection via BDNF/TrkB signalling after MCAO. Salidroside and similar activators of HSC70 might provide clinical therapies for ischemic stroke., (© 2024 John Wiley & Sons Ltd.)

GPR126/ADGRG6, a member of the adhesion G-protein-coupled receptor family, balances cell differentiation and proliferation through fine-tuning of intracellular cAMP levels, which is achieved through coupling to Gs and Gi proteins. While GPR126-mediated cAMP increase has been proven to be essential for differentiation of Schwann cells, adipocytes and osteoblasts, Gi-signaling of the receptor was found to propagate breast cancer cell proliferation. Extracellular ligands or mechanical forces can modulate GPR126 activity but require an intact encrypted agonist sequence, coined the Stachel . Even though coupling to Gi can be seen for constitutively active truncated receptor versions of GPR126 as well as with a peptide agonist derived from the Stachel sequence, all known N-terminal modulators have so far only been shown to modulate Gs coupling. Here, we identified collagen VI as the first extracellular matrix ligand of GPR126 that induces Gi signaling at the receptor, which shows that N-terminal binding partners can mediate selective G protein signaling cascades that are masked by fully active truncated receptor variants.

Src homology 2 domain-containing phosphatase 2 (SHP2) is a cytoplasmic protein tyrosine phosphatase (PTP) that regulates signal transduction of receptor tyrosine kinases (RTKs). Abnormal SHP2 activity is associated with tumorigenesis and metastasis. Because SHP2 contains multiple allosteric sites, identifying inhibitors at specific allosteric binding sites remains challenging. Here, we used structure-based virtual screening to directly search for the SHP2 "tunnel site" allosteric inhibitor. A novel hit (70) was identified as the SHP2 allosteric inhibitor with an IC 50 of 10.2 μM against full-length SHP2. Derivatization of hit compound 70 using molecular modeling-guided structure-based modification allowed the discovery of an effective and selective SHP2 inhibitor, compound 129, with 122-fold improved potency compared to the hit. Further studies revealed that 129 effectively inhibited signaling in multiple RTK-driven cancers and RTK inhibitor-resistant cancer cells. Remarkably, 129 was orally bioavailable (F = 55%) and significantly inhibited tumor growth in haematological malignancy. Taken together, compound 129 developed in this study may serve as a promising lead or candidate for cancers bearing RTK oncogenic drivers and SHP2-related diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lingfeng Chen reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels 1 . Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mitochondrial dynamics and bioenergetics are considered play pivotal roles in the maintenance of mitochondrial function and cell viability. During the widely distributed food contaminant 3-chlorpropane-1,2-diol (3-MCPD) induced nephrotoxicity, mitochondrial morphology and function were impaired, but the specific mechanism responsible for the process has not been fully elucidated. In the present study, using an in vitro human embryonic kidney 293 (HEK293) cell culture model, the role of LKB1/AMPK pathway and mitochondrial fission and fusion dynamics in 3-MCPD-induced cell apoptosis was investigated by using the AMPK inhibitor dorsomorphin and mitochondrial division inhibitor 1 (Mdivi-1), respectively. The results revealed that 3-MCPD significantly decreased the ATP levels, activated the energy-sensing regulator AMPKα and its upstream protein kinase LKB1, disrupted mitochondrial dynamics equilibrium characterized by promoting division and inhibiting fusion, thus inducing cell apoptosis. Notably, suppression of AMPK by dorsomorphin mitigated 3-MCPD-induced cytotoxicity through improvement of the function and dynamics of mitochondria and alleviated apoptosis via the mitochondria-dependent pathway. Moreover, inhibition of mitochondrial fission by Mdivi-1 protected against apoptosis induced by 3-MCPD. Taken together, these results suggest that 3-MCPD triggers apoptosis through activation of LKB1/AMPKα signaling pathway and regulation of mitochondrial fission and fusion dynamics in HEK293 cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: Aberrant microglial responses promote neuroinflammation in neurodegenerative diseases. However, rifampicin's effect on cognitive and motor sequelae of inflammation remains unknown. Therefore, we investigated whether rifampicin exerts neuroprotection against lipopolysaccharide (LPS)-induced cognitive and motor impairments., Methods: A mouse model of LPS-induced cognitive and motor impairment was established. Adult C57BL/6 mice were injected intraperitoneally with 25 mg/kg rifampicin 30 min before intraperitoneal microinjection of LPS (750 μg/kg) daily until study end. Treatments and behavioral experiments were performed once daily for 7 days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced damage to the hippocampus and substantia nigra (SN)., Results: Rifampicin attenuated LPS-induced cognitive and motor impairments, based on performance in the behavioral tests. Rifampicin suppressed the release of pro-inflammatory mediators, including tumor necrosis factor-α, interleukin-1β, and prostaglandin E 2 in the serum and nitric oxide (NO) in brain tissue, and cyclooxygenase-2 and inducible nitric oxide synthase levels. Immunofluorescence revealed that rifampicin inhibited LPS-induced microglial activation in the hippocampus and SN, thus protecting the neurons. Rifampicin inhibited the activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway. Rifampicin downregulated TLR4 and MyD88 protein levels and inhibited NF-κB inhibitor alpha and NF-κB inhibitor kinase beta phosphorylation, thus reducing p65 nuclear transfer by inhibiting NF-κB signaling activation in LPS-treated mice., Conclusion: Rifampicin protects against LPS-induced neuroinflammation and attenuates cognitive and motor impairments by inhibiting the TLR4/MyD88/NF-κB signaling pathway. Our findings might aid the development of novel therapies to treat progressive neurodegenerative diseases.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC., Competing Interests: Declaration of Interests L.F.E.-H. is a consultant for KDx Diagnostics Inc and OncoGenesis Inc. O.A.-W. served as a consultant for H3B Biomedicine, Foundation Medicine Inc, Merck, and Janssen, and is on the scientific advisory board of Envisagenics; O.A.-W. received prior research funding from H3B Biomedicine unrelated to the current manuscript and serves on the Scientific Advisory Board of Envisagenics Inc. S.D.L. is a member of the Scientific Advisory Board for Nybo Pharmaceuticals and co-founder of Episteme Prognostics., (Copyright © 2020. Published by Elsevier Inc.)

