Search Results (255)

Leaf senescence is a developmental process that allows nutrients to be remobilized and transported to sink organs. Previously, papain-like cysteine proteases (PLCPs) have been found to be highly expressed during leaf senescence in different plant species. In this study, we analyzed active PLCPs in barley (Hordeum vulgare L.) leaves during the terminal stage of natural senescence. Anion exchange chromatography of protein extracts from barley leaves, harvested six weeks after anthesis, followed by activity assays using the substrates Z-FR-AMC and Z-RR-AMC, revealed a single prominent peak corresponding to active PLCPs. This hydrolytic activity was completely inhibited by E-64, a potent and irreversible inhibitor of cysteine proteases. Fractions enriched for PLCP activity were affinity-labeled with DCG-04 and subjected to SDS-PAGE fractionation, separating two major bands at 43 and 38 kDa. These bands were analyzed using tandem mass spectrometry, allowing the identification of eleven PLCPs. Identified enzymes belong to eight PLCP subfamilies, including CTB/cathepsin B-like (HvPap-19 and -20), RD19/cathepsin F-like (HvPap-1), ALP/cathepsin H-like (HvPap-12 or aleurain), SAG12/cathepsin L-like A (HvPap-17), CEP/cathepsin L-like B (HvPap-14), RD21/cathepsin L-like D (HvPap-6 and -7), cathepsin L-like E (HvPap-13 and -16), and XBCP3 (HvPap-8). Among the identified PLCPs, HvPap-6 was the most abundant. Peptides corresponding to HvPap-6 were identified in both the 43 kDa and 38 kDa bands in approximately the same quantity based on total spectral count. Thus, our results indicate that two active HvPap-6 isoforms can be isolated from barley leaves at late senescence. Full article

Background/Objectives: Melanoma cells enhance glycolysis and expand lysosomes to support energy metabolism, proliferation, and metastasis. However, lysosomal membrane permeabilization (LMP) causes cathepsin leakage into cytosol triggering cytotoxicity. This study investigated the antimelanoma effect of 2-deoxy-D-glucose (2DG), an inhibitor of glycolytic enzyme hexokinase-2, in combination with cathepsin C-dependent LMP inducer L-leucyl-L-leucine methyl ester (LLOMe) and cathepsin C-independent LMP-inducers mefloquine and siramesine. Methods: The viability of A375 and B16 melanoma cells and primary fibroblasts was measured by crystal violet. Apoptosis, necrosis, and LMP were assessed by flow cytometry; caspase activation, mitochondrial depolarization, superoxide production, and energy metabolism were analyzed by fluorimetry, and expression of cathepsins and hexokinase-2 was evaluated by immunoblot. Appropriate inhibitors, antioxidant, and energy boosters were used to confirm cell death type and mechanism. Results: LLOMe triggered LMP, mitochondrial depolarization, and mitochondrial superoxide production, while suppressing oxidative phosphorylation. 2DG suppressed glycolysis and, together with LLOMe, synergized in ATP depletion, caspase activation, and mixed apoptosis and necrosis in A375 cells. Inhibitors of lysosomal acidification, cysteine cathepsins, and caspases, as well as antioxidant and energy boosters, reduced 2DG+LLOMe-induced toxicity. Cathepsins B, C, and D were lower, while hexokinase-2 was higher in A375 cells than fibroblasts. Accordingly, 2DG exhibited lower while LLOMe exhibited higher toxicity against fibroblasts than A375 and B16 cells. However, mefloquine and siramesine induced stronger LMP in A375 cells than in fibroblasts and showed melanoma-selective toxicity when combined with 2DG. Conclusions: 2DG-mediated glycolysis inhibition in combination with lysosomal destabilization induced by mefloquine and siramesine, but not with non-selectively toxic LLOMe, may be promising antimelanoma strategy. Full article

