Acceleration of bone repairation by BMSCs overexpressing NGF combined with NSA and allograft bone scaffolds.

BACKGROUND: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. METHODS: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague-Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. RESULTS: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. CONCLUSION: NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.

Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation.

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

[Sivelestat sodium inhibits neutrophil elastase to regulate intrahepatic biliary mucin 5AC expression].

OBJECTIVE: To explore whether sivelestat sodium could reduce the expression of mucin 5AC (MUC5AC) in intrahepatic bile duct epithelial cells by inhibiting neutrophil elastase (NE) and thus provide new potential therapeutic ideas for the treatment of intrahepatic bile duct stone (IBDS). METHODS: (1) Bioinformatics analysis: differential gene analysis was performed on gallbladder stone cholecystitis sequencing data based on the gene expression omnibus (GEO) to screen for significantly different genes related to neutrophils and mucins. The search tool for the retrieval of interacting genes database (STRING) was used for protein interaction analysis to predict whether there was an interaction between NE and MUC5AC genes. (2) Animal experiment: a total of 18 male SD rats were divided into the sham-operated group, cholangitis model group and sivelestat sodium treatment group according to the random number table method, with 6 rats in each group. The cholangitis rat model was established by a one-time injection of 1.25 mg/kg lipopolysaccharide (LPS) into the right anterior lobe of the liver of rats in combination with the pre-experiment; the liver of the sham-operated group was injected with an equal volume of saline. After the modelling, 100 mg/kg of sivelestat sodium was injected into the tail vein of the cevalexin treatment group once a day for 5 days, and an equal volume of saline was injected into the tail vein of the sham-operated group and the cholangitis model group. Two weeks later, the rats were euthanized and their liver and bile duct tissues were taken. The pathological changes in the liver and bile duct tissues were observed under the light microscope. Immunohistochemical staining was used to detect the expressions of NE and MUC5AC in liver and bile duct tissues. The protein expressions of NE, MUC5AC and Toll-like receptor 4 (TLR4) were detected by Western blotting. (3) Cell experiment: primary human intrahepatic biliary epithelial cell line (HiBEpiC) was divided into blank control group, NE group (10 nmol/L NE), NE+sivelestat sodium low dose group (10 nmol/L NE+1×10-8 g/L sivelestat sodium 1 mL), NE+sivelestat sodium medium dose group (10 nmol/L NE+1×10-7 g/L sivelestat sodium 1 mL), NE+sivelestat sodium high dose group (10 nmol/L NE+1×10-6 g/L sivelestat sodium 1 mL). Cells were collected after 48 hours of culture, and EdU was performed to detect the proliferative activity of cells; enzyme linked immunosorbent assay (ELISA) and Western blotting were performed to detect the expression of MUC5AC in cells. RESULTS: (1) Bioinformatics analysis: the NE gene (ELANE) had a reciprocal relationship with MUC5AC. (2) Animal experiment: light microscopy showed that hepatocyte edema, hepatocyte diffuse point and focal necrosis, confluent area fibrous tissue and intrahepatic bile ducts hyperplasia and inflammatory cell infiltration in the cholangitis model group; hepatic lobule structure of sivelestat sodium treatment group was clear, and the degree of peripheral inflammatory cell infiltration was reduced compared with the cholangitis model group. Immunohistochemical staining showed that the expressions of NE and MUC5AC were increased in the cholangitis model group compared with the sham-operated group, and the expressions of NE and MUC5AC were decreased in the sivelestat sodium group compared with the cholangitis model group [NE (A value): 5.23±2.02 vs. 116.67±23.06, MUC5AC (A value): 5.40±3.09 vs. 23.81±7.09, both P < 0.05]. Western blotting showed that the protein expressions of NE, MUC5AC, and TLR4 in the hepatic biliary tissues of the cholangitis model group were significantly higher than those of the sham-operated group; and the protein expressions of NE, MUC5AC, and TLR4 in the liver biliary tissues of the sivelestat sodium treatment group were significantly higher than those of the sham-operated group (NE/ß-actin: 0.38±0.04 vs. 0.70±0.10, MUC5AC/ß-actin: 0.37±0.03 vs. 0.61±0.05, TLR4/ß-actin: 0.39±0.10 vs. 0.93±0.15, all P < 0.05). (3) Cell experiment: fluorescence microscopy showed that the proliferation of HiBEpiC cells in each group was good, and there was no significant difference in the proportion of positive cells. ELISA and Western blotting showed that the expressions of MUC5AC in cells of the NE group were significantly higher than those of the blank control group. The expressions of MUC5AC in the NE+different dose of sivelestat sodium group were significantly lower than those in the NE group, and showed a decreasing trend with the increase of sevastatin sodium concentration, especially in the highest dose group [MUC5AC (µg/L): 3.46±0.20 vs. 6.33±0.52, MUC5AC/ß-actin: 0.45±0.07 vs. 1.75±0.10, both P < 0.05]. CONCLUSIONS: LPS can upregulate the expression of NE and MUC5AC in rats with cholangitis, while sodium sivelestat can reduce the expression of MUC5AC in in intrahepatic biliary epithelial cells by inhibiting NE, providing a new direction for the treatment of IBDS.