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na + channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the β-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not β- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na + transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.

The Hippo pathway, which plays a critical role in organ size control and cancer, features numerous WW domain-based protein-protein interactions. However, ~100 WW domains and 2,000 PY motif-containing peptide ligands are found in the human proteome, raising a "WW-PY" binding specificity issue in the Hippo pathway. In this study, we have established the WW domain binding specificity for Hippo pathway components and uncovered a unique amino acid sequence required for it. By using this criterion, we have identified a WW domain-containing protein, STXBP4, as a negative regulator of YAP. Mechanistically, STXBP4 assembles a protein complex comprising α-catenin and a group of Hippo PY motif-containing components/regulators to inhibit YAP, a process that is regulated by actin cytoskeleton tension. Interestingly, STXBP4 is a potential tumor suppressor for human kidney cancer, whose downregulation is correlated with YAP activation in clear cell renal cell carcinoma. Taken together, our study not only elucidates the WW domain binding specificity for the Hippo pathway, but also reveals STXBP4 as a player in actin cytoskeleton tension-mediated Hippo pathway regulation., (© 2019 The Authors.)

Objective To determine the effect of ropivacaine on peripheral neuropathy in diabetic rats and its possible mechanism. Methods Forty-eight Sprague-Dawley rats were randomly divided into six groups: nondiabetic control group, nondiabetic group A (0.25% ropivacaine), nondiabetic group B (0.75% ropivacaine), diabetic control group (diabetic peripheral neuropathy (DPN) +artificial cerebrospinal fluid), diabetic group A (DPN+0.25% ropivacaine), and diabetic group B (DPN + 0.75% ropivacaine), with eight rats in each group. Within an hour of the last administration, the sciatic motor nerve conduction velocity (MNCV) of each group was measured, and the morphological changes of rat sciatic nerve were observed by HE, Weil's staining and electron microscopy. The expression of transient receptor potential vanilloid (TRPV1) in the spinal cord dorsal horn of rats was analyzed by immunohistochemistry, and the expression of Calcitonin gene-related peptide (CGRP) protein in the spinal cord was analyzed by Western blot. Results Compared with the nondiabetic control group, elevated blood glucose, decreased weight and reduced average mechanical withdrawal threshold (MWT), additionally, the sciatic nerves showed significantly slowed conduction velocity (both P<0.001) and damaged pathological structure, the expression of TRPV1 and CGRP were decreased (both P<0.001) in the diabetic groups. Compared with the diabetic control group, down-regulation of TRPV1 and CGRP in spinal cord was significant for the diabetic groups A and B treated with 0.25 and 0.75% ropivacaine, the higher concentration of ropivacaine correlated with a greater change. Conclusion Ropivacaine can significantly block sciatic nerve conduction velocity in DPN rats in a concentration-dependent manner, which may be related to the expression of the TRPV1-CGRP pathway., (© 2019 The Author(s).)

