LRP6 modulates the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction

Background Ventricular tachycardia (VT) and ventricular fibrillation are the most causes of early death in patients with myocardial infarction (MI). This study was aimed to explore whether LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Connexin-43 (Cx43) via Gαs in ventricular tachycardia of MI. Method we constructed the hypoxia cardiomyocyte model and AMI mice, and explored the modulation relationship of LRP6 and its upstream genes circRNA1615 and miR-152-3p. In addition, the immunoblot analysis with monoclonal and polyclonal antibodies were used to detect whether LRP6 and Cx43 were phosphorylated, further investigated that the LRP6 regulated the phosphorylation of its downstream target Cx43 via G-protein alpha subunit Gαs by using cell transfection, FISH assay, HE staining, RT-qPCR, and Western blot techniques. Result LRP6 mRNA expression was significantly reduced in AMI group compared with the control group. Hypoxia could inhibit the protein and phosphorylation levels of LRP6 and Cx43. The expression of circRNA1615 in AMI mice was significantly decreased, but overexpression of circRNA1615 significantly reversed it. Also overexpression of circRNA1615 could weaken the effect of miR-152-3p mimic, and the miR-152-3p mimic increased the hypoxia injury of LRP6 and Cx43, further LRP6 interference fragments could aggravate hypoxia injury of Cx43. The overexpression of LRP6 could significantly increase the protein level and phosphorylation level of Cx43, but the interference with LRP6 showed the opposite trend. Noticeably, the interference with Gαs weakened the protein and phosphorylation levels of Cx43, however, the interference with LRP6 further inhibited the protein and phosphorylation levels of Cx43. Finally, the transcriptions of circRNA1615 and LRP6 were inhibited in AMI, but the transcription of miR-152-3p was promoted, and the overexpression of circRNA1615 could weaken the damage effect and VT of AMI. Conclusion LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction.

Concomitant Activation of OSM and LIF Receptor by a Dual-Specific hlOSM Variant Confers Cardioprotection after Myocardial Infarction in Mice

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR. Cell viability assays, transcriptome and immunoblot analysis revealed increased survival of hypoxic cardiomyocytes by mLIF, mOSM and hlOSM stimulation, associated with increased activation of STAT3. Kinetic expression profiling of infarcted hearts further specified a transient increase of OSM and LIF during the early inflammatory phase of cardiac remodeling. A post-infarction delivery of hlOSM but not mOSM or mLIF within this time period combined with cardiac magnetic resonance imaging-based strain analysis uncovered a global cardioprotective effect on infarcted hearts. Our data conclusively suggest that a simultaneous and rapid activation of OSMR and LIFR after MI offers a therapeutic opportunity to preserve functional and structural integrity of the infarcted heart.

Total Flavone of Abelmoschus manihot Ameliorates TNBS-Induced Colonic Fibrosis by Regulating Th17/Treg Balance and Reducing Extracellular Matrix

Background and Aims: Surgery remains the major available strategy in inflammatory bowel disease (IBD) fibrotic strictures because no available drugs have sufficient prevention and treatment in this complication. This study aimed to evaluate the efficacy of the total flavone of Abelmoschus manihot L. Medic (TFA) on the development of colonic fibrosis in mice and its possible mechanism.Methods: The 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced chronic colonic inflammation-associated fibrosis mice were used to evaluate anti-fibrosis of TFA using macroscopic, histological, immunohistochemical analyses, ELISA, Masson staining, Verhoeff’s von Gieson staining, transcription-quantitative polymerase chain reaction, and immunoblot analysis.Results: Oral administration of TFA attenuated body weight loss, reduced colon length shortening, lowered the morphological damage index score, and notably ameliorated the inflammatory response. TFA downregulated proinflammatory cytokines IL-6, IL-17, TNF-α, IFN-γ productions, and increased the levels of anti-inflammatory cytokine IL-10 and TGF-β. The histological severity of the colonic fibrosis was also notably improved by the TFA treatment and associated with a significant reduction in the colonic expression of col1a2, col3a2, and hydroxyproline. TFA inhibits α-SMA, TGF-β, vimentin, TIMP-1 expression, increasing MMPs, thereby inhibiting activated intestinal mesenchymal cells and extracellular matrix (ECM) deposition.Conclusion: Together, we herein provide the evidence to support that TFA may restore the imbalance of Th17/Treg and decrease the generation of ECM. This may be a potential mechanism by which TFA protects the intestine under inflammatory conditions and acts as a therapeutic agent for the treatment of intestinal fibrosis in Crohn’s disease.