Potato protein (PP) at concentrations of 0.025–0.3% was added to tropical fish surimi, including lizardfish (LZ) and threadfin bream (TB), and cold-water fish, namely Alaska pollock (AP) and Pacific whiting (PW), to examine its effect on proteolytic inhibition and surimi gel texture. Tropical fish surimi, particularly LZ, exhibited the highest degree of autolysis induced by endogenous proteases (p < 0.05), as evidenced by degradation of myosin heavy chain and tropomyosin. PP demonstrated a broad range of proteolytic inhibition activities against chymotrypsin, trypsin, papain, and cathepsin L, with chymotrypsin being the most susceptible. At a PP concentration of 0.3%, the highest autolytic inhibition was obtained in AP (72.24%), followed by LZ surimi (60.44%, p < 0.05). Egg white protein (EW) showed autolytic inhibitory activity at 14.50–50.52% in all species at 0.3%. Surimi gels with only 0.025% PP exhibited breaking forces and distance comparable to those with added 0.3% EW, regardless of the cooking regimes. In tropical surimi, PP at 0.3% increased the breaking force by 4.13–5.38-fold under a setting condition (as the best heating regime) compared with the control. At this concentration, PP decreased the whiteness of LZ and AP in the set surimi gels by 7.03% and 6.42% (p < 0.05), respectively, whereas its effect on TB and PW surimi was negligible. The study demonstrates that PP can be a promising alternative to EW to control proteolytic degradation and improve textural properties of cold-water and tropical surimi. Full article

Background/Objectives: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder marked by insulin resistance, hyperglycemia, systemic inflammation, and immune imbalance. This randomized, double-blind, placebo-controlled, crossover trial investigated the effects of Bifidobacterium animalis subsp. lactis TISTR 2591 (BA-2591), a probiotic strain isolated in Thailand, on metabolic, immunologic, and safety parameters. Methods: A total of 44 Thai adults (aged 35–65) with T2DM receiving metformin monotherapy were administered BA-2591 (1 × 10⁹ CFU/g/day) or placebo for 6 weeks, followed by a 4-week washout and crossover. Results: Compared to placebo, BA-2591 significantly attenuated fasting blood glucose elevation (Δ = +1.143 mg/dL vs. +12.570 mg/dL; p < 0.001), minimized the increase in insulin resistance (HOMA-IR: Δ = +0.567 vs. +0.980; p = 0.006), and enhanced β-cell function (HOMA-β: Δ = +6.791% vs. −8.313%; p < 0.001). It also elevated immunoglobulin levels (IgM: +150.300 mg/dL; IgG: +261.500 mg/dL; p < 0.001), reduced LDL-C (p = 0.009), and decreased cathepsin D activity (p = 0.005), with no significant changes in IL-6, adiponectin, MDA, hs-CRP, or body composition. No severe adverse effects were reported. Conclusions: BA-2591 was safe and demonstrated modest, adjunctive benefits for fasting glycemia and immunologic profiles over 6 weeks, without changes in body weight or fat mass. These findings support BA-2591 as a potential adjunct to standard care in early T2DM; larger and longer-duration trials are needed to define its effects on longer-term outcomes. Full article

A focused library of 1,2,4-thiadiazolidin-3,5-diones (THIA-1–10), previously characterized as hydrogen sulfide (H₂S) donors, was evaluated for inhibitory activity against cysteine proteases. We included two key cysteine proteases aiming at antiviral drug development—SARS-CoV-2 3CLpro (Mpro) and PLpro—alongside reference enzymes Papain and Cathepsin L. The compounds exhibited distinct selectivity profiles and inhibition mechanisms. The ability to act as covalent inhibitors of 3CLpro in the nanomolar range is of particular interest, with compounds THIA-6, -7, and -10 proving to be the most potent inhibitors of the series, and compounds THIA-1, -2, and -8 proving to be the most selective with respect to the other proteases. We explored the molecular bases of the observed activity profile of THIA-1–10 through computational studies, which supported and complemented the experimental findings, paving the way for future structure optimization. The results highlight that inhibitory potency depends not only on electrophilicity but also on the ability to access the catalytic cysteine within the active site. The dual functionality of THIA-1–10 as H₂S donors and selective cysteine protease inhibitors underscores its potential as a promising lead for therapeutic development. Full article