Despite the increasing number of available therapeutic methods, the prognosis of non‑small cell lung cancer (NSCLC) remains poor. Furthermore, side effects are an important limiting factor in the treatment of NSCLC. Therefore, developing an efficacious, safe, affordable and easily accessible chemotherapeutic agent is necessary for NSCLC treatment. As a natural chemical produced by Zingiberaceae plants, curcumin exerts distinct antitumor effects on several tumor types. In the present study, curcumin was observed to inhibit not only cell proliferation and cell cycle transition, but also cell migration in NSCLC, as determined by a series of experiments (such as MTS assay, colony formation assay, flow cytometric analysis, Transwell migration assay and western blotting). Mechanistically, curcumin induced G2/M phase arrest by controlling cell cycle‑ and epithelial‑mesenchymal transition (EMT)‑related checkpoints. Furthermore, curcumin significantly inhibited the expression of Toll‑like receptor 4 (TLR4)/MyD88 and EGFR in a dose‑ and time‑dependent manner. Conversely, EGF reversed the inhibitory action of curcumin on TLR4/MyD88. In clinical specimens, TLR4 and MyD88 were highly expressed in NSCLC tissues, and a significant positive association was observed between TLR4 and MyD88 expression. These data suggested that curcumin may control the EGFR and TLR4/MyD88 pathways to synergistically downregulate downstream cell cycle‑ and EMT‑related regulators, in order to block cell proliferation and metastasis in NSCLC. These findings provide evidence for the clinical application of curcumin.

The activation of primordial follicles is critical to ovarian follicle development, which directly influences female fertility and reproductive life span. Several studies have suggested a role for long noncoding RNAs (lncRNAs) in ovarian function. However, the precise involvement of lncRNAs in the initiation of primordial follicles is still unknown. Here, an in vitro culture model was used to investigate the roles of lncRNAs in primordial follicle activation. We found that primordial follicles in day 3 mouse ovaries were activated after culturing for 8 days in vitro, as indicated by ovarian morphology changes, increases in primary follicle number, and downregulation of mammalian Sterile 20-like kinase messenger RNA (mRNA) and upregulation of growth differentiation factor 9 mRNA. We next examined lncRNA expression profiles by RNA sequencing at the transcriptome level and found that among 60 078 lncRNAs, 6541 lncRNA were upregulated and 2135 lncRNA were downregulated in 3-day ovaries cultured for 8 days in vitro compared with ovaries from day 3 mice. We also found that 4171 mRNAs were upregulated and 1795 were downregulated in the cultured ovaries. Gene ontology and pathway analyses showed that the functions of differentially expressed lncRNA targets and mRNAs were closely linked with many processes and pathways related to ovary development, including cell proliferation and differentiation, developmental processes, and other signaling transduction pathways. Additionally, many novel identified lncRNAs showed inducible expression, suggesting that these lncRNAs may be good candidates for investigating mouse primordial follicle activation. This study provides a foundation for further exploring lncRNA-related mechanisms in the initiation of mouse primordial follicles.

Diabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus, for which no effective treatment currently exists. We tested the hypothesis that Qi-dan-di-huang (QDDH) might have therapuetic effects in an experimental rat model of DN. The levels of I kappa KinaseAlpha and Beta, p-p65, p-IκB alpha, TGF-β1 and Alpha-SMA were significantly increased in kidneys in DN. QDDH decoction only partially reversed the increased Ikappa KinaseAlpha/Beta, p-p65, p-IKappaB alpha, TGF-Beta1 and alpha-SMA in the kidneys in DN. However, treatment of diabetic rats with QDDH decoction significantly inhibited the production and release of inflammatory cytokines IL-6, IL-1 beta and TNF-alpha into the serum. QDDH decoction also significantly improved the physiologic and biochemical indicators of DN, reduced glycogen and protein deposition in DN and prevented renal fibrosis. Together, the data show that QDDH decoction exerts a protective effect on kidneys in diabetic rats and reverses the inflammatory milieu of the serum in DN.

The Hippo pathway plays a crucial role in organ size control and tumor suppression, but its precise regulation is not fully understood. In this study, we discovered that phosphatidic acid (PA)-related lipid signaling is a key regulator of the Hippo pathway. Supplementing PA in various Hippo-activating conditions activates YAP. This PA-related lipid signaling is involved in Rho-mediated YAP activation. Mechanistically, PA directly interacts with Hippo components LATS and NF2 to disrupt LATS-MOB1 complex formation and NF2-mediated LATS membrane translocation and activation, respectively. Inhibition of phospholipase D (PLD)-dependent PA production suppresses YAP oncogenic activities. PLD1 is highly expressed in breast cancer and positively correlates with YAP activation, suggesting their pathological relevance in breast cancer development. Taken together, our study not only reveals a role of PLD-PA lipid signaling in regulating the Hippo pathway but also indicates that the PLD-PA-YAP axis is a potential therapeutic target for cancer treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this study, animal experimentation verified that the canonical Wnt/β-catenin signaling pathway was activated under a reduced activity of p-β-catenin (Ser33/37/Thr41) and an increased accumulation of β-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of H. parasuis . In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of β-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between β-catenin and E-cadherin dependent on Wnt/β-catenin signaling pathway. Moreover, Wnt/β-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/β-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.

Background: The receptors of Notch family play an important role in controlling the development, differentiation, and function of multiple cell types. The aim of this study is to investigate the role of Notch1 signaling upon immune suppression induced by melanoma cells., Methods: Melanoma cell line B16 cells were transfected by lentivirus containing mouse Notch1 gene or Notch1 shRNA to generate B16 cell line that highly or lowly expressed Notch1. Notch1 in anti-tumor immune response was comprehensively appraised in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice. The ratios of CD3 + CD8 + cytotoxic T cells, CD49b + NK cells, CD4 + CD25 + FoxP3 + Tregs and Gr1 + CD11b + MDSCs in tumor-DLN or spleen were examined by flow cytometry. After the co-culture of B16 cells and CD8 + T cells, the effects of Notch1 on the proliferation and activation of T cells were assessed by CCK8 assay, CFSE dilution and Chromium-release test. The mRNA expression and supernatant secretion of immunosuppressive cytokines, TGF-β1, VEGF, IL-10 and IFN-γ were measured by RT-PCR and ELISA, respectively., Results: Downregulation or overexpression of Notch1 in B16 melanoma cells inhibited or promoted tumor growth in immunocompetent mice, respectively. Notch1 expression in B16 melanoma cells inhibited the infiltration of CD8+ cytotoxic T lymphocytes and NK cells and reduced IFN-γ release in tumor tissue. It could also enhance B16 cell-mediated inhibition of T cell proliferation and activation, and upregulate PD-1 expression on CD4 + and CD8 + T cells. The percentage of CD4 + CD25 + FoxP3 + Tregs and Gr1 + CD11b + MDSCs were significantly increased in tumor microenvironment, and all these were attributed to the upregulation of TGF-β1., Conclusion: These findings suggested that Notch1 signaling in B16 melanoma cells might inhibit antitumor immunity by upregulation of TGF-β1.

