Aim of the Study: Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. In this study, we aim to demonstrate that cyclic nucleotide phosphodiesterase 1A (PDE1A), a Ca2+/calmodulin-stimulating PDE family member, plays a critical role in the induction of fibrosis and angiogenesis in the lung. Materials and Methods: To induce pulmonary damage, adult male SD rats were treated with bleomycin in a dose of 6 mg/kg body weight by a single intratracheal instillation. For in vivo silencing of PDE1A in rat lung, a nonspecific control siRNA or PDE1A-specific siRNA was used to treat rat through nasal instillation. Human normal pulmonary fibroblasts MRC-5 and hFL1 and rat lung fibroblasts were used as in vitro model. Immunohistochemistry and immunoflurescence staining were performed to detect PDE1A and α-SMA expression. Reverse transcription-qPCR was performed to detect microRNA and mRNA expression. In vitro wound healing assay was performed to detect pulmonary fibroblasts'mortality ability. Results: In vitro studies showed that PDE1A can stimulate lung fibroblasts to undergo myofibroblastic changes. This led to the identification of miR-541-5p as one of the miRNA candidates associated with bleomycin response. We found that miR-541-5p expression is downregulated in TGF-β-treated lung fibroblasts and the rat pulmonary fibrosis model. Overexpression of miR-541-5p in lung fibroblasts inhibited mortality of human lung fibroblasts. Conclusions: MiR-541-5p is a key effector in lung fibroblastsby by regulating PDE1A expression at protein translation level and its overexpression is protective against bleomycin-induced lung fibrosis.
Urokinase-type plasminogen activator (uPA) has been shown to activate matrix metalloproteinase-2 (MMP-2) that leads to the migration and invasion of breast cancer cells. Overexpressed uPA and MMP-2 are regarded as signs of malignant tumors in clinical practice. Therefore, real-time monitoring of the sequential activation of these two signal molecules may have important implications for the evaluation of the invasive potential and tumor progression of breast cancer. However, due to the complicated intracellular environment, visualizing the dynamic changes of protein expression levels in living cells with a noninvasive method is still a great challenge. Here, a novel gold-selenium (Au-Se) fluorescent nanoprobe with excellent selectivity and strong anti-interference capability was designed for the simultaneous in situ imaging of uPA and MMP-2 and real-time monitoring of their changes in living cells. The imaging results demonstrated that the nanoprobe achieved a better prevention of glutathione interference compared to the conventional Au-S nanoprobe, thus it could be applied to actually reflect the expression level of uPA and MMP-2 in different breast cancer cells. Furthermore, the Au-Se nanoprobe could visually present the activation process of the two signal molecules, which play a dual role of insuring the invasiveness evaluation of breast cancer cells. Overall, our work offers a visual biomarker detection method for the judgment of the degree of breast cancer malignancy, and also provides an effective strategy to investigate the relationships among signal molecules of other signaling pathways in the future.
A growing body of evidence indicates that micropeptides encoded by long noncoding RNAs (lncRNAs) act independently or as regulators of larger proteins in fundamental biological processes, especially in the maintenance of cellular homeostasis. However, due to their small size and low intracellular expression, visual monitoring of micropeptides in living cells is still a challenge. In this work, we have designed and synthesized an aptamer-based near-infrared fluorescence nanoprobe for fluorescence imaging of phospholamban (PLN), which is an intracellular micropeptide that affects calcium homeostasis, and is closely associated with human heart failure in the clinic. The nanoprobe could respond specifically to PLN with excellent selectivity, high sensitivity, good nuclease stability, and biocompatibility, and it was successfully applied for imaging of changes in PLN levels in cardiomyocytes and in frozen sections of heart tissues. Further combined with clinical myocardial biopsy, we believe that the developed nanoprobe should be of great significance in later molecular pathology study of heart failure, which may help with diagnosis of early heart failure in the future. More importantly, for the first time nanoprobes were applied to visually monitor the changes of micropeptides in living cells and in frozen tissue sections, and the design concept of the aptamer-based nanoprobe can be extended to fluorescence detection of other micropeptides.
