Prolonged administration of dexamethasone, a potent anti-inflammatory drug, can lead to steroid-induced diabetes. Imatinib, a medication commonly prescribed for chronic myeloid leukemia (CML), has been shown to improve diabetes in CML patients. Our recent study demonstrated that dexamethasone induces pancreatic β-cell apoptosis by upregulating the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 5 (DR5). We hypothesized that imatinib may protect against dexamethasone-induced pancreatic β-cell apoptosis by reducing the expression of TRAIL and DR5, thereby favorably modulating downstream effectors in apoptotic pathways. We test this hypothesis by assessing the effects of imatinib on dexamethasone-induced apoptosis in rat insulinoma cell line cells. As anticipated, dexamethasone treatment led to increased TRAIL and DR5 expression, as well as an elevation in superoxide production. Conversely, expression of the TRAIL decoy receptor (DcR1) was decreased. Moreover, key effectors in the extrinsic and intrinsic apoptosis pathways, such as B-cell lymphoma 2 (BCL-2) associated X (BAX), nuclear factor kappa B (NF-κb), P73, caspase 8, and caspase 9, were upregulated, while the antiapoptotic protein BCL-2 was downregulated. Interestingly and importantly, imatinib at a concentration of 10 µM reversed the effect of dexamethasone on TRAIL, DR5, DcR1, superoxide production, BAX, BCL-2, NF-κB, P73, caspase 3, caspase 8, and caspase 9. Similar effects of imatinib on dexamethasone-induced TRAIL and DR5 expression were also observed in isolated mouse islets. Taken together, our findings suggest that imatinib protects against dexamethasone-induced pancreatic β-cell apoptosis by reducing TRAIL and DR5 expression and modulating downstream effectors in the extrinsic and intrinsic apoptosis pathways.

Elsinochrome A (EA) is a perylene quinone natural photosensitizer, photosensitizer under light excitation generates reactive oxygen species (ROS) to induce apoptosis, so can be used for treating tumors, that is so-called photodynamic therapy (PDT). However, the molecular mechanism, especially related to apoptosis and autophagy, is still unclear. In this study, we aimed to explore the mechanism of EA-PDT-induced B16 cells apoptosis and autophagy. The action of EA-PDT on mitochondrial permeability transition pore (MPTP), mitochondrial membrane potential (MMP) and the mitochondrial function were researched by fluorescence technique and Extracellular Flux Analyzer. Illumina sequencing, tandem mass tags Quantitative Proteomics and Western Blot studied the mechanism at the gene and protein levels. The results indicated that EA-PDT had excellent phototoxicity in vitro. EA could bind to the mitochondria. EA-PDT for 5 min caused MPTP opening, MMP decreasing and abnormal mitochondrial function with a concentration-dependent characteristic. EA-PDT resulted in an increase intracellular ROS and the number of autophagosomes. Caspase2, caspase9 and tnf were upregulated, and bcl2, prkn, atg2, atg9 and atg10 were downregulated. Our results indicated that EA-PDT induced cell apoptosis and autophagy through the mediation of ROS/Atg/Parkin. This study can provide enlightenment for exploring potential targets of drug development for the PDT of melanoma.