Targeting BRD4 mitigates hepatocellular lipotoxicity by suppressing the NLRP3 inflammasome activation and GSDMD-mediated hepatocyte pyroptosis.

Nod-like receptor family pyrin-containing protein 3 (NLRP3) inflammasome plays a pathologic role in metabolic dysfunction-associated steatohepatitis (MASH), but the molecular mechanism regulating the NLRP3 inflammasome activation in hepatocellular lipotoxicity remains largely unknown. Bromodomain-containing protein 4 (BRD4) has emerged as a key epigenetic reader of acetylated lysine residues in enhancer regions that control the transcription of key genes. The aim of this study is to investigate if and how BRD4 regulated the NLRP3 inflammasome activation and pyroptosis in MASH. Using the AML12 and primary mouse hepatocytes stimulated by palmitic acid (PA) as an in vitro model of hepatocellular lipotoxicity, we found that targeting BRD4 by genetic knockdown or a selective BRD4 inhibitor MS417 protected against hepatosteatosis; and this protective effect was attributed to inhibiting the activation of NLRP3 inflammasome and reducing the expression of Caspase-1, gasdermin D (GSDMD), interleukin (IL)-1ß and IL-6. Moreover, BRD4 inhibition limited the voltage-dependent anion channel-1 (VDAC1) expression and oligomerization in PA-treated AML12 hepatocytes, thereby suppressing the NLRP3 inflammasome activation. Additionally, the expression of BRD4 enhanced in MASH livers of humans. Mechanistically, BRD4 was upregulated during hepatocellular lipotoxicity that in turn modulated the active epigenetic mark H3K27ac at the promoter regions of the Vdac and Gsdmd genes, thereby enhancing the expression of VDAC and GSDMD. Altogether, our data provide novel insights into epigenetic mechanisms underlying BRD4 activating the NLRP3 inflammasome and promoting GSDMD-mediated pyroptosis in hepatocellular lipotoxicity. Thus, BRD4 might serve as a novel therapeutic target for the treatment of MASH.

Promising anti-proliferative indolic benzenesulfonamides alter mechanisms with sulfonamide nitrogen substituents.

Agents that cause apoptotic cell death by interfering with tubulin dynamics, such as vinblastine and paclitaxel, are an important class of chemotherapeutics. Unfortunately, these compounds are substrates for multidrug resistance (MDR) pumps, allowing cancer cells to gain resistance to these chemotherapeutics. The indolesulfonamide family of tubulin inhibitors are not excluded by MDR pumps and have a promising activity profile, although their high lipophilicity is a pharmacokinetic limitation for their clinical use. Here we present a new family of N-indolyl-3,4,5-trimethoxybenzenesulfonamide derivatives with modifications on the indole system at positions 1 and 3 and on the sulfonamide nitrogen. We synthesized and screened against HeLa cells 34 novel indolic benzenesulfonamides. The most potent derivatives (1.7-109 nM) were tested against a broad panel of cancer cell lines, which revealed that substituted benzenesulfonamides analogs had highest potency. Importantly, these compounds were only moderately toxic to non-tumorigenic cells, suggesting the presence of a therapeutic index. Consistent with known clinical anti-tubulin agents, these compounds arrested the cell cycle at G2/M phase. Mechanistically, they induced apoptosis via caspase 3/7 activation, which occurred during M arrest. The substituents on the sulfonamide nitrogen appeared to determine different mechanistic results and cell fates. These results suggest that the compounds act differently depending on the bridge substituents, thus making them very interesting as mechanistic probes as well as potential drugs for further development.