ASAP1 regulates F-actin-based structures and functions, including focal adhesions (FAs) and circular dorsal ruffles (CDRs), cell spreading and migration. ASAP1 function requires its N-terminal BAR domain. We discovered that nonmuscle myosin 2A (NM2A) directly bound the BAR-PH tandem of ASAP1in vitro ASAP1 and NM2A co-immunoprecipitated and colocalized in cells. Knockdown of ASAP1 reduced colocalization of NM2A and F-actin in cells. Knockdown of ASAP1 or NM2A recapitulated each other's effects on FAs, cell migration, cell spreading, and CDRs. The NM2A-interacting BAR domain contributed to ASAP1 control of cell spreading and CDRs. Exogenous expression of NM2A rescued the effect of ASAP1 knockdown on CDRs but ASAP1 did not rescue NM2A knockdown defect in CDRs. Our results support the hypothesis that ASAP1 is a positive regulator of NM2A. Given other binding partners of ASAP1, ASAP1 may directly link signaling and the mechanical machinery of cell migration., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Viral infection triggers a series of signaling cascades, which converge to activate the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3), thereby inducing the transcription of type I interferons (IFNs). Although not fully characterized, these innate antiviral responses are fine-tuned by dynamic ubiquitination and deubiquitination processes. In this study, we report ubiquitin-specific protease (USP) 15 is involved in regulation of the retinoic acid-inducible gene I (RIG-I)-dependent type I IFN induction pathway. Knockdown of endogenous USP15 augmented cellular antiviral responses. Overexpression of USP15 inhibited the transcription of IFN-β. Further analyses identified histidine 862 as a critical residue for USP15's catalytic activity. Interestingly, USP15 specifically removed lysine 63-linked polyubiquitin chains from RIG-I among the essential components in RIG-I-like receptor-dependent pathway. In addition, we demonstrated that in contrast to USP15 de-ubiquitinating (DUB) activity, USP15-mediated inhibition of IFN signaling was not abolished by mutations eliminating the catalytic activity, indicating that a fraction of USP15-mediated IFN antagonism was independent of the DUB activity. Catalytically inactive USP15 mutants, as did the wild-type protein, disrupted virus-induced interaction of RIG-I and IFN-β promoter stimulator 1. Taken together, our data demonstrate that USP15 acts as a negative regulator of RIG-I signaling via DUB-dependent and independent mechanisms.

Transmissible gastroenteritis virus (TGEV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea and severe dehydration in piglets. In this study, liquid chromatography-tandem mass spectrometry coupled to isobaric tags for relative and absolute quantification labeling was used to quantitatively identify differentially expressed cellular proteins after TGEV infection in PK-15 cells. In total, 162 differentially expressed cellular proteins were identified, including 60 upregulated proteins and 102 downregulated proteins. These differentially expressed proteins were involved in the cell cycle, cellular growth and proliferation, the innate immune response, etc. Interestingly, many upregulated proteins were associated with interferon signaling, especially signal transducer and activator of transcription 1 (STAT1) and interferon-stimulated genes (ISGs). Immunoblotting and real-time quantitative reverse transcription polymerase chain reaction demonstrated that TGEV infection induces STAT1 phosphorylation and nuclear translocation, as well as ISG expression. This study for the first time reveals that TGEV induces interferon signaling from the point of proteomic analysis.

Breast cancer already taken the first place of incidence in Chinese female cancer patients. TRPM8 is found to be over-expressed in breast cancer, but whether it promotes breast cancer aggressiveness remains unknown. In our study, TRPM8 was identified highly expressing in all the tested breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3 and ZR-75-30, while it just could be detected in MCF-10A, the normal breast epithelial cell. Then four pairs of clinical samples were analyzed using Western blotting and the result showed that TRPM8 expression is higher in tumor tissues than in adjacent nontumor tissues. Subsequently, we established TRPM8 high-expressing MCF-7 cell line and TRPM8 knockout MDA-MB-231 cell line to explore expression status of cancer-related proteins. The Western blotting and immunofluorescence analysis outcomes demonstrated that TRPM8 might influence cancer cell metastasis by regulating the EMT phenotype via activating AKT/GSK-3β pathway, and the hypothesis had been supported by cell function tests. All the results demonstrated that TRPM8 significantly up-expressed in breast cancer cells and promoted their metastasis by regulating EMT via activating AKT/GSK-3β pathway, indicating TRPM8 gets the prospects of to be developed as medication or diagnostic indicator to be applied in clinical work.