Electrical stimulation (ES) influences neural regeneration and functionality. We here investigate whether ES regulates DNA demethylation, a critical epigenetic event known to influence nerve regeneration. Retinal ganglion cells (RGCs) have long served as a standard model for central nervous system neurons, whose growth and disease development are reportedly affected by DNA methylation. The current study focuses on the ability of ES to rescue RGCs and preserve vision by modulating DNA demethylation. To evaluate DNA demethylation pattern during development, RGCs from mice at different stages of development, were analyzed using qPCR for ten-eleven translocation (TETs) and immunostained for 5 hydroxymethylcytosine (5hmc) and 5 methylcytosine (5mc). To understand the effect of ES on neurite outgrowth and DNA demethylation, cells were subjected to ES at 75 µAmp biphasic ramp for 20 min and cultured for 5 days. ES increased TETs mediated neurite outgrowth, DNA demethylation, TET1 and growth associated protein 43 levels significantly. Immunostaining of PC12 cells following ES for histone 3 lysine 9 trimethylation showed cells attained an antiheterochromatin configuration. Cultured mouse and human retinal explants stained with β-III tubulin exhibited increased neurite growth following ES. Finally, mice subjected to optic nerve crush injury followed by ES exhibited improved RGCs function and phenotype as validated using electroretinogram and immunohistochemistry. Our results point to a possible therapeutic regulation of DNA demethylation by ES in neurons.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is still one of the global health burdens. The occurrence of various cases and multidrug resistance confirm that TB has not been completely conquered. For these reasons, the present research has been conducted to explore TB vaccine and drug candidate possibility using Mtb-secreted proteins. Among these proteins, MPT32 is known to have antigenicity and immunogenicity. There has not been a report on the host immune responses and regulation in macrophage cells. The present study was conducted with MPT32 in RAW 264.7 murine macrophage cells that control immune responses by sensing pathogen invasion and environmental change. We have found that MPT32 could activate lipopolysaccharide (LPS)-induced gene expression of metalloproteinase-9 (MMP-9) and inflammation in RAW 264.7 cells. After treating cells with MPT32, the increase in pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β (IL-1β) and IL-6, was observed. In addition, activated macrophages expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to generate various inflammatory mediator molecules, such as nitric oxide (NO). The increase in iNOS and COX-2 levels, which are up-regulators of MMP-9 expression, was also confirmed. The biochemical events are involved in the downstream of activated MAPK signaling and translocation of NF-κ B transcription factor. The present results prove the immunomodulatory effect of MPT32 in the RAW 264.7 murine macrophage cells. it claims the possibility of a TB vaccination and drug candidate using MPT32, contributing to the prevention of TB.

Antimicrobial resistance (AMR) poses a significant threat to global public health, with multidrug-resistant Pseudomonas aeruginosa being a leading cause of mortality, accounting for 18%-61% of deaths annually. The quorum sensing (QS) systems of P. aeruginosa, particularly the LasI-LasR system, play a crucial role in promoting biofilm formation and expression of virulent genes, which contribute to the development of AMR. This study focuses on LasI, the mediator of biofilm formation for identifying its inhibitors from a marine compound database comprising of 32 000 compounds using molecular docking and molecular simulation techniques. The virtual screening and docking experiments demonstrated that the top 10 compounds exhibited favorable docking scores of <-7.19 kcal/mol compared to the reported inhibitor 3,5,7-Trihydroxyflavone with a docking score of -3.098 kcal/mol. Additionally, molecular mechanics/Poisson-Boltzmann generalized born surface area (MM-GBSA) analyses were conducted to assess these compounds' suitability for further investigation. Out of 10 compounds, five compounds demonstrated high MM-GBSA binding energy (<-35.33 kcal/mol) and were taken up for molecular dynamics simulations to evaluate the stability of the protein-ligand complex over a 100 ns period. Based on root mean square deviation, root mean square fluctuation, radius of gyration, and hydrogen bond interactions analysis, three marine compounds, namely MC-2 (CMNPD13419) and MC-3 (CMNPD1068), exhibited consistent stability throughout the simulation. Therefore, these compounds show potential as promising LasI inhibitors and warrant further validation through in vitro and in vivo experiments. By exploring the inhibitory effects of these marine compounds on P. aeruginosa's QS system, this research aims to contribute to the development of novel strategies to combat AMR.

