Background: The outbreak of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China has been declared a public health emergency of international concern. The cardiac injury is a common condition among the hospitalized patients with COVID-19. However, whether N terminal pro B type natriuretic peptide (NT-proBNP) predicted outcome of severe COVID-19 patients was unknown. Methods: The study initially enrolled 102 patients with severe COVID-19 from a continuous sample. After screening out the ineligible cases, 54 patients were analyzed in this study. The primary outcome was in-hospital death defined as the case fatality rate. Research information and following-up data were obtained from their medical records.Results: The best cut-off value of NT-proBNP for predicting in-hospital death was 88.64 pg/mL with the sensitivity for 100% and the specificity for 66.67%. Patients with high NT-proBNP values (> 88.64 pg/mL) had a significantly increased risk of death during the days of following-up compared with those with low values (≤88.64 pg/mL). After adjustment for potential risk factors, NT-proBNP was independently correlated with in-hospital death. Conclusion: NT-proBNP might be an independent risk factor for in-hospital death in patients with severe COVID-19.
The outbreak of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China has been declared a public health emergency of international concern. The cardiac injury was dominate in the process. However, whether N terminal pro B type natriuretic peptide (NT-proBNP) predicted outcome of COVID-19 patients was unknown. The study initially enrolled 102 patients with severe COVID-19 pneumonia from a continuous sample. After screening out the ineligible cases, 54 patients were analyzed in this study. Results found that patients with higher NT-proBNP (above 88.64 pg/mL) level had more risks of inhospital death. After adjusting for potential cofounders in separate modes, NT-proBNP presented as an independent risk factor of in-hospital death in patients with severe COVID-19. : medRxiv preprint Figure 1. The NT-proBNP for in-hospital death of coronavirus disease 2019 (COVID-19) by receiver operating characteristic (ROC) curves. The area under the curve (AUC) of NT-proBNP was 0.909. The best cutoff of NT-proBNP for prediction in-hospital death was 88.64 pg/mL with the sensitivity of 100% and the specificity of 66.67%. 95%CI, 95% confidence interval. All rights reserved. No reuse allowed without permission.the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10. 1101 Figure 2. Kaplan-Meier plots showing the cumulative survival rate of COVID-19 patients who were stratified into two groups according to plasma NT-proBNP cutoff point at baseline (Dotted line, NT-proBNP ≤88.64 pg/ml, n=24; Solid line, NT-proBNP >88.64 pg/ml, n=30; log-rank test for trend, P<0.001).All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
Myofibroblasts are effector cells in fibrotic disorders that synthesize and remodel the extracellular matrix (ECM). This study investigated the role of the Src kinase pathway in myofibroblast activation in vitro and fibrogenesis in vivo. The profibrotic cytokine, transforming growth factor b1 (TGF-b1), induced rapid activation of Src kinase, which led to myofibroblast differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-b1-induced Src kinase activation in a dose-dependent manner. Inhibition of Src kinase significantly reduced a-smooth muscle actin (a-SMA) expression, a marker of myofibroblast differentiation, in TGFb1-treated lung fibroblasts. In addition, the induced expression of collagen and fibronectin and three-dimensional collagen gel contraction were also significantly inhibited in AZD0530-treated fibroblasts. The therapeutic efficiency of Src kinase inhibition in vivo was tested in the bleomycin murine lung fibrosis model. Src kinase activation and collagen accumulation were significantly reduced in the lungs of AZD0530-treated mice when compared with controls. Furthermore, the total fibrotic area and expression of a-SMA and ECM proteins were significantly decreased in lungs of AZD0530-treated mice. These results indicate that Src kinase promotes myofibroblast differentiation and activation of lung fibroblasts. Additionally, these studies provide proof-ofconcept for targeting the noncanonical TGF-b signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.
Myofibroblasts are implicated in pathological stromal responses associated with lung fibrosis. One prominent phenotypic marker of fully differentiated myofibroblasts is the polymerized, thick cytoplasmic filaments containing newly synthesized α-smooth muscle actin (α-SMA). These α-SMA-containing cytoplasmic filaments are important for myofibroblast contractility during tissue remodeling. However, the molecular mechanisms regulating the formation and maturation of α-SMA-containing filaments have not been defined. This study demonstrates a critical role for neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating the formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and in myofibroblast contractility. Focal adhesion kinase (FAK) is activated by transforming growth factor-β1 (TGF-β1) and is required for phosphorylation of tyrosine residue 256 (Y256) of N-WASP. Phosphorylation of Y256 of N-WASP is essential for TGF-β1-induced formation of α-SMA-containing cytoplasmic filaments in primary human lung fibroblasts. In addition, we demonstrate that actin-related protein (Arp) 2/3 complex is downstream of N-WASP and mediates the maturation of α-SMA-containing cytoplasmic filaments. Together, this study supports a critical role of N-WASP in integrating FAK and Arp2/3 signaling to mediate formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and maturation.