Nucleotide-binding oligomerization domains (NOD)-like receptors (NLRs) evolve as a group of germline-encoded receptors that detect cytosolic pathogen-associated molecular patterns. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide. By examining the expression kinetics of ten selected NLRs, NOD2 and NLRP3 were found to be continuously up-regulated in PRRSV-infected MARC-145 cells during 48 h of post-infection. Further study revealed that PRRSV infection enhanced the expression and phosphorylation of RIP2. Knockdown of NOD2 and RIP2 by siRNA significantly decreased PRRSV-induced phosphorylation of NF-κB subunit p65, JNK, Erk and p38 MAPK, as well as the expression of IL-6, IL-8, TNF-α, and RANTES in MARC-145 cells. Moreover, increased expression of NOD2 and RIP2 mRNA were observed in alveolar macrophages isolated from PRRSV-challenged piglets at 3, 7 and 10 day post-challenge. Collectively, our results revealed that PRRSV infection activates NOD2-RIP2 signaling pathway to induce pro-inflammatory response., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Previously, we found that sperm-associated antigen 5 (SPAG5) was upregulated in pelvic lymph node metastasis-positive cervical cancer. The aim of this study is to examine the role of SPAG5 in the proliferation and tumorigenicity of cervical cancer and its clinical significance in tumor progression. In our study, SPAG5 expression in cervical cancer patients was detected using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry; cervical cancer cell function with downregulated SPAG5 in vitro was explored using tetrazolium assay, flow cytometry, and colony formation and Transwell assays. SPAG5 was upregulated in tumor tissue compared with paired adjacent noncancerous tissues; SPAG5 upregulation in tumor tissues indicated poor disease-free survival, which was also an independent prognostic indicator for cervical cancer patients. In vitro study demonstrated that SPAG5 downregulation inhibited cell proliferation and growth significantly by G2/M arrest and induction of apoptosis, and hindered cell migration and invasion. Under SPAG5 downregulation, the sensitivity of cervical cancer cells differed according to taxol dose, which correlated with mammalian target of rapamycin (mTOR) signaling pathway activity. In general, SPAG5 upregulation relates to poor prognosis in cervical cancer patients, and SPAG5 is a regulator of mTOR activity during taxol treatment in cervical cancer.

Ubiquitination and deubiquitination have emerged as critical regulatory processes in the virus-triggered type I interferon (IFN) induction pathway. In this study, we carried out a targeted siRNA screen of 54 ubiquitin-specific proteases (USPs) and identified USP25 as a negative regulator of the virus-triggered type I IFN signaling pathway. Overexpression of USP25 inhibited virus-induced activation of IFN-β, interferon regulation factor 3 (IRF3) and nuclear factor-kappa B (NF-κB), as well as the phosphorylation of IRF3 and NF-κB subunit p65. Furthermore, Knockdown of USP25 potentiated virus-induced induction of the IFN-β. In addition, detailed analysis demonstrated that USP25 cleaved lysine 48- and lysine 63-linked polyubiquitin chains in vitro and in vivo, and its deubiquitinating enzyme (DUB) activity, were dependent on a cysteine residue (Cys178) and a histidine residue (His607). USP25 mutants lacking DUB activity lost the ability to block virus-induced type I IFN to some degree. Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling. Our findings suggest that USP25 is a novel DUB negatively regulating virus-induced type I IFN production.