The study investigated the effects of chloride salts (control: 100% NaCl, salt mixture I: NaCl/KCl (50/50), salt mixture II: NaCl/KCl/CaCl₂ (40/40/20), salt mixture III: NaCl/KCl/CaCl₂/MgCl₂ (30/40/20/10)) on enzymatic activity, lipid oxidation, and volatile compounds in reduced-sodium salt pastırma, a Turkish dry-cured meat product. Lipid oxidation and instrumental color values were not affected by different salt mixtures. Salt mixtures II and III decreased pH value (p < 0.05). However, the mean pH value did not fall below 5.5 in any sample. The salt mixture treatment had significant effect on water activity, cathepsin B, and cathepsin B + L. In contrast, a_w value was under 0.90 in all treatments. The highest mean values for cathepsin B and B + L were determined in the control group with 11.69 ± 2.73 and 85.82 ± 12.65 U g⁻¹ × 10⁻³ dry matter, respectively. The closest correlation for lipolytic enzyme activities was determined by the mixture II and III groups, while a closer correlation was observed between salt mixtures I and III in terms of proteolytic enzyme activities. With regard to volatile compounds, there was a closer relationship between the control and salt mixture I. As a result, it can be concluded that salt mixture I in reduced-sodium salt pastırma showed closer results to the control group. Full article

The sea cucumber (Apostichopus japonicus) is highly susceptible to environmental stress during aquaculture, storage, and transportation, often resulting in autolysis and considerable economic losses. UVA irradiation and Vibrio splendidus infection were used to induce skin ulceration in A. japonicus. In this study, UVA irradiation and V. splendidus infection were used to induce skin ulceration, and the effectiveness of a compound inhibitor in delaying its onset was evaluated. The degree of skin ulceration in A. japonicus was evaluated. Body wall tissues were collected to measure the activities of self-digesting enzymes, AchE, cathepsin L, SOD, and CAT. Caspase-3 expression was also analyzed to assess apoptosis and tissue damage. The results indicated that soaking A. japonicus in the inhibitor composition significantly delayed the onset of skin ulceration. After 72 h of UVA irradiation, the skin ulceration in group Eg was 0.55%, which was significantly lower than that in groups Cg and Wg. In the V. splendidus infection model, group Eg showed a 4-day delay in the onset of skin ulceration, compared to group Cg. Enzyme activity and gene expression analysis revealed that the inhibitor composition significantly reduced self-digesting enzyme expression in the A. japonicus body wall, increased SOD and CAT activities, and inhibited Caspase-3 expression. This study provides valuable theoretical insights into controlling skin ulceration in A. japonicus during aquaculture, preservation, and transportation. Full article

Cathepsin L (CatL) is a critical protease involved in cleaving the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), facilitating viral entry into host cells. Inhibition of CatL is essential for preventing SARS-CoV-2 cell entry, making it a potential therapeutic target for drug development. Six QSAR models were established to predict the inhibitory activity (expressed as IC₅₀ values) of candidate compounds against CatL. These models were developed using statistical method heuristic methods (HMs), the evolutionary algorithm gene expression programming (GEP), and the ensemble method random forest (RF), along with the kernel-based machine learning algorithm support vector regression (SVR) configured with various kernels: radial basis function (RBF), linear-RBF hybrid (LMIX2-SVR), and linear-RBF-polynomial hybrid (LMIX3-SVR). The particle swarm optimization algorithm was applied to optimize multi-parameter SVM models, ensuring low complexity and fast convergence. The properties of novel CatL inhibitors were explored through molecular docking analysis. The LMIX3-SVR model exhibited the best performance, with an $R^2$ of 0.9676 and 0.9632 for the training set and test set and RMSE values of 0.0834 and 0.0322. Five-fold cross-validation $R_{5-fold}^2$ = 0.9043 and leave-one-out cross-validation $R_{loo}^2$ = 0.9525 demonstrated the strong prediction ability and robustness of the model, which fully proved the correctness of the five selected descriptors. Based on these results, the IC₅₀ values of 578 newly designed compounds were predicted using the HM model, and the top five candidate compounds with the best physicochemical properties were further verified by Property Explorer Applet (PEA). The LMIX3-SVR model significantly advances QSAR modeling for drug discovery, providing a robust tool for designing and screening new drug molecules. This study contributes to the identification of novel CatL inhibitors, which aids in the development of effective therapeutics for SARS-CoV-2. Full article

