The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.

UNASSIGNED: This study aimed to explore the serum levels of gasdermin D (GSDMD) in uremic (end-stage kidney disease, ESKD) patients and their correlation with vascular calcification (VC) and clinical results.
UNASSIGNED: This prospective observational cohort study enrolled 213 ESKD patients who were undergoing regular maintenance hemodialysis (MHD) for > 3 months in our hospital from August 2019 to July 2022. The abdominal aortic calcification score (AACS) was used to assess the VC condition of patients with ESKD. Serum GSDMD, caspase-1, interleukin (IL)-6, IL-1β, IL-18 and C-reactive protein (CRP) levels were measured using enzyme-linked immunosorbent assay (ELISA). Demographic and clinical data were obtained. All patients were followed up for 1 year, and patients with major adverse cardiovascular events (MACE) were defined as having a poor prognosis. All data used SPSS 26.0 to statistical analyses.
UNASSIGNED: The serum total cholesterol (TC) levels of patients in the AACS > 4 group were significantly elevated compared with those in the AACS ≤ 4 group. In addition, ESKD patients with an AACS > 4 had significantly higher serum levels of GSDMD, caspase-1, IL-6, IL-18 and IL-1β. Moreover, Pearson\'s analysis supported a positive correlation between GSDMD and caspase-1, IL-6, and IL-1β. In addition, we found that GSDMD levels were positively correlated with the clinical data (AACS scores and serum TC levels) of patients with ERSD. Additionally, ROC curves showed that the serum levels of GSDMD could be a potential predictive biomarker of moderate/severe VC and prognosis in patients with ESKD. Finally, the results of logistic regression indicated that GSDMD and AACS scores were risk factors for poor prognosis in patients with ESKD.
UNASSIGNED: Serum GSDMD levels were remarkably elevated in patients with ESKD with moderate/severe calcification. In addition, serum levels of GSDMD could be a potential predictive biomarker of moderate/severe VC and prognosis in patients with ESKD.

JNK3, a neuronal kinase activated by stress, plays a role in stress-induced apoptosis, leading to neuronal cell death following cerebral ischemia. This study investigates the neuroprotective effects of piceatannol (PCT) in SHSY-5Y neuroblastoma cells after hypoxic injury and its interaction with JNK3. We analyzed the crystal coordinates, interaction energies, and amino acid interactions to determine PCT\'s selectivity for JNK3. The electrostatic potential was computed using density functional theory, while molecular dynamics assessed the stability and structural consistency of the JNK3-PCT complex. We used SP600125 (SP6), a JNK3 inhibitor, as a reference compound. Additionally, we performed cell-free JNK 1, 2, and 3 kinase assays to evaluate the isoform selectivity of PCT. Cytotoxicity and cell viability were determined by an MTT test. To assess apoptosis, we used acridine orange/ethidium bromide dual fluorescent labeling and ANNEXIN A5-FITC flow cytometry. Western blot was used to evaluate the attenuation of JNK3 and apoptotic proteins. In silico studies revealed a stronger binding affinity between PCT and JNK3 compared to JNK1 and JNK2, which was further supported by the in vitro kinase assay. PCT-treated cells exhibited a decrease in Cyt-c and caspase-3 expression and an increase in Bcl-2 level, compared to hypoxic control (p < .001). PCT also demonstrated superior efficacy over SP6 in inhibiting JNK3 phosphorylation (p < .001). Furthermore, PCT significantly increased the expression of neuronal genes, including NgN1, neuroD2, and survivin (p < .001). In conclusion, PCT is a potential JNK3 inhibitor, since it inhibited phosphorylation and the Bcl-2/Cyt-C/caspase-3-dependent apoptotic pathway after ischemic/hypoxic insult.

At the molecular level, several developmental signaling pathways, such as Wnt/β-catenin, have been associated with the initiation and subsequent progression of prostate carcinomas. The present report elucidated the anti-cancerous attributes of an anthraquinone, aloe-emodin (AE), against androgen-independent human prostate cancer DU145 cells. The cytotoxicity profiling of AE showed that it exerted significant cytotoxic effects and increased lactose dehydrogenase levels in DU145 cells (p < 0.01 and p < 0.001). AE also induced considerable reactive oxygen species (ROS)-mediated oxidative stress, which escalated at higher AE concentrations of 20 and 25 μM. AE also efficiently instigated nuclear fragmentation and condensation concomitantly, followed by the activation of caspase-3 and -9 within DU145 cells. AE further reduced the viability of mitochondria with increased cytosolic cytochrome-c levels (p < 0.01 and p < 0.001) in DU145 cells. Importantly, AE exposure was also correlated with reduced Wnt2 and β-catenin mRNA levels along with their target genes, including cyclin D1 and c-myc. Furthermore, the molecular mechanism of AE was evaluated by performing molecular docking studies with Wnt2 and β-catenin. Evidently, AE exhibited good binding energy scores toward Wnt2 and β-catenin comparable with their respective standards, CCT036477 (Wnt2 inhibitor) and FH535 (β-catenin inhibitor). Thus, it may be considered that AE was competent in exerting anti-growth effects against DU145 androgen-independent prostate cancer cells plausibly by modulating the expression of Wnt/β-catenin signaling.