Recent studies recognize that Hypocretin system (also known as Orexin) plays a critical role in sleep/wake disorders and feeding behaviors. However, little is known about the regulation of the Hypocretin system. It is also known that tumor necrosis factor alpha (TNF-α) is involved in regulation of sleep/wake cycle. Here, we test our hypothesis that the Hypocretin system is regulated by TNF-α. Prepro-Hypocretin and Hypocretin receptor 2 (HcrtR2) can be detected at a very low level in rat B35 neuroblastoma cells. In response to TNF-α, Prepro-Hypocretin mRNA and protein levels are down-regulated, and also HcrtR2 protein level is down-regulated in B35 cells. To investigate the mechanism, exogenous rat Prepro-Hypocretin and rat HcrtR2 were overexpressed in B35 cells. In response to TNF-α, protein and mRNA of Prepro-Hypocretin are significantly decreased (by 93% and 94%, respectively), and the half-life of Prepro-Hypocretin mRNA is decreased in a time- and dose-dependent manner. The level of HcrtR2 mRNA level is not affected by TNF-α treatment; however, HcrtR2 protein level is significantly decreased (by 86%) through ubiquitination in B35 cells treated with TNF-α. Downregulation of cellular inhibitor of apoptosis protein-1 and -2 (cIAP-1 and -2) abrogates the HcrtR2 ubiquitination induced by TNF-α. The control green fluorescent protein (GFP) expression is not affected by TNF-α treatment. These studies demonstrate that TNF-α can impair the function of the Hypocretin system by reducing the levels of both Prepro-Hypocretin and HcrtR2.
S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation, and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-b1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27 Kip1 expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events, and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway. S 100A4, also known as metastasin (Mts1) 1,2 , belongs to the S100 family, and it is a calcium-binding protein with two EF-hands 3-5 . S100A4 is involved in a variety of physiological functions, such as cell motility, adhesion, proliferation, invasion, and metastasis 3,4,6,15 . S100A4 is considered as a mediator of tumor progression and metastasis 3,6 . S100A4 can suppress the BNIP3 expression and contributes to chemoresistance and survival in pancreatic cancer cells 16 . S100A4 is involved in epithelial mesenchymal transition mediated by the Shh-Gli1 signaling pathway 17 , and S100A4 promotes cell invasion in pancreatic cancer cells 18 . S100A4 is overexpressed in pancreatic cancer 7 , and is also frequently overexpressed in other metastatic cancers, including prostate 8,9 , ovarian 10 , and breast carcinomas 11,12 . Increased S100A4 expression has been strongly associated with poor clinical outcomes of the pancreatic cancer 7,13,14 . Although aberrant S100A4 expression is an independent biomarker of poor outcome, the molecular mechanisms by which S100A4 regulates pancreatic cancer progression in vivo are not completely understood. Whether S100A4 directly contributes to pancreatic cancer progression in vivo or is just a secondary effect of other changes during pancreatic cancer progression remains to be answered.Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase, that is upregulated in many types of cancers, including pancreatic cancers 19,20 . FAK is activated when its tyrosine-397 (Y397) is phosphorylated, and maximal FAK activation requires binding of Src kinase 21,22 . Increased FAK activation is posit...
The cellular membrane transporter for the inhibitory neurotransmitter c-aminobutyric acid (GABA) belongs to a family of secondary active systems that are driven by electrochemica1 gradients of Na + and Cl - [1]. The main physiological function of the transporter is believed to be the control of the concentration and dwell time of GABA in the synaptic cleft. Because the transport of one molecule of GABA is coupled to the Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft. The GABA transporter GAT1 belongs to the family of Na + -and Cl --coupled transport proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain 3 and 4. To study the significance of N-glycosylation, green fluorescence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells. Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM). This indicates that N-glycosylation, but not terminal trimming of the N-glycans is involved in the attainment of a correctly folded and stable conformation of GAT1. All N-glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA-uptake activity. Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity. Further experiments showed that both N-glycosylation mutations and dMM reduced the V max value, while not increasing the K m value for GABA uptake. Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na + and slowed kinetics of the transport cycle. This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1. They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding.Abbreviations CHO, Chinese hamster ovary; dMM, 1-deoxymannojirimycin; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; GABA, c-aminobutyric acid; GAT1, GABA transporter type I; GFP, green fluorescence protein.
TGF-b1 induces an increase in paracellular permeability and actin stress fiber formation in lung microvascular endothelial and alveolar epithelial cells via small Rho GTPase. The molecular mechanism involved is not fully understood. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) has an essential role in actin structure dynamics. We hypothesized that N-WASP plays a critical role in these TGF-b1-induced responses. In these cell monolayers, we demonstrated that N-WASP down-regulation by short hairpin RNA prevented TGF-b1-mediated disruption of the cortical actin structure, actin stress filament formation, and increased permeability. Furthermore, N-WASP down-regulation blocked TGF-b1 activation mediated by IL-1b in alveolar epithelial cells, which requires actin stress fiber formation. Control short hairpin RNA had no effect on these TGF-b1-induced responses. TGF-b1-induced phosphorylation of Y256 of N-WASP via activation of small Rho GTPase and focal adhesion kinase mediates TGF-b1-induced paracellular permeability and actin cytoskeleton dynamics. In vivo, compared with controls, N-WASP down-regulation increases survival and prevents lung edema in mice induced by bleomycin exposure-a lung injury model in which TGF-b1 plays a critical role. Our data indicate that N-WASP plays a crucial role in the development of TGF-b1-mediated acute lung injury by promoting pulmonary edema via regulation of actin cytoskeleton
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