Aim of the Study: Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. In this study, we aim to demonstrate that cyclic nucleotide phosphodiesterase 1A (PDE1A), a Ca2+/calmodulin-stimulating PDE family member, plays a critical role in the induction of fibrosis and angiogenesis in the lung. Materials and Methods: To induce pulmonary damage, adult male SD rats were treated with bleomycin in a dose of 6 mg/kg body weight by a single intratracheal instillation. For in vivo silencing of PDE1A in rat lung, a nonspecific control siRNA or PDE1A-specific siRNA was used to treat rat through nasal instillation. Human normal pulmonary fibroblasts MRC-5 and hFL1 and rat lung fibroblasts were used as in vitro model. Immunohistochemistry and immunoflurescence staining were performed to detect PDE1A and α-SMA expression. Reverse transcription-qPCR was performed to detect microRNA and mRNA expression. In vitro wound healing assay was performed to detect pulmonary fibroblasts'mortality ability. Results: In vitro studies showed that PDE1A can stimulate lung fibroblasts to undergo myofibroblastic changes. This led to the identification of miR-541-5p as one of the miRNA candidates associated with bleomycin response. We found that miR-541-5p expression is downregulated in TGF-β-treated lung fibroblasts and the rat pulmonary fibrosis model. Overexpression of miR-541-5p in lung fibroblasts inhibited mortality of human lung fibroblasts. Conclusions: MiR-541-5p is a key effector in lung fibroblastsby by regulating PDE1A expression at protein translation level and its overexpression is protective against bleomycin-induced lung fibrosis.
ABSTRACT Toll-like receptors (TLRs) are evolutionarily conserved host proteins that are essential for effective host defense against pathogens. However, recent studies suggest that some TLRs can negatively regulate immune responses. We observed here that TLR2 and TLR9 played opposite roles in regulating innate immunity against oral infection of Salmonella enterica serovar Typhimurium in mice. While TLR9 −/− mice exhibited shortened survival, an increased cytokine storm, and more severe Salmonella hepatitis than wild-type (WT) mice, TLR2 −/− mice exhibited the opposite phenomenon. Further studies demonstrated that TLR2 deficiency and TLR9 deficiency in macrophages both disrupted NK cell cytotoxicity against S . Typhimurium-infected macrophages by downregulating NK cell degranulation and gamma interferon (IFN-γ) production through decreased macrophage expression of the RAE-1 NKG2D ligand. But more importantly, we found that S . Typhimurium-infected TLR2 −/− macrophages upregulated inducible nitric oxide synthase (iNOS) expression, resulting in a lower bacterial load than that in WT macrophages in vitro and livers in vivo as well as low proinflammatory cytokine levels. In contrast, TLR9 −/− macrophages showed decreased reactive oxygen species (ROS) expression concomitant with a high bacterial load in the macrophages and in livers of TLR9 −/− mice. TLR9 −/− macrophages were also more susceptible than WT macrophages to S . Typhimurium-induced necroptosis in vitro , likely contributing to bacterial spread and transmission in vivo . Collectively, these findings indicate that TLR2 negatively regulates anti- S . Typhimurium immunity, whereas TLR9 is vital to host defense and survival against S . Typhimurium invasion. TLR2 antagonists or TLR9 agonists may thus serve as potential anti- S . Typhimurium therapeutic agents.
A novel dual-aptamer activated proximity-induced qPCR assay was developed for the quantitative analysis of exosomal PD-L1 on T cell-exosome complexes in blood samples.
TLRs are key sensors for conserved bacterial molecules and play a critical role in host defense against invading pathogens. Although the roles of TLRs in defense against pathogen infection and in maintaining gut immune homeostasis have been studied, the precise functions of different TLRs in response to pathogen infection in the gut remain elusive. The present study investigated the role of TLR signaling in defense against the Gram-negative bacterial pathogen Salmonella typhimurium The results indicated that TLR9-deficient mice were more susceptible to S. typhimurium infection compared with wild-type and TLR2- or TLR4-deficient mice, as indicated by more severe intestinal damage and the highest bacterial load. TLR9 deficiency in intestinal epithelial cells (IECs) augmented the activation of NF-κB and NLRP3 inflammasomes significantly, resulting in increased secretion of IL-1β. IL-1β increased the expression of NKG2D on intestinal intraepithelial lymphocytes and NKG2D ligands on IECs, resulting in higher susceptibility of IECs to cytotoxicity of intestinal intraepithelial lymphocytes and damage to the epithelial barrier. We proposed that TLR9 regulates the NF-κB-NLRP3-IL-1β pathway negatively in Salmonella-induced NKG2D-mediated intestinal inflammation and plays a critical role in defense against S. typhimurium infection and in the protection of intestinal integrity.
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