Targeting RBM39 through indisulam induced mis-splicing of mRNA to exert anti-cancer effects in T-cell acute lymphoblastic leukemia.

BACKGROUND: Despite the use of targeted therapeutic approaches, T-cell acute lymphoblastic leukemia (T-ALL) is still associated with a high incidence of complications and a poor prognosis. Indisulam (also known as E7070), a newly identified molecular glue compound, has demonstrated increased therapeutic efficacy in several types of cancer through the rapid degradation of RBM39. This study aimed to evaluate the therapeutic potential of indisulam in T-ALL, elucidate its underlying mechanisms and explore the role of the RBM39 gene. METHODS: We verified the anticancer effects of indisulam in both in vivo and in vitro models. Additionally, the construction of RBM39-knockdown cell lines using shRNA confirmed that the malignant phenotype of T-ALL cells was dependent on RBM39. Through RNA sequencing, we identified indisulam-induced splicing anomalies, and proteomic analysis helped pinpoint protein changes caused by the drug. Comprehensive cross-analysis of these findings facilitated the identification of downstream effectors and subsequent validation of their functional roles. RESULTS: Indisulam has significant antineoplastic effects on T-ALL. It attenuates cell proliferation, promotes apoptosis and interferes with cell cycle progression in vitro while facilitating tumor remission in T-ALL in vivo models. This investigation provides evidence that the downregulation of RBM39 results in the restricted proliferation of T-ALL cells both in vitro and in vivo, suggesting that RBM39 is a potential target for T-ALL treatment. Indisulam's efficacy is attributed to its ability to induce RBM39 degradation, causing widespread aberrant splicing and abnormal translation of the critical downstream effector protein, THOC1, ultimately leading to protein depletion. Moreover, the presence of DCAF15 is regarded as critical for the effectiveness of indisulam, and its absence negates the ability of indisulam to induce the desired functional alterations. CONCLUSION: Our study revealed that indisulam, which targets RBM39 to induce tumor cell apoptosis, is an effective drug for treating T-ALL. Targeting RBM39 through indisulam leads to mis-splicing of pre-mRNAs, resulting in the loss of key effectors such as THOC1.

Senescent cancer-associated fibroblasts in pancreatic adenocarcinoma restrict CD8+ T cell activation and limit responsiveness to immunotherapy in mice.

Senescent cells within tumors and their stroma exert complex pro- and anti-tumorigenic functions. However, the identities and traits of these cells, and the potential for improving cancer therapy through their targeting, remain poorly characterized. Here, we identify a senescent subset within previously-defined cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinomas (PDAC) and in premalignant lesions in mice and humans. Senescent CAFs isolated from mouse and humans expressed elevated levels of immune-regulatory genes. Depletion of senescent CAFs, either genetically or using the Bcl-2 inhibitor ABT-199 (venetoclax), increased the proportion of activated CD8+ T cells in mouse pancreatic carcinomas, whereas induction of CAF senescence had the opposite effect. Combining ABT-199 with an immune checkpoint therapy regimen significantly reduced mouse tumor burden. These results indicate that senescent CAFs in PDAC stroma limit the numbers of activated cytotoxic CD8+ T cells, and suggest that their targeted elimination through senolytic treatment may enhance immunotherapy.

Determining M2 macrophages content for the anti-tumor effects of metal-organic framework-encapsulated pazopanib nanoparticles in breast cancer.