BACKGROUND: High mobility group box-1 (HMGB1) is an endogenous danger signal that mediates activation of the innate immune response including NLR pyrin domain containing 3 (NLRP3) inflammasome activation and proinflammatory cytokine release. Although HMGB1 and NLRP3 have been implicated in the pathophysiology of seizures, the correlation between HMGB1 and NLRP3 expression has not been determined in children with febrile seizures (FS). To explore the relationship between extra-cellular HMGB1 and NLRP3 in children with FS, we analyzed serum HMGB1, NLRP3, caspase-1, and proinflammatory cytokines in patients with FS.
METHODS: Thirty children with FS and thirty age-matched febrile controls were included in this study. Blood was obtained from the children with FS within 1 h of the time of the seizure; subsequently, the serum contents of HMGB1, NLRP3, caspase-1, interleukin (IL)-1β, interleukin (IL)-6, and tumour necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay. The Mann‒Whitney U test was used to compare serum cytokine levels between FS patients and controls. Spearman\'s rank correlation coefficient was calculated to detect significant correlations between cytokine levels.
RESULTS: Serum levels of HMGB1, NLRP3, caspase-1, IL-1β, IL-6, and TNF-α were significantly higher in FS patients than in febrile controls (p < 0.05). Serum levels of HMGB1 were significantly correlated with levels of NLRP3 and caspase-1 (both, p < 0.05). Serum levels of caspase-1 were significantly correlated with levels of IL-1β (p < 0.05). Serum levels of IL-1β were significantly correlated with levels of IL-6 and TNF-α (p < 0.05).
CONCLUSIONS: HMGB1 is up-regulated in the peripheral serum of FS patients, which may be responsible, at least in part, for the increased expression of NLRP3 and Caspase-1. Increased expression of caspase-1 was significantly associated with elevated serum levels of IL-1β. Given that activated Caspase-1 directly regulates the expression of mature IL-1β and positively correlates with activation of the NLRP3 inflammasome, our data suggest that increased levels of peripheral HMGB1 possibly mediate IL-1β secretion through the activation of the NLRP3 inflammasome in children with FS. Thus, both HMGB1 and NLRP3 might be potential targets for preventing or limiting FS.

Non-alcoholic steatohepatitis (NASH) is a progressive, inflammatory liver disease with no approved pharmacological treatment. This Phase IIa, double-blind, placebo-controlled, multicentre trial (ClinicalTrials.gov: NCT03166735) investigated pharmacodynamics and safety of BI 1467335, an amine oxidase copper-containing 3 (AOC3) inhibitor, in adults with NASH from Europe and North America. Participants from 44 centres across the US, Germany, Spain, Belgium, the UK, Netherlands, Canada, France and Ireland were randomised (2:1:1:1:2; 27 July 2017 to 14 June 2019) to daily oral BI 1467335 1 mg (n = 16), 3 mg (n = 16), 6 mg (n = 17), 10 mg (n = 32) or placebo (n = 32) for 12 weeks, with follow-up to Week 16. Primary endpoint was AOC3 activity relative to baseline (%), 24 hours post-dose after 12 weeks\' treatment. Secondary biomarker endpoints included changes from baseline at Week 12 in alanine aminotransferase (ALT) and caspase-cleaved cytokeratin 18 (CK-18 caspase). Mean AOC3 activities relative to baseline at Week 12: 90.4% (placebo; n = 32), 26.5% (1 mg; n = 16), 10.4% (3 mg; n = 16), 5.0% (6 mg; n = 16), 3.3% (10 mg; n = 32). These changes indicated that BI 1467335 dose-dependently inhibited AOC3 activity; ≥3 mg doses achieved >80% inhibition ( < 20% activity) at Week 4. At Week 12 following doses of BI 1467335 ≥ 3 mg, ALT and CK-18 caspase decreased dose-dependently. All tested BI 1467335 doses were well tolerated, with no clinically relevant treatment-emergent safety signals. BI 1467335 strongly inhibited AOC3 in participants with NASH, with doses ≥3 mg dose-dependently reducing the levels of liver injury biomarkers, ALT and CK-18. This trial was registered with ClinicalTrials.gov (NCT03166735) and the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT 2016-000499-83).

OBJECTIVE: In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells.
METHODS: Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco\'s Modified Eagle\'s Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent.
RESULTS: MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells\' migration capability and proliferation for 24 hours.
CONCLUSIONS: Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair.
Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis.
A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIβ but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51.
The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

OBJECTIVE: To investigate the GSDMD, CASP1, CASP4 and CASP5 expression in peripheral blood mononuclear cells of non-small cell lung cancer patients and analyze their clinical significance.
METHODS: 71 non-small cell lung cancer patients were selected as the study group and 50 healthy individuals as the control group. The GSDMD, CASP1, CASP4 and CASP5 expression in peripheral blood mononuclear cells of the two groups were detected by real-time fluorescence quantitative PCR. The GSDMD, CASP1, CASP4, CASP5 expression and their relationship with the clinical characteristics of the patients were analyzed.
RESULTS: Compared with the control group, the GSDMD, CASP4 and CASP5 expression in PBMCs of lung cancer patients was significantly higher(P < 0.05). Lymph node metastasis had significant difference with the CASP4 and GSDMD expression (P < 0.05); tumor volume had significant difference with CASP1 and CASP5 expression (P < 0.05). The areas under predictive ROC curve of the GSDMD, CASP1, CASP4, and CASP5 mRNA expression were 0.629(P < 0.05), 0.574(p > 0.05), 0.701(P < 0.05) and 0.628(P < 0.05), the sensitivity values were 84.5%, 67.6% 43.7%, and 84.3%;the specificity values were 42%, 52%, 84% and 64%, respectively.
CONCLUSIONS: GSDMD, CASP1, CASP4 and CASP5 gene expression are highly increased in PBMCs of non-small cell lung cancer patients and their expression are closely related to the clinical characteristics of patients. The early enhanced pyroptosis-related gene expression may be potential molecular markers for early diagnosis of non-small cell lung cancer.