Identification of Mycoplasma pneumoniae proteins interacting with NOD2 and their role in macrophage inflammatory response.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is a cell wall-deficient microorganism known to cause chronic respiratory infections in both children and adults. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor primarily responsible for identifying muramyl dipeptide (MDP) found in bacterial cell walls. Previous experiments have demonstrated that Mycoplasma ovipneumoniae induces macrophage autophagy through NOD2. In this study, we conducted RNA-seq analysis on macrophages infected with M. pneumoniae and observed an up-regulation in the expression of genes associated with the NOD2 signaling pathway. Mechanistic investigations further revealed the involvement of the NOD2 signaling pathway in the inflammatory response of macrophages activated by M. pneumoniae. We utilized GST pull-down technology in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to pinpoint the M. pneumoniae proteins that interact with NOD2. Additionally, co-immunoprecipitation (Co-IP) and immunofluorescence co-localization techniques were used to confirm the interaction between DUF16 protein and NOD2. We found that DUF16 protein can enter macrophages and induce macrophage inflammatory response through the NOD2/RIP2/NF-κB pathway. Notably, the region spanning amino acids 13-90 was identified as a critical region necessary for DUF16-induced inflammation. This research not only broadens our comprehension of the recognition process of the intracellular receptor NOD2, but also deepens our understanding of the development of M. pneumoniae infection.

Glutathione Induces Keap1 S-Glutathionylation and Mitigates Oscillating Glucose-Induced ß-Cell Dysfunction by Activating Nrf2.

Glutathione (GSH), a robust endogenous antioxidant, actively participates in the modulation of the redox status of cysteine residues in proteins. Previous studies have indicated that GSH can prevent ß-cell failure and prediabetes caused by chronic oscillating glucose (OsG) administration. However, the precise mechanism underlying the protective effect is not well understood. Our current research reveals that GSH is capable of reversing the reduction in Nrf2 levels, as well as downstream genes Grx1 and HO-1, in the islet ß-cells of rats induced by chronic OsG. In vitro experiments have further demonstrated that GSH can prevent ß-cell dedifferentiation, apoptosis, and impaired insulin secretion caused by OsG. Additionally, GSH facilitates the translocation of Nrf2 into the nucleus, resulting in an upregulation of Nrf2-targeted genes such as GCLC, Grx1, HO-1, and NQO1. Notably, when the Nrf2 inhibitor ML385 is employed, the effects of GSH on OsG-treated ß-cells are abrogated. Moreover, GSH enhances the S-glutathionylation of Keap1 at Cys273 and Cys288, but not Cys151, in OsG-treated ß-cells, leading to the dissociation of Nrf2 from Keap1 and facilitating Nrf2 nuclear translocation. In conclusion, the protective role of GSH against OsG-induced ß-cell failure can be partially attributed to its capacity to enhance Keap1 S-glutathionylation, thereby activating the Nrf2 signaling pathway. These findings provide novel insights into the prevention and treatment of ß-cell failure in the context of prediabetes/diabetes, highlighting the potential of GSH.

VGLL4 promotes vascular endothelium specification via TEAD1 in the vascular organoids and human pluripotent stem cells-derived endothelium model.

BACKGROUND: We have shown that Hippo-YAP signaling pathway plays an important role in endothelial cell differentiation. Vestigial-like family member 4 (VGLL4) has been identified as a YAP inhibitor. However, the exact function of VGLL4 in vascular endothelial cell development remains unclear. In this study, we investigated the role of VGLL4, in human endothelial lineage specification both in 3D vascular organoid and 2D endothelial cell differentiation. METHODS AND RESULTS: In this study, we found that VGLL4 was increased during 3D vascular organoids generation and directed differentiation of human embryonic stem cells H1 towards the endothelial lineage. Using inducible ectopic expression of VGLL4 based on the piggyBac system, we proved that overexpression of VGLL4 in H1 promoted vascular organoids generation and endothelial cells differentiation. In contrast, VGLL4 knockdown (heterozygous knockout) of H1 exhibited inhibitory effects. Using bioinformatics analysis and protein immunoprecipitation, we further found that VGLL4 binds to TEAD1 and facilitates the expression of endothelial master transcription factors, including FLI1, to promote endothelial lineage specification. Moreover, TEAD1 overexpression rescued VGLL4 knockdown-mediated negative effects. CONCLUSIONS: In summary, VGLL4 promotes EC lineage specification both in 3D vascular organoid and 2D EC differentiation from pluripotent stem cell, VGLL4 interacts with TEAD1 and facilitates EC key transcription factor, including FLI1, to enhance EC lineage specification.