Pazopanib (PAZ), an oral multi-tyrosine kinase inhibitor, demonstrates promising cytostatic activities against various human cancers. However, its clinical utility is limited by substantial side effects and therapeutic resistance. We developed a nanoplatform capable of delivering PAZ for enhanced anti-breast cancer therapy. Nanometer-sized PAZ@Fe-MOF, compared to free PAZ, demonstrated increased anti-tumor therapeutic activities in both syngeneic murine 4T1 and xenograft human MDA-MB-231 breast cancer models. High-throughput single-cell RNA sequencing (scRNAseq) revealed that PAZ@Fe-MOF significantly reduced pro-tumorigenic M2-like macrophage populations at tumor sites and suppressed M2-type signaling pathways, such as ATF6-TGFBR1-SMAD3, as well as chemokines including CCL17, CCL22, and CCL24. PAZ@Fe-MOF reprogramed the inhibitory immune microenvironment and curbed tumorigenicity by blocking the polarization of M2 phenotype macrophages. This platform offers a promising and new strategy for improving the cytotoxicity of PAZ against breast cancers. It provides a method to evaluate the immunological response of tumor cells to PAZ-mediated treatment.

Synthesis and Enzymatic Evaluation of a Small Library of Substituted Phenylsulfonamido-Alkyl Sulfamates towards Carbonic Anhydrase II.

A small library of 79 substituted phenylsulfonamidoalkyl sulfamates, 1b-79b, was synthesized starting from arylsulfonyl chlorides and amino alcohols with different numbers of methylene groups between the hydroxyl and amino moieties yielding intermediates 1a-79a, followed by the reaction of the latter with sulfamoyl chloride. All compounds were screened for their inhibitory activity on bovine carbonic anhydrase II. Compounds 1a-79a showed no inhibition of the enzyme, in contrast to sulfamates 1b-79b. Thus, the inhibitory potential of compounds 1b-79b towards this enzyme depends on the substituent and the substitution pattern of the phenyl group as well as the length of the spacer. Bulkier substituents in the para position proved to be better for inhibiting CAII than compounds with the same substituent in the meta or ortho position. For many substitution patterns, compounds with shorter spacer lengths were superior to those with long chain spacers. Compounds with shorter spacer lengths performed better than those with longer chain spacers for a variety of substitution patterns. The most active compound held inhibition constant as low as Ki = 0.67 µM (for 49b) and a tert-butyl substituent in para position and acted as a competitive inhibitor of the enzyme.

Synthesis, Docking, and Machine Learning Studies of Some Novel Quinolinesulfonamides-Triazole Hybrids with Anticancer Activity.

In the presented work, a series of 22 hybrids of 8-quinolinesulfonamide and 1,4-disubstituted triazole with antiproliferative activity were designed and synthesised. The title compounds were designed using molecular modelling techniques. For this purpose, machine-learning, molecular docking, and molecular dynamics methods were used. Calculations of the pharmacokinetic parameters (connected with absorption, distribution, metabolism, excretion, and toxicity) of the hybrids were also performed. The new compounds were synthesised via a copper-catalysed azide-alkyne cycloaddition reaction (CuAAC). 8-N-Methyl-N-{[1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl]methyl}quinolinesulfonamide was identified in in silico studies as a potential strong inhibitor of Rho-associated protein kinase and as a compound that has an appropriate pharmacokinetic profile. The results obtained from in vitro experiments confirm the cytotoxicity of derivative 9b in four selected cancer cell lines and the lack of cytotoxicity of this derivative towards normal cells. The results obtained from silico and in vitro experiments indicate that the introduction of another quinolinyl fragment into the inhibitor molecule may have a significant impact on increasing the level of cytotoxicity toward cancer cells and indicate a further direction for future research in order to find new substances suitable for clinical applications in cancer treatment.

Effect of NLRP3 gene knockdown on pyroptosis and ferroptosis in diabetic cardiomyopathy injury.

