Ballingkuhn9399

The cell wall is crucial for fungal growth, proliferation and interaction with the environment and host. Understanding the regulatory mechanism of cell wall integrity may help with improvement of fungal biocontrol agents. Here, a putative target of the cell wall integrity pathway-involved Slt2 MAP kinase, Mb1, an orthologue of MADS-box transcription factor Rlm1, was characterized in an economically important insect fungal pathogen, Beauveria bassiana. Mb1 disruption mutant (ΔMb1) displayed reduced growth and increased conidial production on minimal medium but not on rich-nutrient media, which is different from ΔSlt2 to a great extent. Loss of Mb1 resulted in a significant increase in sensitivity to cell wall-perturbing agents (Congo red and calcofluor white), with alteration in cell composition that was inconsistent with ΔSlt2 strain, including increased chitin content and reduced chitin-binding β-1, 3/1,6-glucan levels in the absence of any stress. Transcription levels of 15 chitin synthesis and metabolism-associated and 17 Pkc1-Slt2 CWI (cell wall integrity) pathway, glucan synthesis, and cell wall remodeling enzyme synthesis-involved genes were significantly increased and repressed in ΔMb1 strain, respectively, some of which were verified to be the targets of Mb1. Insect bioassays revealed decreased virulence for the ΔMb1 strain in both topical and intrahemocoel injection assays. Our results demonstrated that Mb1 control fungal biocontrol potential-associated traits, including growth, conidiation and cell wall integrity, in B. bassiana. The difference of Mb1 and Slt2 in contribution to cell wall integrity is discussed. OBJECTIVE Toll-like receptor (TLR)-mediated catabolic responses are implicated to contribute to osteoarthritis (OA). However, deficiency of TLRs has little chondroprotection in mice in vivo. Here, we studied the effect of deficiency of TLR2 and TLR4 in articular chondrocytes on cellular stress responses in vitro. DESIGN Chondrocytes isolated from TLR2 and TLR4 double knockout (TLR2/4dKO) and wild type (WT) mice and recombinant HMGB1 (rHMGB1) and LPS were used. Expression of anti-oxidant and DNA repair enzymes including SOD1, SOD2 and OGG1, and phosphorylation of H2AX (a marker for DNA damage) were examined by Western blotting. MitoSOX Red staining was used for assessing mitochondrial superoxide generation. Autophagic activity was monitored by flow cytometry analysis of mean fluorescence intensity (MFI) of GFP and RFP in chondrocytes transfected with a tandem GFP-mRFP-LC3 plasmid, and by Western blot analysis of expression of LC3 and p62, a selective autophagy adaptor. RESULTS Basal expression of SOD2 but not SOD1 was largely reduced in TLR2/4dKO compared to WT chondrocytes, correlated with significantly enhanced menadione-induced mitochondrial superoxide generation (2.85-3.92 and 3.39 to 8.97 with mean difference 3.39 and 6.18 for 25 and 50μM menadione, respectively) and phosphorylation of H2AX. LPS and rHMGB1 induced expression of SOD2, OGG1 and p62 in WT but not TLR2/4dKO chondrocytes. Autophagy flux was impaired in TLR2/4dKO chondrocytes after acute nutrient stress and by LPS and rHMGB1. CONCLUSIONS TLR2 and TLR4 deficiency appears to reduce chondrocyte anti-oxidative stress and autophagy flux capacity, which may compromise cartilage homeostasis as a result of chondrocyte dysfunction. Published by Elsevier Ltd.INTRODUCTION Coronary microvascular dysfunction (CMD) is a complex disease, difficult to diagnose and often requires advanced imaging. We used mass spectrometry (MS) using discovery approach to search for serum proteins as potential biomarkers in these patients. METHODS We used serum samples from 10 patients with CMD and 10 with normal coronary flow reserve (CFR) admitted to an observation unit where acute myocardial infarction was excluded. We identified CMD using 82Rb positron emission tomography/computed tomography as CFR less then 2 in response to regadenoson, in the absence of coronary calcification or regional perfusion defects. We used MS to identify potential protein biomarkers that were differentially expressed in cases and controls. RESULTS Baseline characteristics were not different between cases and controls, except for beta-blocker use and which was higher in cases, and mean (SD) CFR which was lower in cases [1.19 (0.23) and 2.78 (0.78) in cases and controls respectively; p  less then  0.01]. We identified 5345 peptides corresponding to 209 proteins, and identified 197 proteins by peptides with suitable properties to infer relative quantitation values. While the calculated values for some proteins (e.g. vascular cell adhesion molecule-1, apolipoprotein C and Von Willebrand Factor) indicate fold-differences between groups, these are most likely a result of high values in only 1-2 patients and are not statistically significant. CONCLUSION Mass spectrometry using discovery approach may not be an adequate method for quantitative assessment of serum proteins in CMD patients. Future MS studies should evaluate other approaches including tissue samples or serial measurements. Preclinical and clinical studies have indicated that antidepressants can promote inflammation and fibrogenesis, particularly in the lung, by mechanisms not fully elucidated. We have previously shown that different classes of antidepressants can activate the lysophosphatidic acid (LPA) receptor LPA1, a major pathogenetic mediator of tissue fibrosis. The aim of the present study was to investigate whether in cultured human dermal and lung fibroblasts antidepressants could trigger LPA1-mediated profibrotic responses. In both cell types amitriptyline, clomipramine and mianserin mimicked the ability of LPA to induce the phosphorylation/activation of extracellular signal -regulated kinases 1 and 2 (ERK1/2), which was blocked by the selective LPA1 receptor antagonist AM966 and the LPA1/3 antagonist Ki16425. Antidepressant-induced ERK1/2 stimulation was absent in fibroblasts stably depleted of LPA1 by short hairpin RNA transfection and was prevented by pertussis toxin, an uncoupler of receptors from Gi/o proteins. Like LPA, antidepressants stimulated fibroblasts proliferation and this effect was blocked by either AM966 or the MEK1/2 inhibitor PD98059. Moreover, by acting through LPA1 antidepressants induced the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, and caused an ERK1/2-dependent increase in the cellular levels of transforming growth factor-β (TGF-β)1, a potent fibrogenic cytokine. Pharmacological blockade of TGF-β receptor type 1 prevented antidepressant- and LPA-induced α-SMA expression. Gefitinib inhibitor These data indicate that in human dermal and lung fibroblasts different antidepressants can induce proliferative and differentiating responses by activating the LPA1 receptor coupled to ERK1/2 signalling and suggest that this property may contribute to the promotion of tissue fibrosis by these drugs.