Hypochlorite-Modified LDL Induces Arrhythmia and Contractile Dysfunction in Cardiomyocytes

Neutrophil-derived myeloperoxidase (MPO) and its potent oxidant, hypochlorous acid (HOCl), gained attention as important oxidative mediators in cardiac damage and dysfunction. As cardiomyocytes generate low-density lipoprotein (LDL)-like particles, we aimed to identify the footprints of proatherogenic HOCl-LDL, which adversely affects cellular signalling cascades in various cell types, in the human infarcted myocardium. We performed immunohistochemistry for MPO and HOCl-LDL in human myocardial tissue, investigated the impact of HOCl-LDL on electrophysiology and contractility in primary cardiomyocytes, and explored underlying mechanisms in HL-1 cardiomyocytes and human atrial appendages using immunoblot analysis, qPCR, and silencing experiments. HOCl-LDL reduced ICa,L and IK1, and increased INaL, leading to altered action potential characteristics and arrhythmic events including early- and delayed-afterdepolarizations. HOCl-LDL altered the expression and function of CaV1.2, RyR2, NCX1, and SERCA2a, resulting in impaired contractility and Ca2+ homeostasis. Elevated superoxide anion levels and oxidation of CaMKII were mediated via LOX-1 signaling in HL-1 cardiomyocytes. Furthermore, HOCl-LDL-mediated alterations of cardiac contractility and electrophysiology, including arrhythmic events, were ameliorated by the CaMKII inhibitor KN93 and the INaL blocker, ranolazine. This study provides an explanatory framework for the detrimental effects of HOCl-LDL compared to native LDL and cardiac remodeling in patients with high MPO levels during the progression of cardiovascular disease.

Plastid Deficient 1 Is Essential for the Accumulation of Plastid-Encoded RNA Polymerase Core Subunit β and Chloroplast Development in Arabidopsis

Plastid-encoded RNA polymerase (PEP)-dependent transcription is an essential process for chloroplast development and plant growth. It is a complex event that is regulated by numerous nuclear-encoded proteins. In order to elucidate the complex regulation mechanism of PEP activity, identification and characterization of PEP activity regulation factors are needed. Here, we characterize Plastid Deficient 1 (PD1) as a novel regulator for PEP-dependent gene expression and chloroplast development in Arabidopsis. The PD1 gene encodes a protein that is conserved in photoautotrophic organisms. The Arabidopsis pd1 mutant showed albino and seedling-lethal phenotypes. The plastid development in the pd1 mutant was arrested. The PD1 protein localized in the chloroplasts, and it colocalized with nucleoid protein TRXz. RT-quantitative real-time PCR, northern blot, and run-on analyses indicated that the PEP-dependent transcription in the pd1 mutant was dramatically impaired, whereas the nuclear-encoded RNA polymerase-dependent transcription was up-regulated. The yeast two-hybrid assays and coimmunoprecipitation experiments showed that the PD1 protein interacts with PEP core subunit β (PEP-β), which has been verified to be essential for chloroplast development. The immunoblot analysis indicated that the accumulation of PEP-β was barely detected in the pd1 mutant, whereas the accumulation of the other essential components of the PEP complex, such as core subunits α and β′, were not affected in the pd1 mutant. These observations suggested that the PD1 protein is essential for the accumulation of PEP-β and chloroplast development in Arabidopsis, potentially by direct interaction with PEP-β.

A Novel Pseudoalteromonas xiamenensis Marine Isolate as a Potential Probiotic: Anti-Inflammatory and Innate Immune Modulatory Effects against Thermal and Pathogenic Stresses

A marine bacterial strain was isolated from seawater and characterized for it beneficial probiotic effects using zebrafish as a model system. The strain was identified by morphological, physiological, biochemical, and phylogenetic analyses. The strain was most closely related to Pseudoalteromonas xiamenensis Y2, with 99.66% similarity; thus, we named it Pseudoalteromonas xiamenensis S1131. Improvement of host disease tolerance for the P. xiamenensis isolate was adapted in a zebrafish model using Edwardsiella piscicida challenge. The larvae were pre-exposed to P. xiamenensis prior to E. piscicida challenge, resulting in a 73.3% survival rate compared to a 46.6% survival for the control. The treated larvae tolerated elevated temperatures at 38 °C, with 85% survival, compared to 60% survival for the control. Assessment of immunomodulatory responses at the mRNA level demonstrated the suppression of pro-inflammatory markers tnfα and il6, and upregulation of heat shock protein hsp90 and mucin genes. The same effect was corroborated by immunoblot analysis, revealing significant inhibition of Tnfα and an enhanced expression of the Hsp90 protein. The antibacterial activity of P. xiamenensis may be related to mucin overexpression, which can suppress bacterial biofilm formation and enhance macrophage uptake. This phenomenon was evaluated using nonstimulated macrophage RAW264.7 cells. Further studies may be warranted to elucidate a complete profile of the probiotic effects, to expand the potential applications of the present P. xiamenensis isolate.