Diabetic cardiomyopathy (DCM) is a chronic disease caused by diabetes mellitus, which is recognized as a worldwide challenging disease. This study aimed to investigate the role and the potential mechanism of knocking down the NACHT-, LRR- and PYD domains-containing protein 3 (NLRP3), an inflammasome associated with onset and progression of various diseases, on high glucose or diabetes -induced cardiac cells pyroptosis and ferroptosis, two regulated non-necrosis cell death modalities discovered recent years. In the present study, both in vivo and in vitro studies were conducted simultaneously. Diabetic rats were induced by 55 mg/kg intraperitoneal injection of streptozotocin (STZ). Following the intraperitoneal injection of MCC950 (10 mg/kg), On the other hand, the DCM model in H9C2 cardiac cells was simulated with 35 mmol/L glucose and a short hairpin RNA vector of NLRP3 were transfected to cells. The results showed that in vivo study, myocardial fibers were loosely arranged and showed inflammatory cell infiltration, mitochondrial cristae were broken and the GSDMD-NT expression was found notably increased in the DM group, while the protein expressions of xCT and GPX4 was significantly decreased, both of which were reversed by MCC950. High glucose reduced the cell viability and ATP level in vitro, accompanied by an increase in LDH release. All of the above indicators were reversed after NLRP3 knockdown compared with the HG treated alone. Moreover, the protein expressions of pyroptosis- and ferroptosis-related fators were significantly decreased or increased, consistent with the results shown by immunofluorescence. Furthermore, the protective effects of NLRP3 knockdown against HG were reversed following the mtROS agonist rotenone (ROT) treatment. In conclusion, inhibition of NLRP3 suppressed DM-induced myocardial injury. Promotion of mitochondrial ROS abolished the protective effect of knockdown NLRP3, and induced the happening of pyroptosis and ferroptosis. These findings may present a novel therapeutic underlying mechanism for clinical diabetes-induced myocardial injury treatment.

Recent development of azole-sulfonamide hybrids with the anticancer potential.

Cancer exhibits heterogeneity that enables adaptability and remains grand challenges for effective treatment. Chemotherapy is a validated and critically important strategy for the treatment of cancer, but the emergence of multidrug resistance which may lead to recurrence of disease or even death is a major hurdle for successful chemotherapy. Azoles and sulfonamides are important anticancer pharmacophores, and azole-sulfonamide hybrids have the potential to simultaneously act on dual/multiple targets in cancer cells, holding great promise to overcome drug resistance. This review outlines the current scenario of azole-sulfonamide hybrids with the anticancer potential, and the structure-activity relationships as well as mechanisms of action are also discussed, covering articles published from 2020 onward.

[Box: see text].

Inhibition of NLRP3 inflammasome ameliorates LPS-induced neuroinflammatory injury in mice via PINK1/Parkin pathway.

Parkinson's disease (PD) is characterized by the severe loss of dopaminergic neurons in the substantia nigra pars compacta, leading to motor dysfunction. The onset of PD is often accompanied by neuroinflammation and α-Synuclein aggregation, and extensive research has focused on the activation of microglial NLRP3 inflammasomes in PD, which promotes the death of dopaminergic neurons. In this study, a model of cerebral inflammatory response was constructed in wild-type and Parkin＋/－ mice through bilateral intraventricular injection of LPS. LPS-induced activation of the NLRP3 inflammasome in wild-type mice promotes the progression of PD. The use of MCC950 in wild mice injected with LPS induces activation of Parkin/PINK and improves autophagy, which in turn improves mitochondrial turnover. It also inhibits LPS-induced inflammatory responses, improves motor function, protects dopaminergic neurons, and inhibits microglia activation. Furthermore, Parkin＋/－ mice exhibited motor dysfunction, loss of dopaminergic neurons, activation of the NLRP3 inflammasome, and α-Synuclein aggregation beginning at an early age. Parkin ± mice exhibited more pronounced microglia activation, greater NLRP3 inflammasome activation, more severe autophagy dysfunction, and more pronounced motor dysfunction after LPS injection compared to wild-type mice. Notably, the use of MCC950 in Parkin ± mice did not ameliorate NLRP3 inflammasome activation, autophagy dysfunction, or α-synuclein aggregation. Thus, MCC950 can only exert its effects in the presence of Parkin/PINK1, and targeting Parkin-mediated NLRP3 inflammasome activation is expected to be a potential therapeutic strategy for Parkinson's disease.