Extracellular Vesicles Released from Macrophages Infected with Mycoplasma pneumoniae Stimulate Proinflammatory Response via the TLR2-NF-κB/JNK Signaling Pathway.

Mycoplasma pneumoniae (M. pneumoniae, Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae-infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae-infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae-infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae-infected macrophages are pure, with a 30-200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection.

Viscous, Hofmeister Effect-Enhanced Macromolecular Adhesives for Effective Bonding in Seawater.

Deployment of adhesives in natural seawater to in situ bonds is urgently needed in engineering fields. However, stable adhesion in natural seawater remains a challenge due to the turbulent environment and high ion concentration. Herein, we reported a viscous, macromolecular underwater adhesive enhanced by Hofmeister effect (EHUA) for practical application in dynamic seawater. EHUA was synthesized via a facile one-step copolymerization. After transferred into seawater, the solvent of EHUA was exchanged to seawater, and thereby hydrogen bonds inside the adhesive were activated and enhanced by Hofmeister effect. We demonstrated EHUA can adhere on the surface in turbulent seawater, and the adhesive strength could reach 1.691 MPa. In addition, the adhesives also exhibited long-term storage stability and convenient recyclability. These fascinating properties enable adhesives to seal leaky pipelines, repair damaged ships and construct buildings in turbulent seawater. This work may open an avenue for the design of adhesives for seawater environments.

VGLL4-TEAD1 promotes vascular smooth muscle cell differentiation from human pluripotent stem cells via TET2.

The Hippo signaling pathway plays a critical role in cardiovascular development and stem cell differentiation. Using microarray profiling, we found that the Hippo pathway components vestigial-like family member 4 (VGLL4) and TEA domain transcription factor 1 (TEAD1) were upregulated during vascular smooth muscle cell (VSMC) differentiation from H1 ESCs (H1 embryonic stem cells). To further explore the role and molecular mechanisms of VGLL4 in regulating VSMC differentiation, we generated a VGLL4-knockdown H1 ESC line (heterozygous knockout) using the CRISPR/Cas9 system and found that VGLL4 knockdown inhibited VSMC specification. In contrast, overexpression of VGLL4 using the PiggyBac transposon system facilitated VSMC differentiation. We confirmed that this effect was mediated via TEAD1 and VGLL4 interaction. In addition, bioinformatics analysis revealed that Ten-eleven-translocation 2 (TET2), a DNA dioxygenase, is a target of TEAD1, and a luciferase assay further verified that TET2 is the target of the VGLL4-TEAD1 complex. Indeed, TET2 overexpression promoted VSMC marker gene expression and countered the VGLL4 knockdown-mediated inhibitory effects on VSMC differentiation. In summary, we revealed a novel role of VGLL4 in promoting VSMC differentiation from hESCs and identified TET2 as a new target of the VGLL4-TEAD1 complex, which may demethylate VSMC marker genes and facilitate VSMC differentiation. This study provides new insights into the VGLL4-TEAD1-TET2 axis in VSMC differentiation and vascular development.

Glutathione prevents high glucose-induced pancreatic fibrosis by suppressing pancreatic stellate cell activation via the ROS/TGFß/SMAD pathway.