AXL Regulates Neuregulin1 Expression Leading to Cetuximab Resistance in Head and Neck Cancer

Background The receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) is overexpressed and an important therapeutic target in Head and Neck cancer (HNC). Cetuximab is currently the only EGFR-targeting agent approved by the FDA for treatment of HNC; however, intrinsic and acquired resistance to cetuximab is a major problem in the clinic. Our lab previously reported that AXL leads to cetuximab resistance via activation of HER3. Methods In this study, we investigate the connection between AXL, HER3, and neuregulin1 (NRG1) gene expression with a focus on understanding how their interdependent signaling promotes resistance to cetuximab in head and neck cancer. Plasmid or siRNA transfections, cell proliferation assays, and clonogenic assays were conducted to test cetuximab sensitivity. Quantitative PCR and immunoblot analysis were used to analyze gene expression levels. Seven HNC patient-derived xenografts (PDXs) were evaluated for protein expression levels. Results We found that HER3 expression was necessary but not sufficient for cetuximab resistance without AXL expression. Our results demonstrated that addition of the HER3 ligand NRG1 to cetuximab-sensitive HNC cells leads to cetuximab resistance. Further, AXL-overexpressing cells regulate NRG1 at the level of transcription, thereby promoting cetuximab resistance. Immunoblot analysis revealed that NRG1 expression was relatively high in cetuximab-resistant HNC PDXs compared to cetuximab-sensitive HNC PDXs. Finally, genetic inhibition of NRG1 resensitized AXL-overexpressing cells to cetuximab. Conclusions The results of this study indicate that AXL may signal through HER3 via NRG1 to promote cetuximab resistance and that targeting of NRG1 could have significant clinical implications for HNC therapeutic approaches.

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.

Eosinophilic pneumonia induced by lettuce: A case report

Background: Lettuce (Lactuca sativa) belongs to the Composite family and is a vegetable widely consumed globally. Although lettuce is extensively cultivated and consumed, lettuce-associated occupational allergy is rarely reported. Herein, we are reporting a case of eosinophilic pneumonia induced by lettuce for the first time.Case presentation: A 56-year-old female lettuce farmer was admitted to the hospital with a low-grade fever, worsening cough, and dyspnoea. A blood test revealed eosinophilia and a high serum IgE concentration. A chest X-ray taken on admission showed an infiltrative shadow in the upper lung field. Chest CT revealed patchy ground glass opacity on the upper lung field and thickening of the bronchial wall. The bronchoalveolar lavage fluid contained 8% eosinophils. The IgG-binding proteins that reacted with the patient’s sera were identified by immunoblot analysis. She was diagnosed as lettuce induced eosinophilic pneumonia and was treated with prednisolone, and her symptoms and radiological findings improved. Wearing a mask and reducing the amount of the crop improved her symptoms the following year.Conclusions: This is the first case report about lettuce-induced eosinophilic pneumonia which occurred in a lettuce farmer. The avoidance from antigen is quite useful in this patient.

Obesity and Oral Contraceptives Synergistically Increase Thrombotic Risk By Downregulating Protein S

Background: The increased thrombotic effects of estrogen-based oral contraceptives and obesity have been documented independently. However, obesity and oral contraceptives combined are associated with a far greater thrombotic risk, but we have a poor understanding of the mechanism of this greater effect. Increasingly, women are using oral contraceptives, and the national obesity rate has been skyrocketing. Thus, it is imperative to explain how obesity and oral contraceptives work together to significantly elevate thrombotic risk. We know that hypoxia-inducible factor 1-ɑ (HIF-1ɑ) and the estrogen receptor (Erɑ) bind to the promoter of the Protein S gene; binding occurs at sites within ~450 nucleotides of each other, and the two transcription factors downregulate Protein S expression independently. We hypothesize that the two factors, by binding the promoter simultaneously, synergistically downregulate Protein S to a degree much greater than the downregulation mediated by each factor separately. Aims: The goal of this project is determining whether estrogen and obesity-induced hypoxia work synergistically to downregulate Protein S transcription and increase thrombotic risk. Methods: We measured the effects of obesity and oral contraceptives on hepatocarcinoma (HEP G2) cells because the liver is the major producer of Protein S. Estrogen of varying concentrations (25-150 μM) was used to mimic the effects of oral contraceptives, and cobalt chloride of varying concentrations (25-150 μM) was used to stimulate hypoxia and HIF-1ɑ expression. Cells were exposed to estrogen only, cobalt chloride only, and estrogen and cobalt chloride together for 24 hours, after which the cells were harvested and subjected to q-PCR and immunoblot blot analyses to measure Protein S transcription and protein expression. Although cobalt chloride is a reliable inducer of hypoxia and HIF-1ɑ expression, we also performed hypoxia experiments by incubating cells in a chamber with varying O 2 concentrations (20%, 15%, 10%, 5%, 1%). Results and Conclusions: Immunoblot analysis of cells treated with either CoCl 2 and estrogen supplementation revealed a ~20% reduction in Protein S levels compared to control conditions and a more significant reduction in (~60%) Protein S expression in cells treated with estrogen and CoCl 2 together. These results supported our hypothesis that obesity and estrogen-based contraceptives increase thrombotic risk by downregulating anticoagulant Protein S transcription and subsequently decreasing Protein S level. Disclosures No relevant conflicts of interest to declare.