Background: Recent studies have demonstrated that myocardial calpain triggers caspase-3 activation and myocardial apoptosis in models of sepsis, whereas the inhibition of calpain activity down-regulates myocardial caspase-3 activation and apoptosis. However, the mechanism underlying this pathological process is unclear. Therefore, in this study, our aim was to explore whether the Hsp90/Akt signaling pathway plays a role in the induction of myocardial calpain activity, caspase-3 activation and apoptosis in the septic mice., Methods: Adult male C57 mice were injected with lipopolysaccharide (LPS, 4 mg/kg, i.p.) to induce sepsis. Next, myocardial caspase-3 activity and the levels of Hsp90/p-Akt (phospho-Akt) proteins were detected, and apoptotic cells were assessed by performing the TUNEL assay., Results: In the septic mice, there was an increase in myocardial calpain and caspase-3 activity in addition to an increase in the number of apoptotic cells; however, there was a time-dependent decrease in myocardial Hsp90/p-Akt protein levels. The administration of calpain inhibitors (calpain inhibitor-Ш or PD150606) prevented the LPS-induced degradation of myocardial Hsp90/p-Akt protein and its expression in cardiomyocytes in addition to inhibiting myocardial caspase-3 activation and apoptosis. The inhibition of Hsp90 by pretreatment with 17-AAG induced p-Akt degradation, and the inhibition of Akt activity by pretreatment with wortmannin resulted in caspase-3 activation in wildtype C57 murine heart tissues., Conclusions: Myocardial calpain induces myocardial caspase-3 activation and apoptosis in septic mice via the activation of the Hsp90/Akt pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. To investigate the impact of cellular microRNAs (miRNAs) on the replication of PRRSV, we screened 10 highly conserved miRNAs implicated in innate immunity or antiviral function and identified miR-125b as an inhibitor of PRRSV replication. Virus titer and western blot assays demonstrated that miR-125b reduced PRRSV replication and viral gene expression in a dose-dependent manner in both MARC-145 cell line and primary porcine alveolar macrophages. Mechanistically, miR-125b did not target the PRRSV genome. Rather, it inhibited activation of NF-κB, which we found to be required for PRRSV replication. PRRSV, in turn, down-regulated miR-125b expression post-infection to promote viral replication. Collectively, miR-125b is an antiviral host factor against PRRSV, but it is subject to manipulation by PRRSV. Our study reveals an example of manipulation of a cellular miRNA by an arterivirus to re-orchestrate host gene expression for viral propagation and sheds new light on targeting host factors to develop effective control measures for PRRS.

Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-α/β response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3C(pro) specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.

To identify unique biochemical pathways in embryonic stem cell-derived insulin-producing cells as potential therapeutic targets to prevent or delay beta-cell dysfunction or death in diabetic patients, comparative genome-wide gene expression studies of recently derived mouse insulin-producing cell lines and their progenitor cell lines were performed using microarray technology. Differentially expressed genes were functionally clustered to identify important biochemical pathways in these insulin-producing cell lines. Biochemical or cellular assays were then performed to assess the relevance of these pathways to the biology of these cells. A total of 185 genes were highly expressed in the insulin-producing cell lines, and computational analysis predicted the pentose phosphate pathway (PPP), clathrin-mediated endocytosis, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway as important pathways in these cell lines. Insulin-producing ERoSHK cells were more resistant to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Inhibition of PPP by dehydroepiandrosterone and 6-aminonicotinamide abrogated this H(2)O(2) resistance with a concomitant decrease in PPP activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Clathrin-mediated endocytosis, which is essential in maintaining membrane homeostasis in secreting cells, was up-regulated by glucose in ERoSHK but not in their progenitor ERoSH cells. Its inhibition by chlorpromazine at high glucose concentration was toxic to the cells. Troglitazone, a PPARG agonist, up-regulated expression of Ins1 and Ins2 but not Glut2. Gene expression analysis has identified the PPP, clathrin-mediated endocytosis, and the PPAR signaling pathway as the major delineating pathways in these insulin-producing cell lines, and their biological relevance was confirmed by biochemical and cellular assays.

Cellular movement is mainly orchestrated by actin-dependent cytoskeleton in which Rho GTPase Rac1 or vasodilator-stimulated phosphoprotein (VASP) closely collaborates. In the present in vitro study, we investigated the inhibitory effect and underlying molecular mechanism of icariin, a pure extract of the traditional Chinese medicine Herba epimedii, on the invasive and migration properties of human gastric cancer cell line BGC-823. At 50% growth-inhibiting concentration, icariin significantly suppressed tumor cells migration and invasion, which were traceable to down-regulation of Rac1 and VASP. Together with icariin, the selected siRNA targeting Rac1 or VASP reinforced these inhibitory effects. Rac1-siRNA-dependent down-regulation of Rac1 led to a large drop in VASP expression, whereas VASP-siRNA led to a slight fall in Rac1 expression, implying that the amount of Rac1 may influence VASP expression level. Moreover, transfection with Rac1 plasmids pcDNA3-EGFP-Rac1-Q61L led to the enhancement in expression level of both Rac1 and VASP. These results indicate that icariin exerts negative effects on tumor cell invasion and migration via the Rac1-dependent VASP pathway and may be a potential anti-cancer drug.