Impact of monocytic differentiation on acute myeloid leukemia patients treated with venetoclax and hypomethylating agents.

INTRODUCTION: Although the combination of venetoclax (VEN) and hypomethylating agents (HMAs) results in impressive efficacy in acute myeloid leukemia (AML), there is still a subset of patients who are refractory. We investigated the outcomes of AML patients with monocytic differentiation who were treated with frontline VEN/HMA. METHODS: A total of 155 patients with newly diagnosed AML treated with frontline VEN/HMA were enrolled in the study. Monocyte-like AML was identified by flow cytometry with typical expression of monocytic markers, and M5 was identified according to French, American, and British category. We compared the outcomes of patients with different characteristics. RESULTS: The rate of complete remission (CR) and CR with incomplete recovery of blood counts (CRi), progression-free survival (PFS), and overall survival (OS) in monocyte-like AML were inferior to those in nonmonocyte-like AML (CR/CRi rates, 26.7% vs. 80.0%, p < 0.001; median PFS, 2.1 vs. 8.8 months, p < 0.001; median OS, 9.2 vs. 19 months, p = 0.013). CR/CRi rate in M5 was lower than that in non-M5 (60.7% vs. 75.5%, p = 0.049). Multivariate analyses showed that monocyte-like AML was associated with lower odds of CR/CRi and higher risk of progression. CONCLUSION: Our study suggested that newly diagnosed AML with a monocytic immunophenotype had a poor prognosis with VEN/HMA treatment.

Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.

Antiplasmodial action of 4-nitrobenzenesulfonamide chalcones: Design, synthesis, characterisation, in vitro and in silico evaluation against blood stages of Plasmodium falciparum 3D7.

Malaria is an intracellular protozoan parasitic disease caused by Plasmodium species with significant morbidity and mortality in endemic regions. The complex lifecycle of the parasite and the emergence of drug-resistant Plasmodium falciparum have hampered the efficacy of current anti-malarial agents. To circumvent this situation, the present study attempts to demonstrate the blood-stage anti-plasmodial action of 26 hybrid compounds containing the three privileged bioactive scaffolds (sulfonamide, chalcone, and nitro group) with synergistic and multitarget action. These three parent scaffolds exhibit divergent activities, such as antibacterial, anti-malarial, anti-fungal, anti-inflammatory, and anticancer. All the synthesised compounds were characterised using various spectroscopic techniques. The in vitro blood-stage inhibitory activity of 26 hybrid compounds was evaluated against mixed-stage culture (asynchronize) of human malarial parasite P. falciparum, Pf 3D7 at different concentrations ranging from 25.0 µg/mL to 0.78 µg/mL using SYBR 1 green assay, with IC50 values determined after 48 h of treatment based on the drug-response curves. Two potent compounds (11 and 10), with 2-Br and 2,6-diCl substitutions, showed pronounced activity with IC50 values of 5.4 µg/mL and 5.6 µg/mL, whereas others displayed varied activity with IC50 values ranging from 7.0 µg/mL to 22.0 µg/mL. Both 11 and 10 showed greater susceptibility towards mature-stage trophozoites than ring-stage parasites. The hemolytic and in vitro cytotoxicity assays revealed that compounds 11 and 10 did not cause any toxic effects on host red blood cells (uninfected), human-derived Mo7e cells, and murine-derived BA/F3 cells. The in vitro observations are consistent with the in silico studies using P. falciparum-dihydrofolate reductase, where 11 and 10 showed a binding affinity of -10.4 Kcal/mol. This is the first report of the hybrid scaffold, 4-nitrobenzenesulfonamide chalcones, demonstrating its potential as an anti-plasmodial agent.

Combined Insults of a MASH Diet and Alcohol Binges Activate Intercellular Communication and Neutrophil Recruitment via the NLRP3-IL-1ß Axis in the Liver.

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1ß via NLRP3 activation and that IL-1ß acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.

Repositioning of baricitinib for management of memory impairment in ovariectomized/D-galactose treated rats: A potential role of JAK2/STAT3-PI3K/AKT/mTOR signaling pathway.