This study investigated the effects of integrating biofloc with microalgae on growth performance and immune gene expression in red tilapia (Oreochromis sp.). The experiment consisted of four treatments: C (Biofloc), T1 (Chlorella vulgaris and Nannochloropsis sp.; 1:1), T2 (Biofloc + Chlorella vulgaris and Nannochloropsis sp.; 1:1), T3 (Biofloc + Chlorella vulgaris and Nannochloropsis sp.; 2:1) in 500 L plastic tanks for 60 days. T2 and T3 exhibited the lowest ammonia and nitrite levels, respectively. T3 exhibited the highest chlorophyll a and chlorophyll b levels, while T2 showed the highest carotenoid content. T2 showed the highest weight gain (142 ± 0.7 g) and SGR (1.61 ± 0.02) and the lowest FCR (1.79 ± 0.009). T2 exhibited the highest gene expression levels in the intestine, with 7.8-fold upregulation of the cathepsin L (ctsl) gene, 3-fold upregulation of toll-like receptor 7 (tlr7), 6.7-fold upregulation of interleukin-1 b (il-1b), 4.7-fold upregulation of tumor necrosis factor-alpha (tnf-a), and 2.8-fold upregulation of metallothionein (mt). In the head kidney, the mt upregulation was highest in T3 (7.2-fold), while tnf-a and tlr7 upregulations were highest in T2 (5.9-fold and 5-fold, respectively). In the liver, the gene expressions were highest in T3, with 6.4-fold upregulation of mt, 5-fold upregulation of ctsl, 2.7-fold upregulation of tlr7, 3-fold upregulation of il-1b, and 5.4-fold upregulation of tnf-a. These results suggest a synergistic effect of algae and bacteria on immune and antioxidative capacity in red tilapia. Full article

Global warming is increasing in severity, affecting insects across various biological species. This study investigated the heat resistance ability of the small hive beetle (Aethina tumida) by studying gene expression under heat stress and showed that A. tumida exhibits strong heat resistance and transcriptomic plasticity under heat stress. RNA-seq analysis identified 547, 1127, and 866 differentially expressed genes (DEGs) at 38 °C, 42 °C, and 46 °C, respectively, compared to 25 °C. Among them, 16, 25, and 5 heat shock protein (HSP) genes were differentially expressed under the three heat stress conditions. Specifically, one HSP70 gene (Loc109602670) was consistently upregulated across all temperatures. Furthermore, the lysosome-related pathway was the top enriched pathway under heat treatments, with key genes such as lysosomal aspartic protease-like, cathepsin L1-like, and lipase 3-like significantly upregulated. Overall, these findings suggest that A. tumida exhibits transcriptomic plasticity under sublethal heat stress, and key HSP genes with genes from lysosome pathways are likely to contribute to heat resistance. This study provides novel insights into the molecular basis of thermotolerance in A. tumida, contributing to our understanding of how this invasive pest adapts to high-temperature environments. Full article

Introduction: Here we consider the collision of a genetic factor and an essential instigator in Alzheimer’s neuropathogenesis: (i) the Alzheimer’s gene (APOEε4), which downregulates lysosomal autophagy and induces synthesis of (ii) the instigator, interleukin-1β (IL-1β), which drives synthesis of βAPP for Aβ plaques and of MAPKp38 for phosphorylation of tau for formation of neurofibrillary tangles (NFTs), the two cardinal features of AD. Methods: RT-PCR, immunoblotting and immunohistochemistry techniques were used to assess the levels of IL-1β and its signaling cascade in ADε4,4, ε3,3, and age-matched controls (AMC3,3) in hippocampal regions of the brain. Results: IL-1β and its downstream signaling proteins TLR-2, MyD88, NFκB, COX-1, and COX-2 were greater in tissues from ADε4,4 than ADε3,3 or AMC3,3. Cathepsin B, D, and L levels, which play a pivotal role and are necessary for lysosomal autophagy, were lower in ADε4,4 than in ADε3,3 or AMC ε3,3. IL-1β and its downstream signaling cascade TLR-2, MyD88, NFκB, COX-1, and COX-2 expression levels were high in SH-SY5Y and T98G cells transfected with APOεE4. Conclusions: APOEε4 causes Alzheimer’s by downregulating autophagy, thus inducing IL-1β for Aβ plaque and neurofibrillary tangle formation. Full article