The DNA-dependent activator of IFN-regulatory factors (DAI) is a recently identified DNA sensor for intracellular DNA that triggers a signal for the production of type I IFN. Here we report the cloning and characterization of porcine DAI (poDAI). The full-length of poDAI encodes 439 amino acids, contains two N-terminal DNA-binding domains and shows similarity to mouse, rat, dog, monkey, human, horse and cattle counterparts ranging from 44% to 67%. poDAI mRNA expression was mainly detected in spleen, lung, kidney and small intestine. Over-expression of poDAI activated transcription factors IRF3 and NF-kappaB and induced IFN-beta in different porcine cell lines, but to varying degrees. Deletion mutant analysis revealed that both the DNA-binding domains and the C-terminus are required for full activation of IFN-beta. siRNA targeting poDAI significantly decreased poly(dAT:dAT)- or Pseudorabies virus (PRV)-induced IFN-beta activation. These results indicate that DAI is an important immuno-regulator of the porcine innate immune system., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Objective: To investigate the relationship between Wnt pathway and intrauterine hypoxia-ischemia brain damage., Methods: The gestational rats (15-17days) were randomly divided into sham operation group and transient intrauterine ischemia group. For transient intrauterine ischemia group, the gestational rats' bilateral uterine arteries were clamped for 30 min, followed by reperfusion. For sham operation, animals were subjected to the same surgical procedures, except that bilateral uterine arteries were not clamped. Samples were collected at 24, 48, and 72 h after reperfusion. The whole brain tissues were dissected for Nissl stain and Caspase-3 immumohistochemical. The expressions of GSK-3 beta and beta-catenin protein in the two groups were analyzed with Western Blotting., Results: The Nissl body of the transient intrauterine ischemia group decreased and the masculine cell of Caspase-3 was significantly higher than sham operation group at each time point (P < 0.05). The sham operation group is 12.71 +/- 1.42 (24 h), 11.45 +/- 1.50 (48 h), 14.10 +/- 1.29 (72 h) and the transient intrauterine ischemia group is 26.29 +/- 1.64 (24 h), 32.38 +/- 1.05 (48 h), 39.20 +/- 1.88 (72 h). beta-catenin expression between the two groups was no statistically significant difference. However, expression of GSK-3 beta in transient intrauterine ischemia group increased (P < 0.05). The sham operation group is 1.75 +/- 1.07 (24 h), 1.34 +/- 1.03 (48 h), 1.48 +/- 1.10 (72 h) and the transient intrauterine ischemia group is 3.25 +/- 1.34 (24 h), 2.99 +/- 1.26 (48 h), 3.10 +/- 1.35 (72 h)., Conclusion: Wnt pathway may play an important part in intrarterine hypoxia-ischemia brain damage.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the swine industry since the late 1980s. Accumulating evidences have revealed that PRRSV infection fails to induce type I interferon (IFN-alpha/beta), which are normally induced rapidly during virus replication in virus-infected cells. However, the potential mechanisms remain largely unclear. In this study, we showed that PRRSV infection activated the signal transduction components of NF-kappaB and AP-1, but not of interferon regulatory factor 3 (IRF3), an essential IFN-beta transcription factor. Furthermore, PRRSV infection significantly blocked synthetic dsRNA-induced IFN-beta production and IRF3 nuclear translocation. To better understand the upstream signaling events that suppress IRF3 activation, we further investigated the roles of individual components of the retinoic acid-inducible gene I (RIG-I)- and Toll-like receptor 3 (TLR3)-mediated signaling pathway for IFN-beta production during PRRSV infection. We observed that PRRSV infection significantly inhibited dsRNA-induced IRF3 activation and IFN-beta generation by inactivating IFN-beta promoter stimulator 1 (IPS-1), an adaptor molecule of RIG-I. In contrast, PRRSV infection only partially reduced the activation of TIR domain-containing adaptor inducing IFN-beta (TRIF), an adaptor molecule of TLR3. Our results suggest that PRRSV infection suppresses production of IFN-beta primarily by interfering with the IPS-1 activation in the RIG-I signaling pathway.

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.