Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that ...the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development.
Neurofibromatosis type I (NF1) is characterized by prominent skeletal manifestations caused by NF1 loss. While inhibitors of the ERK activating kinases MEK1/2 are promising as a means to treat NF1, ...the broad blockade of the ERK pathway produced by this strategy is potentially associated with therapy limiting toxicities. Here, we have sought targets offering a more narrow inhibition of ERK activation downstream of NF1 loss in the skeleton, finding that MEKK2 is a novel component of a noncanonical ERK pathway in osteoblasts that mediates aberrant ERK activation after NF1 loss. Accordingly, despite mice with conditional deletion of Nf1 in mature osteoblasts (Nf1
;Dmp1-Cre) and Mekk2
each displaying skeletal defects, Nf1
;Mekk2
;Dmp1-Cre mice show an amelioration of NF1-associated phenotypes. We also provide proof-of-principle that FDA-approved inhibitors with activity against MEKK2 can ameliorate NF1 skeletal pathology. Thus, MEKK2 functions as a MAP3K in the ERK pathway in osteoblasts, offering a potential new therapeutic strategy for the treatment of NF1.
Neutrophils cast neutrophil extracellular traps (NETs) to ensnare microbial pathogens. Nevertheless, the molecular rheostats that regulate NETosis in response to bacteria are not clearly established. ...We hypothesized that stress-activated protein kinase or c-Jun N-terminal Kinase (SAPK/JNK) is a molecular switch that turns on NETosis in response to increasing concentrations of lipopolysaccharide (LPS)- and Gram-negative bacteria. Here we show that Escherichia coli LPS (0111:B4; 10-25 μg/ml), but not phorbol myristate acetate (PMA), activates JNK in human neutrophils in a dose-dependent manner. JNK inhibitors SP600125 and TCSJNK6o, and a TLR4 inhibitor TAK242 suppress reactive oxygen species production and NETosis in LPS-, but not PMA-treated neutrophils. Diphenyleneiodonium suppresses LPS-induced NETosis, confirming that endotoxin induces NADPH oxidase-dependent NETosis. Immunoblots, Sytox Green assays, and confocal microscopy of cleaved caspase-3 and nuclear morphology show that JNK inhibition does not induce apoptosis in LPS-stimulated neutrophils. JNK inhibition also suppresses NETosis induced by two typical Gram-negative bacteria, E. coli and Pseudomonas aeruginosa. Therefore, we propose that neutrophils use a TLR4-dependent, JNK-mediated molecular sensing mechanism to initiate NADPH oxidase-dependent suicidal NETosis in response to increasing concentrations of LPS, and Gram-negative bacteria. The LPS-TLR4-JNK activation axis determines the fate of these cells: to be or not to be NETotic neutrophils.
STAR/StarD1, part of a protein complex, mediates the transport of cholesterol from the outer to inner mitochondrial membrane, which is the rate-limiting step for steroidogenesis, and where steroid ...hormone synthesis begins. Herein, we examined the role of oxidant-sensitive p38 MAPKs in the regulation of STAR gene transcription, using model steroidogenic cell lines. Our data indicate that oxidant activation of p38 MAPK exhibits a negative regulatory role in the induction of functional expression of STAR, as evidenced by enhanced induction of STAR (mRNA/protein) expression and increased steroidogenesis during pharmacological inhibition of p38 MAPK or in cells with increased transient overexpression of a dominant-negative (dn) form of p38 MAPKα or p38 MAPKβ. Studies with rat Star-promoter demonstrated that overexpression of p38 MAPKα-wt, -β, or -γ significantly reduced both basal and cAMP-sensitive promoter activity. In contrast, overexpression of p38 MAPKα-dn, -β, or -γ enhanced the Star promoter activity under basal conditions and in response to cAMP stimulation. Use of various constitutively active and dn constructs and designer knock-out cell lines demonstrated that MKK3 and MKK6, the upstream activators of p38 MAPKs, play a role in p38 MAPKα-mediated inhibition of Star promoter activity. In addition, our studies raised the possibility of CREB being a potential target of the p38 MAPK inhibitory effect on Star promoter activity. Collectively, these data provide novel mechanistic information about how oxidant-sensitive p38 MAPKs, particularly p38 MAPKα, contribute to the negative regulation of Star gene expression and inhibit steroidogenesis.
Visualizing flows between origins and destinations can be straightforward when dealing with small numbers of journeys or simple geographies. Lines embedded in geographic space have commonly been used ...in mapping transport flows, especially when geographic patterns are important, as they are when characterizing cities or managing transportation. For larger numbers of flows, however, this approach requires careful design to avoid problems of occlusion, salience bias, and information overload. Driven by the requirements identified by users and managers of the London Bicycle Hire scheme, we present three methods of representation of bicycle-hire use and travel patterns. Flow maps with curved flow symbols are used to show overviews in flow structures. Gridded views of docking-station locations that preserve geographic relationships are used to explore docking-station status over space and time in a graphically efficient manner. Origin–Destination maps that visualize the OD matrix directly while maintaining geographic context are used to provide visual details on demand. We use these approaches to identify changes in travel behaviour over space and time, to aid station rebalancing, and to provide a framework for incorporating travel modelling and simulation.