The activation of pancreatic stellate cells (PSCs) is the key mechanism of pancreatic fibrosis, which can lead to ß-cell failure. Oxidative stress is an important risk factor for PSC activation. There is no direct evidence proving if administration of glutathione can inhibit fibrosis and ß-cell failure. To explore the role of glutathione in pancreatic fibrosis and ß-cell failure induced by hyperglycaemia, we established a rat model of pancreatic fibrosis and ß-cell failure. The model was founded through long-term oscillating glucose (LOsG) intake and the setup of a sham group and a glutathione intervention group. In vitro, rat PSCs were treated with low glucose, high glucose, or high glucose plus glutathione to explore the mechanism of high glucose-induced PSC activation and the downstream effects of glutathione. Compared with sham rats, LOsG-treated rats had higher reactive oxygen species (ROS) levels in peripheral leukocytes and pancreatic tissue while TGFß signalling was upregulated. In addition, as the number of PSCs and pancreatic fibrosis increased, ß-cell function was significantly impaired. Glutathione evidently inhibited the upregulation of TGFß signalling and several unfavourable outcomes caused by LOsG. In vitro treatment of high glucose for 72 h resulted in higher ROS accumulation and potentiated TGFß pathway activation in PSCs. PSCs showed myofibroblast phenotype transformation with upregulation of α-SMA expression and increased cell proliferation and migration. Treatment with either glutathione or TGFß pathway inhibitors alleviated these changes. Together, our findings suggest that glutathione can inhibit PSC activation-induced pancreatic fibrosis via blocking ROS/TGFß/SMAD signalling in vivo and in vitro.

Protocol for inducible piggyBac transposon system for efficient gene overexpression in human pluripotent stem cells.

In human pluripotent stem cells (hPSCs), traditional approaches for gene overexpression have low efficiency and are often laborious. Here, we provide a relatively simple protocol for gene overexpression with the Dox-inducible PiggyBac transposon system. We detail the steps for overexpression of FLI1 and/or YAP in H1 embryonic stem cells (H1 ESCs) as an example. Our protocol can be applied to any gene of interest in a variety of hPSCs. For complete details on the use and execution of this protocol, please refer to Quan et al. (2021).

Updated Overall Survival of Ribociclib plus Endocrine Therapy versus Endocrine Therapy Alone in Pre- and Perimenopausal Patients with HR+/HER2- Advanced Breast Cancer in MONALEESA-7: A Phase III Randomized Clinical Trial.

PURPOSE: Ribociclib plus endocrine therapy (ET) demonstrated a statistically significant progression-free survival and overall survival (OS) benefit in the phase III MONALEESA-7 trial of pre-/perimenopausal patients with hormone receptor (HR)-positive (HR+), HER2-negative (HER2-) advanced breast cancer (ABC). The median OS was not reached in the ribociclib arm in the protocol-specified final analysis; we hence performed an exploratory OS and additional outcomes analysis with an extended follow-up (median, 53.5 months). PATIENTS AND METHODS: Patients were randomized to receive ET [goserelin plus nonsteroidal aromatase inhibitor (NSAI) or tamoxifen] with ribociclib or placebo. OS was evaluated with a stratified Cox proportional hazard model and summarized with Kaplan-Meier methods. RESULTS: The intent-to-treat population included 672 patients. Median OS was 58.7 months with ribociclib versus 48.0 months with placebo [hazard ratio = 0.76; 95% confidence interval (CI), 0.61-0.96]. Kaplan-Meier estimated OS at 48 months was 60% and 50% with ribociclib and placebo, respectively. Subgroup analyses were generally consistent with the OS benefit, including patients who received NSAI and patients aged less than 40 years. Subsequent antineoplastic therapies following discontinuation were balanced between the ribociclib (77%) and placebo (78%) groups. Use of cyclin-dependent kinase 4/6 inhibitors after discontinuation was higher with placebo (26%) versus ribociclib (13%). Time to first chemotherapy was significantly delayed with ribociclib versus placebo. No drug-drug interactions were observed between ribociclib and either NSAI. CONCLUSIONS: Ribociclib plus ET continued to show significantly longer OS than ET alone in pre-/perimenopausal patients, including patients aged less than 40 years, with HR+/HER2- ABC with 53.5 months of median follow-up (ClinicalTrials.gov, NCT02278120).

YAP inhibition promotes endothelial cell differentiation from pluripotent stem cell through EC master transcription factor FLI1.