AIMS: Neuroinflammation plays a pivotal role in amyloid ß (Aß) plaques formation which is among the hallmarks of Alzheimer's disease (AD). The present study investigated the potential therapeutic effects of baricitinib (BAR), a selective JAK2/ STAT3 inhibitor, in ovariectomized/ D-galactose (OVX/D-gal) treated rats as a model for AD. MAIN METHODS: To induce AD, adult female rats (130-180 g) underwent bilateral ovariectomy and were injected daily with 150 mg/kg, i.p. D-gal for 8 consecutive weeks. BAR (10 and 50 mg/kg/day) was then given orally for 14 days. KEY FINDINGS: BAR in a dose-dependent effect mitigated OVX/D-gal-induced aberrant activation of JAK2/STAT3 signaling pathway resulting in significant decreases in the expression of p-JAK 2, and p-STAT3 levels, along with deactivating AKT/PI3K/mTOR signaling as evidenced by deceased protein expression of p-AKT, p-PI3K, and p-mTOR. As a result, neuroinflammation was diminished as evidenced by decreased NF-κß, TNF-α, and IL-6 levels. Moreover, oxidative stress biomarkers levels as iNOS, and MDA were reduced, whereas GSH was increased by BAR. BAR administration also succeeded in reverting histopathological alterations caused by OVX/D-gal, increased the number of intact neurons (detected by Nissl stain), and diminished astrocyte hyperactivity assessed as GFAP immunoreactivity. Finally, treatment with BAR diminished the levels of Aß. These changes culminated in enhancing spatial learning and memory in Morris water maze, and novel object recognition test. SIGNIFICANCE: BAR could be an effective therapy against neuroinflammation, astrogliosis and cognitive impairment induced by OVX/ D-gal where inhibiting JAK2/STAT3- AKT/PI3K/mTOR seems to play a crucial role in its beneficial effect.

Lysosome-Disrupting Agents in Combination with Venetoclax Increase Apoptotic Response in Primary Chronic Lymphocytic Leukemia (CLL) Cells Mediated by Lysosomal Cathepsin D Release and Inhibition of Autophagy.

Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.

Synergistic Effect of Venetoclax and Bendamustine in Early T-cell Precursor Acute Lymphoblastic Leukemia.

BACKGROUND/AIM: To date, therapeutic options for T-cell acute lymphoblastic leukemia (T-ALL) remain very limited. This study evaluated the efficacy of monotherapies and combination therapies including a selective BCL-2 inhibitor for T-ALL cell lines, namely Jurkat, CCRF-CEM, and Loucy. MATERIALS AND METHODS: Loucy is an early T-precursor ALL (ETP-ALL) cell line characterized by an immature phenotype, whereas Jurkat and CCRF-CEM are late T-cell progenitor ALL (LTP-ALL) cell lines. Monotherapy was conducted with venetoclax, cytarabine, bendamustine, or azacytidine, whereas combination therapy was performed with venetoclax plus cytarabine, venetoclax plus bendamustine, or venetoclax plus azacytidine. Cell viability assay was conducted after 48 h using Trypan blue and the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Statistical analysis for evaluating synergistic interactions between anticancer drugs was performed by using the SynergyFinder Plus and drc R package. RESULTS: Adding venetoclax to cytarabine, bendamustine, or azacitidine achieved an additive effect, with Loewe synergic scores ranging from -10 to 10 in Jurkat and CCRF-CEM. Conversely, the combination of venetoclax and cytarabine displayed an additive effect (Loewe synergic score: 8.45 and 5.82 with MTS and Trypan blue assays, respectively), whereas venetoclax plus bendamustine or azacitidine exhibited a synergistic effect (Loewe synergic score >10 with MTS assay) in Loucy. Remarkably, the Bliss/Loewe score revealed that the combination of venetoclax and bendamustine was the most synergistic, yielding a score of 13.832±0.55. CONCLUSION: The combination of venetoclax and bendamustine demonstrated the greatest synergistic effect in suppressing ETP-ALL cell proliferation. Further studies are warranted to determine the mechanisms for the synergism between venetoclax and bendamustine in high-risk T-ALL.