Map matching is to track the positions of vehicles on the road network based on the positions provided by GPS (Global Positioning System) devices. Balancing localization accuracy and computation ...efficiency is a key problem in map matching. Existing methods mainly use Hidden Markov Model (HMM) or historical transportation data to learn the transitional probabilities among road segments. Although the roads to explore can be remarkably reduced by the Markov assumption, miss-of-match and matching breaks may occur if the GPS data is highly noisy, and the transitional model needs to be learned offline. To address these problems, this paper presents Multiple Candidate Matching (MCM) to improve the robustness of map matching. MCM doesn't need to pre-train the transitional model nor the historical transportation information. MCM memorizes multiple historical matching candidates in the map matching process. It votes among historical matchings and current matchings, but generates limited number of road candidates in real-time to restrict the computation complexity. MCM for both online map matching and offline map matching are presented and their properties are analyzed theoretically and experimentally. Numerical experiments in large-scale data sets show that MCM is very promising in terms of accuracy, computational efficiency, and robustness. The matching break and miss-of-match problems can be resolved effectively when compared with the state-of-the-art map matching methods. Codes are outsourced at https://github.com/lindalee-inlab/MCM.
Authenticated image encryption is crucial in handling top-secret images. To ensure confidentiality, authenticated encryption supplies legal validity for the image data. We propose a novel modified ...logistic map with an expanded key space, an integer-based key generation algorithm and an authenticated image encryption algorithm for block ciphers using a modified logistic map and a butterfly-like structure. The key that is used to encrypt images also aids in detecting content tampering of the images. The performance evaluations demonstrate that the proposed encryption method is fast and invulnerable to attacks and has a large key space. Additionally, the designed algorithm can assess the credibility of the data. Moreover, eight keys are used in decryption compared to four in encryption, which strengthens the security. Therefore, this algorithm can be used in real-time scenarios.
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► Codon harmonization was used as a precautionary measure toward soluble JNK1β1 overexpression. ► JNK1β1 and ATF2 were purified in high yields to >99% purity using GST-tags. ► JNK1β1 ...was activated by reconstituting the MEKK1→MKK4→JNK MAPK pathway in vitro. ► Phosphorylated JNK1β1 had high specific activity toward its substrate, ATF2.
The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His6-tagged proteins and purified using urea refolding and Ni2+-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.
Indonesia is one of the most seismically active countries in the world, and its large, vulnerable population makes reliable seismic hazard assessment an urgent priority. In 2016, the Indonesian ...Ministry of Public Works and Housing established a team of earthquake scientists and engineers tasked with improving the input data available for revising the national seismic hazard map. They compiled results of recent active fault studies using geological, geophysical, and geodetic observations, as well as a new comprehensive earthquake catalog including hypocenters relocated in a three-dimensional velocity model. Seismic hazard analysis was undertaken using recently developed ground motion prediction equations (GMPEs), and logic trees for the inclusion of epistemic uncertainty associated with different choices for GMPEs and earthquake recurrence models. The new seismic hazard maps establish the importance of active faults and intraslab seismicity, as well as the subduction megathrust, in determining the level of seismic hazard, especially in onshore, populated areas. The new Indonesian hazard maps will be used to update national standards for design of earthquake-resilient buildings and infrastructure.
Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibrotic destruction of normal lung architecture. Due to a lack of effective treatment options, new treatment approaches are ...needed. We previously identified transglutaminase (TG)2, a multifunctional protein expressed by human lung fibroblasts (HLFs), as a positive driver of fibrosis. TG2 catalyzes crosslinking of extracellular matrix proteins, enhances cell binding to fibronectin and integrin, and promotes fibronectin expression. We investigated whether the small electrophilic molecules 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2) inhibit the expression and profibrotic functions of TG2. CDDO and 15d-PGJ2 reduced expression of TG2 mRNA and protein in primary HLFs from control donors and donors with IPF. CDDO and 15d-PGJ2 also decreased the in vitro profibrotic effector functions of HLFs including collagen gel contraction and cell migration. The decrease in TG2 expression did not occur through activation of the peroxisome proliferator activated receptor γ or generation of reactive oxidative species. CDDO and 15d-PGJ2 inhibited the extracellular signal-regulated kinase pathway, resulting in the suppression of TG2 expression. This is the first study to show that small electrophilic compounds inhibit the expression and profibrotic effector functions of TG2, a key promoter of fibrosis. These studies identify new and important antifibrotic activities of these two small molecules, which could lead to new treatments for fibrotic lung disease.
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