Endothelial cells (ECs) derived from pluripotent stem cells (PSCs) provide great resource for vascular disease modeling and cell-based regeneration therapy. However, the molecular mechanisms of EC differentiation are not completely understood. In this study, we checked transcriptional profile by microarray and found Hippo pathway is changed and the activity of YAP decreased during mesoderm-mediated EC differentiation from human embryonic stem cells (hESCs). Knockdown of YAP in hESCs promoted both mesoderm and EC differentiation indicating by mesodermal- or EC-specific marker gene expression increased both in mRNA and protein level. In contrast, overexpression of YAP inhibited mesoderm and EC differentiation. Microarray data showed that several key transcription factors of EC differentiation, such as FLI1, ERG, SOX17 are upregulated. Interestingly, knockdown YAP enhanced the expression of these master transcription factors. Bioinformation analysis revealed that TEAD, a YAP binds transcription factors, might regulate the expression of EC master TFs, including FLI1. Luciferase assay confirmed that YAP binds to TEAD1, which would inhibit FLI1 expression. Finally, FLI1 overexpression rescued the effects of YAP overexpression-mediated inhibition of EC differentiation. In conclusion, we revealed the inhibitory effects of YAP on EC differentiation from PSCs, and YAP inhibition might promote expression of master TFs FLI1 for EC commitment through interacting with TEAD1, which might provide an idea for EC differentiation and vascular regeneration via manipulating YAP signaling.

Critical role of VGLL4 in the regulation of chronic normobaric hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension (PH), a rare but deadly cardiopulmonary disorder, is characterized by extensive remodeling of pulmonary arteries resulting from enhancement of pulmonary artery smooth muscle cell proliferation and suppressed apoptosis; however, the underlying pathophysiological mechanisms remain largely unknown. Recently, epigenetics has gained increasing prominence in the development of PH. We aimed to investigate the role of vestigial-like family member 4 (VGLL4) in chronic normobaric hypoxia (CNH)-induced PH and to address whether it is associated with epigenetic regulation. The rodent model of PH was established by CNH treatment (10% O2 , 23 hours/day). Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, immunoprecipitation, and adeno-associated virus tests were performed to explore the potential mechanisms involved in CNH-induced PH in mice. VGLL4 expression was upregulated and correlated with CNH in PH mouse lung tissues in a time-dependent manner. VGLL4 colocalized with α-smooth muscle actin in cultured pulmonary arterial smooth muscle cells (PASMCs), and VGLL4 immunoactivity was increased in PASMCs following hypoxia exposure in vitro. VGLL4 knockdown attenuated CNH-induced PH and pulmonary artery remodeling by blunting signal transducer and activator of transcription 3 (STAT3) signaling; conversely, VGLL4 overexpression exacerbated the development of PH. CNH enhanced the acetylation of VGLL4 and increased the interaction of ac-H3K9/VGLL4 and ac-H3K9/STAT3 in the lung tissues, and levels of ac-H3K9, p-STAT3/STAT3, and proliferation-associated protein levels were markedly up-regulated, whereas apoptosis-related protein levels were significantly downregulated, in the lung tissues of mice with CNH-induced PH. Notably, abrogation of VGLL4 acetylation reversed CNH-induced PH and pulmonary artery remodeling and suppressed STAT3 signaling. Finally, STAT3 knockdown alleviated CNH-induced PH. In conclusion, VGLL4 acetylation upregulation could contribute to CNH-induced PH and pulmonary artery remodeling via STAT3 signaling, and abrogation of VGLL4 acetylation reversed CNH-induced PH. Pharmacological or genetic deletion of VGLL4 might be a potential target for therapeutic interventions in CNH-induced PH.

Generation of human embryonic stem cell lines (WAe001-A-67,WAe001-A-68) with TEAD1 and TEAD4 expression by the PiggyBac transposon system.