Glabridin, a polyphenolic flavonoid derived from Glycyrrhiza glabra (licorice) roots, has shown anti-inflammatory and antioxidant properties. The current study sought to investigate glabridin's immunomodulatory effect in ovalbumin induced allergic asthma. Healthy male Wistar rats were divided into five groups. Group I served as a control group. Asthma was induced in groups II- IV. Groups III and IV were treated with glabridin (40 mg/kg) and methylprednisolone (15 mg/kg), respectively. Inflammatory cells counts were determined in blood and bronchoalveolar lavage fluid (BALF). Serum IgE levels and levels of catalase, superoxide dismutase and glutathione peroxidase in lung homogenate were measured. The levels of mRNA expression of pro-inflammatory, anti-inflammatory and oxidative stress markers were analysed. Delayed type hypersensitivity (DTH) and acute toxicity of glabridin were also checked. Glabridin significantly decreased inflammatory cells in the blood and BALF. It increased the concentration of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase. Glabridin markedly decreased serum IgE levels and DTH when compared to asthmatic rats. It significantly alleviated the expression of TNF-α, IL-4, IL-5, CXCL1, iNOS, and NF-κB. Administering 10 times the therapeutic dose of glabridin did not show any signs of acute toxicity. Findings suggest that glabridin has the potential to ameliorate allergic asthma and its effects are comparable to those of methylprednisolone. The immunomodulatory effect of glabridin might be contributed by the suppression of pro-inflammatory cytokines, oxidative stress markers, IgE antibodies, and elevation of antioxidant enzymes, suggesting future study and clinical trials to propose it as a candidate to treat allergic asthma.

The emergence of multiple drug resistance and extreme drug resistance pathogens necessitates the continuous evaluation of the pathogenic genome to identify conserved molecular targets and their respective inhibitors. In this study, we mapped the global mutational landscape of Neisseria gonorrhoeae (an intracellular pathogen notoriously known to cause the sexually transmitted disease gonorrhoea). We identified highly variable amino acid positions in the antibiotic target genes like the penA, ponA, 23s rRNA, rpoB, gyrA, parC, mtrR and porB. Some variations are directly reported to confer resistance to the currently used front-line drugs like ceftriaxone, cefixime, azithromycin and ciprofloxacin. Further, by whole genome comparison and Shannon entropy analysis, we identified a completely conserved protein HtpX in the drug-resistant as well as susceptible isolates of N. gonorrhoeae (NgHtpX). Comparison with the only available information of Escherichia coli HtpX suggested it to be a transmembrane metalloprotease having a role in stress response. The critical zinc-binding residue of NgHtpX was mapped to E141. By applying composite high throughput screening followed by MD simulations, we identified pemirolast and thalidomide as high-energy binding ligands of NgHtpX. Following cloning and expression of the purified metal-binding domain of NgHtpX (NgHtpXd), its Zn2+ -binding (Kd  = 0.4 µM) and drug-binding (pemirolast, Kd  = 3.47 µM; and thalidomide, Kd  = 1.04 µM) potentials were determined using in-vitro fluorescence quenching experiment. When tested on N. gonorrhoeae cultures, both the ligands imposed a dose-dependent reduction in viability. Overall, our results provide high entropy positions in the targets of presently used antibiotics, which can be further explored to understand the AMR mechanism. Additionally, HtpX and its specific inhibitors identified can be utilised effectively in managing gonococcal infections.

Skeletal muscle is composed of multinucleated myotubes formed by the fusion of mononucleated myoblasts. Skeletal muscle differentiation, termed as myogenesis, have been investigated using the mouse skeletal myoblast cell line C2C12. It has been reported that several "small" Rab proteins, major membrane-trafficking regulators, possibly regulate membrane protein transport in C2C12 cells; however, the role of Rab proteins in myogenesis remains unexplored. Rab44, a member of "large" Rab GTPases, has recently been identified as a negative regulator of osteoclast differentiation. In this study, using C2C12 cells, we found that Rab44 expression was upregulated during myoblast differentiation into myotubes. Knockdown of Rab44 enhanced myoblast differentiation and myotube formation. Consistent with these results, Rab44 knockdown in myoblasts increased expression levels of several myogenic marker genes. Rab44 knockdown increased the surface accumulation of myomaker and myomixer, two fusogenic proteins required for multinucleation, implying enhanced cell fusion. Conversely, Rab44 overexpression inhibited myoblast differentiation and tube formation, accompanied by decreased expression of some myogenic markers. Furthermore, Rab44 was found to be predominantly localized in lysosomes, and Rab44 overexpression altered the number and size of lysosomes. Considering the underlying molecular mechanism, Rab44 overexpression impaired the signaling pathway of the mechanistic target of rapamycin complex1 (mTORC1) in C2C12 cells. Namely, phosphorylation levels of mTORC1 and downstream mTORC1 substrates, such as S6 and P70-S6K, were notably lower in Rab44 overexpressing cells than those in control cells. These results indicate that Rab44 negatively regulates myoblast differentiation into myotubes by controlling fusogenic protein transport and mTORC1 signaling.