Transcription factors TEAD1 and TEAD4 play an important role in development, differentiation, cell growth and proliferation. To further understand the exact role of TEAD1 and TEAD4 in these processes. We generated TEAD1 and TEAD4 doxycycline-inducible expression human embryonic stem cell lines (WAe001-A-67 and WAe001-A-68) by PiggyBac transposon system. These cell lines retained normal morphology and karyotype, normal expression of pluripotent markers, and differentiation potential. These cell lines can be used to verify whether the TEAD1 and TEAD4 play a role in stem cell and cell lineage differentiation.

Upregulation of KDM6B contributes to lipopolysaccharide-induced anxiety-like behavior via modulation of VGLL4 in mice.

Histone H3K27me3 demethylase KDM6B (also known as Jumonji domain-containing protein D3, JMJD3) plays vital roles in the etiology of inflammatory responses; however, little is known about the role of KDM6B in neuroinflammation-induced anxiety-like behavior. The present study aimed to investigate the potential role of KDM6B in lipopolysaccharide (LPS)-induced anxiety-like behavior and to evaluate whether it is associated with the modulation of vestigial-like family member 4 (VGLL4). The elevated plus maze, light-dark box, and open-field test were performed to test the anxiety-like behavior induced by LPS in C57BL/6 J male mice. Levels of relative protein expression in the hippocampus were quantified by western blotting. KDM6B inhibitor GSK-J4 and microglia inhibitor minocycline as well as adeno-associated virus of Vgll4 shRNA were used to explore the underlying mechanisms. We found that KDM6B, VGLL4, interleukin-1ß (IL-1ß), and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) protein levels were increased in LPS-dose dependent manner in the hippocampus but not in prefrontal cortex. GSK-J4 treatment attenuated LPS-induced VGLL4, the signal transducer and activator of transcription 3 (STAT3), IL-1ß and Iba-1 upregulation and anxiety-like behavior. Knockdown VGLL4 with Vgll4 shRNA prevented the increase of anxiety-like behavior and levels of STAT3, IL-1ß, and Iba-1 expression in the hippocampus of LPS-treated mice. Moreover, minocycline, an inhibitor of microglia treatment blunted LPS-induced anxiety-like behavior. Collectively, these results demonstrate that the induction of neuroinflammation by LPS promotes KDM6B activation in the hippocampus, and LPS-induced anxiety-like behavior is associated with upregulation of VGLL4 by KDM6B in the hippocampus.

Establishment and characterization of human induced pluripotent stem cell line (WMUi020-A) from a patient with bicuspid aortic valve aortopathy.

A human induced pluripotent stem cell (hiPSC) line (WMUi020-A) was generated from the aortic smooth muscle cells of a 56-year-old donor with bicuspid aortic valve and ascending aortic aneurysm. Episomal vector-mediated Non-integration iPSC reprogramming was used for this iPSC line generation. The established iPSC line highly expressed pluripotency markers with three germ-layer differentiation potential in vitro, as well as a normal karyotype. We further found that this iPSC line has a potential mutation of ROBO4 (c.161 T>C, p.Q54R), which may be useful for the disease modeling of bicuspid aortic valve aortopathy.

Resveratrol ameliorates lipopolysaccharide-induced anxiety-like behavior by attenuating YAP-mediated neuro-inflammation and promoting hippocampal autophagy in mice.

Resveratrol, a type of natural polyphenol mainly extracted from the skin of grapes, has been reported to protect against inflammatory responses and exert anxiolytic effect. Yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, plays a critical role in inflammation. The present study aimed to explore whether YAP pathway was involved in the anxiolytic effect of resveratrol in lipopolysaccharide (LPS)-treated C57BL/6J male mice. LPS treatment induced anxiety-like behavior and decreased sirtuin 1 while increased YAP expression in the hippocampus. Resveratrol attenuated LPS-induced anxiety-like behavior, which was blocked by EX-527 (a sirtuin 1 inhibitor). Mechanistically, the anxiolytic effects of resveratrol were accompanied by a marked decrease in YAP, interleukin-1ß and ionized calcium binding adaptor molecule 1 (Iba-1) while a significant increase in autophagic protein expression in the hippocampus. Pharmacological study using XMU-MP-1, a YAP activator, showed that activating YAP could induce anxiety-like behavior and neuro-inflammation as well as decrease hippocampal autophagy. Moreover, activation of YAP by XMU-MP-1 treatment attenuated the ameliorative effects of resveratrol on LPS-induced anxiety-like behavior, while blockade of YAP activation with verteporfin, a YAP inhibitor, attenuated LPS-induced anxiety-like behavior and neuro-inflammation as well as hippocampal autophagy. Finally, rapamycin-mediated promotion of autophagy attenuated LPS-induced anxiety-like behavior and decreased interleukin-1ß and Iba-1 expression in the hippocampus. Collectively, these results indicate that amelioration by resveratrol in LPS-induced anxiety-like behavior is through attenuating YAP-mediated neuro-inflammation and promoting hippocampal autophagy, and suggest that inhibition of YAP pathway could be a potential therapeutic target for anxiety-like behavior induced by neuro-inflammation.

YAP activity protects against endotoxemic acute lung injury by activating multiple mechanisms.

The roles of the Hippo­Yes­associated protein (YAP) pathway in lung injury and repair remain elusive. The present study examined the effects of systemic inhibition or stimulation of YAP activity on lung injury, repair and inflammation in a mouse model of lipopolysaccharide (LPS)­induced lung injury. Mice were treated with or without YAP inhibitor, verteporfin, or with or without YAP stimulator, XMU­MP­1, and intraperitoneally injected with LPS (7.5 mg/kg). Lung injury and repair were evaluated by histological analysis and by testing for markers of lung injury. Lung inflammation was assessed by measuring tissue levels of inflammatory mediators. Lung injury was associated with a decreased, whereas lung repair was associated with an increased YAP activity evidenced by nuclear translocation. Lung injury was associated with a high level of lung inflammation and epithelial adherens junction disassembly, but not with cell proliferation or epithelial cell regeneration. The injury phase was defined as 0­48 h post­LPS injection, and the 48­168 h time period was considered the repair phase. Inhibition of YAP activity at the injury phase, using verteporfin, exacerbated, whereas its stimulation, using XMU­MP­1, alleviated lung injury, lung inflammation and epithelial adherens junction disassembly. Inhibition or stimulation of YAP activity at the injury phase had no effects on cell proliferation or epithelial regeneration. By contrast, lung repair was associated with inflammation resolution, increased cell proliferation, epithelial regeneration and reassembly of epithelial adherens junctions. Inhibition of YAP activity at the repair phase delayed inflammation resolution, impeded lung recovery, inhibited cell proliferation and epithelial regeneration, and inhibited epithelial adherens junction reassembly. Stimulation of YAP activity at the repair phase reversed all these processes. The results of the current study demonstrated that the Hippo­YAP activity serves a protective role against endotoxemic lung injury. The Hippo­YAP activity alleviated lung inflammation and injury at the injury phase and promoted inflammation resolution and lung repair at the repair phase.

A Comprehensive Investigation on the Fire Hazards and Environmental Risks in a Commercial Complex Based on Fault Tree Analysis and the Analytic Hierarchy Process.

This paper focuses on the fire risk assessment for commercial complex, as the variety of fire accidental triggers inside could be a big threat to the public fire safety, leading to catastrophic loss in human lives and properties. Both the qualitative and quantitative analysis were imposed on a typical large commercial complex to recognize the potential fire-causative factors in this paper. Applying the fault tree analysis, the basic events leading to fire are acquired, and they are then further reclassified based on the analytic hierarchy process. Taking the damage of the accident as the target layer and the fire-causative factors, the equipment operation factors and firefighting factors as the criterion layer, the assessment index is well established. The risk of each factor is quantitatively evaluated, and the effect of each factor on the target layer is analyzed. The result of the fault tree analysis and analytic hierarchy process shows good consistency, in which human behavior is the main factor leading to the fire occurrence, followed by the combustible material, the rescue speed and the staff assignment factors. The results are beneficial for general decisions and measures in public fire safety management.

NOD2/c-Jun NH2-Terminal Kinase Triggers Mycoplasma ovipneumoniae-Induced Macrophage Autophagy.