In this study, we have screened a large number of Food and Drug Administration-approved compounds for novel anti-leishmanial molecules targeting the citrate synthase enzyme of the parasite. Based on their docking and molecular dynamic simulation statistics, five compounds were selected. These compounds followed Lipinski's rule of five. Additionally, in vitro, antileishmanial and cytotoxicity studies were performed. The three compounds, Abemaciclib, Bazedoxifene, and Vorapaxar, had shown effective anti-leishmanial activities with IC50 values of 0.92 ± 0.02, 0.65 ± 0.09, and 6.1 ± 0.91 against Leishmania donovani promastigote and with EC50 values of 1.52 ± 0.37, 2.11 ± 0.38, 10.4 ± 1.27 against intramacrophagic amastigote without significantly harming macrophage cells. Among them, from in silico and antileishmanial activities studies, Abemaciclib had been selected based on their less binding energy, good antileishmanial activities, and also a significant difference in their binding energy with human citrate synthase for cell death mechanistic studies using flow cytometry and a DNA fragmentation assay. The action of this compound resulted in an increased reactive oxygen species production, depolarization of mitochondrial membrane potential, DNA damage, and an increase in the sub-G1 cell population. These properties are the hallmarks of apoptosis which were further confirmed by apoptotic assay. Based on the above result, this anticancer compound Abemaciclib could be employed as a potential treatment option for leishmaniasis after further confirmation.

Numerous studies have revealed the profound impact of microRNAs on regulating skeletal muscle development and regeneration. However, the biological function and regulation mechanism of miR-222-3p in skeletal muscle remains largely unknown. In this study, miR-222-3p was found to be abundantly expressed in the impaired skeletal muscles, indicating that it might have function in the development and regeneration process of the skeletal muscle. MiR-222-3p overexpression impeded C2C12 myoblast proliferation and myogenic differentiation, whereas inhibition of miR-222-3p got the opposite results. The dual-luciferase reporter assay showed that insulin receptor substrate-1 (IRS-1) was the target gene of miR-222-3p. We next found that knockdown of IRS-1 could obviously suppress C2C12 myoblast proliferation and differentiation. Additionally, miR-222-3p-induced repression of myoblast proliferation and differentiation was verified to be associated with a decrease in phosphoinositide 3-kinase (PI3K)-Akt signaling. Overall, we demonstrated that miR-222-3p inhibited C2C12 cells myogenesis via IRS-1/PI3K/Akt pathway. Therefore, miR-222-3p may be used as a therapeutic target for alleviating muscle loss caused by inherited and nonhereditary diseases.

Our previous studies have demonstrated that macrophages (RAW264.7) have a special ability for sensing the gradient of fluid shear stress (FSS) and migrate toward the low-FSS region. However, the molecular mechanism regulating this phenomenon is still unclear. In this study, we examined the transcriptome genes in RAW264.7 cells, MC3T3-E1 osteoblasts, mesenchymal stem cells, canine renal epithelial cells, and periodontal ligament cells. The expression levels of genes related to cell migration, force transfer, and force sensitivity in the Ca2+ signaling pathway were analyzed. We observed that the transient receptor potential cation channel type 2 (TRPV2) was highly expressed in RAW264.7 cells. Furthermore, we used lentiviral transfection to knockdown TRPV2 expression in RAW264.7 cells and studied the effect of TRPV2 on the migration of RAW264.7 cells under a gradient FSS field. The results showed that compared with normal cells, TRPV2-knockdown cells had impaired ability for sensing FSS gradient to migrate toward the low-FSS region and lower intracellular calcium response to FSS stimulation. This study may reveal the molecular mechanism of regulating the directional migration of macrophages under a gradient FSS field.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.