Mycoplasma ovipneumoniae belongs to Mycoplasma, a genus containing the smallest self-replicating microorganisms, and causes infectious pleuropneumonia in goats and sheep. Nucleotide-binding oligomerization domain-containing protein (NOD2), an intracellular pattern recognition receptor, interacts with muramyl dipeptide (MDP) to recognize bacterial peptidoglycans and is involved in autophagy induction. However, there have been no reports about NOD recognition of mycoplasmas or M. ovipneumoniae-induced autophagy. In this study, we sought to determine the role of NOD2 in M. ovipneumoniae-induced autophagy using Western blotting, immunofluorescence, real-time PCR (RT-PCR), and color-changing unit (CCU) analysis. M. ovipneumoniae infection markedly increased NOD2 but did not increase NOD1 expression in RAW 264.7 cells. Treating RAW 264.7 cells with MDP significantly increased colocalization of M. ovipneumoniae and LC3, whereas treatment with NOD inhibitor, NOD-IN-1, decreased colocalization of M. ovipneumoniae and LC3. Furthermore, suppressing NOD2 expression with small interfering RNA (siRNA)-NOD2 failed to trigger M. ovipneumoniae-induced autophagy by detecting autophagy markers Atg5, beclin1, and LC3-II. In addition, M. ovipneumoniae infection significantly increased the phosphorylated c-Jun NH2-terminal kinase (p-JNK)/JNK, p-Bcl-2/Bcl-2, beclin1, Atg5, and LC3-II ratios in RAW 264.7 cells. Treatment with JNK inhibitor, SP600126, or siRNA-NOD2 did not increase this reaction. These findings suggested that M. ovipneumoniae infection activated NOD2, and both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy. This study provides new insight into the NOD2 reorganization mechanism and the pathogenesis of M. ovipneumoniae infection.IMPORTANCEM. ovipneumoniae, which lacks a cell wall, causes infectious pleuropneumonia in goats and sheep. In the present study, we focused on the interaction between NOD and M. ovipneumoniae, as well as its association with autophagy. We showed for the first time that NOD2 was activated by M. ovipneumoniae even when peptidoglycans were not present. We also observed that both NOD2 and JNK pathway activation promoted M. ovipneumoniae-induced autophagy.

YAP promotes endothelial barrier repair by repressing STAT3/VEGF signaling.

AIMS: Endothelial barrier dysfunction is associated with multiple diseases, and barrier repair may be a possible therapeutic target. Yes-associated protein and its pathway have been implicated in organ repair after injury. However, the mechanisms underlying barrier repair and any role YAP plays in the process are unclear. This study aimed to explore the role and mechanism of YAP in the repair of endothelial cell permeability after TNF-α-induced injury. MAIN METHODS: A trans-endothelial electrical resistance assay was performed to investigate changes in endothelial cell permeability. Lentivirus packaging by calcium phosphate transfection was used to construct endothelial cell lines with knocked down or overexpressed YAP. Western blotting, immunofluorescence, CO-IP, and real-time PCR were used to detect related protein and gene expression. KEY FINDINGS: YAP is involved in the repair process of TNF-α-induced endothelial cell permeability injury; its overexpression promotes repair of endothelial cell permeability, and knockdown weakens repair ability. Moreover, YAP may promote repair by down-regulating STAT3 activity, thereby inhibiting VEGF expression. SIGNIFICANCE: Elucidating the role of YAP in endothelial cell permeability repair process after injury might reveal mechanisms of endothelial barrier repair and provide therapeutic targets for treatment of vascular hyper-permeability disease.

Generation of human induced pluripotent stem cell line (WMUi001-A) from a patient with aortic dissection.

A human induced pluripotent stem cell (hiPSC) line (WMUi001-A) was generated from the aortic tissue of a 47-year-old donor with aortic dissection and normal blood pressure. Integration-free episomal vector-mediated reprogramming was used for the generation of this iPSC line. The established iPSC line was found to express pluripotency markers, exhibit a differentiation potential in vitro, as well as display a normal karyotype. We further identified that this iPSC line contained a mutation in collagen type IV (COL4A2, R131M), which may serve as a useful tool for the disease modeling of aortic dissection.