Introduction: Low-grade oncocytic tumor (LOT) is a recently described renal neoplasm characterized by indolent clinical behavior, a small nested architecture, and distinctive immunophenotypic features. Its distinction from other eosinophilic renal tumors, such as oncocytoma, eosinophilic chromophobe renal cell carcinoma (E-chRCC), and eosinophilic vacuolated tumor (EVT), can be challenging due to overlapping features. The L1 cell adhesion molecule (L1CAM) is being increasingly recognized as a potential diagnostic marker for LOT. Aims: To evaluate the diagnostic performance of L1CAM in distinguishing LOT from morphologically and immunophenotypically similar eosinophilic renal neoplasms. Methods: A total of 54 eosinophilic renal tumors (10 LOTs, 22 oncocytomas, 18 E-chRCCs, and 4 EVTs) were retrospectively collected from five academic institutions and reclassified according to the 2022 WHO criteria. All cases underwent histopathologic review and immunohistochemical analysis for CK7, CD117, GATA3, cathepsin K, and L1CAM. Results: L1CAM showed strong membranous expression in all LOTs (100%) and was negative in oncocytoma, E-chRCC, and EVT, yielding 100% sensitivity and specificity. Traditional markers exhibited overlapping patterns among tumor types. Conclusions: Our findings confirm L1CAM as a highly sensitive and specific marker for LOT, effectively distinguishing it from other eosinophilic renal neoplasms. Incorporating L1CAM into diagnostic panels may enhance accuracy, particularly in challenging cases. Full article

Interleukin-1 beta(IL-1β) is the major driving force in neuroinflammation. Here, we report on (i) the role of (IL-1β) in activating a signaling cascade that leads to proliferation and metastasis in glioblastoma cancer pathogenesis as well as (ii) the therapeutic role for IL-1 Receptor Antagonist (IL-1RA) and Tolcapone against untoward aspects of tumor pathogenesis. Here, we report that IL-1β treatment at 50 ng/mL for 48 h increased proliferation and metastasis by 30-fold (p ≤ 0.05), leading to the formation of clones of rapidly dividing cancer cells, leading to the formation of organized glial fibrillary acid protein (GFAP)-immunoreactive, clone-like structures with protruding spikes. Further, IL-1β treatment significantly increased the expression of mRNA levels of the IL-1β-driven pathway TLR-MyD88-NF-κB-TNFα and IL-6 (p ≤ 0.05). IL-1β also increased autophagy via elevation of mRNA and protein levels of cathepsin B, LAMP-2, and LC3B. In contrast, IL-1RA and Tolcapone inhibited this proliferation and the expression of these mRNAs and proteins, inhibiting autophagy by downregulating these autophagy proteins and inducing apoptosis by upregulating the expression of pro-apoptotic proteins like caspase-8 and caspase-3. IL-1β and its receptor can be targeted for successful anticancer therapy, as shown here with the use of IL-1RA and/or Tolcapone. Full article

Background: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease characterized by the proteolytic breakdown of the extracellular matrix. A clinical biomarker is needed for risk stratification and prognosis. Methods: In this single-center, 5-year observational study, 452 patients were enrolled: 343 with AAA (≥3 cm), and 109 controls (<3 cm). Plasma levels of six inflammatory proteins (human epididymis protein 4 (HE4), matrix metalloproteinase (MMP) 1 and 3, cathepsin S, chitinase 3 like-1, cathepsin S, and B-cell activating factor (BAFF)) were quantified at baseline. Patients were followed for a total of 5 years (60 months), and major adverse cardiovascular events (MACEs, defined as the composite of myocardial infarction, cerebrovascular attack, and cardiovascular-related death) were recorded. A Cox proportional hazard model was created using biomarker levels, age, sex, hypertension, hypercholesterolemia, diabetes mellitus, smoking status, and coronary artery disease to determine whether the baseline levels of these proteins were associated with MACEs over 5 years. Results: HE4, MMP-3, BAFF, and cathepsin S levels were significantly elevated in AAA patients compared to controls (all p < 0.05). HE4/WFDC2, MMP-3, and Chitinase 3-like 1 were significantly linearly associated with AAA diameter at baseline. With every normalized unit increase in HE4/WFDC2, MMP-3, and Chitinase 3-like 1, there was an increase in abdominal aortic diameter by 0.154 (95% CI: 0.032–0.276, p = 0.013), 0.186 (95% CI: 0.064–0.309, p = 0.003), and 0.231 (0.110–0.353, p < 0.001) centimeters, respectively. Among patients with AAA, elevated HE4 was associated with higher risk of MACEs (adjusted HR 1.249; 95% CI: 1.057–1.476; p = 0.009). Patients with high baseline HE4 (≥9.338 ng/mL) had significantly lower freedom from MACEs at 5 years (76.7% vs. 84.8%, p = 0.022). Conclusions: HE4 may be a potential prognostic biomarker that can be used to risk stratify patients with AAA to better personalize treatment strategies to reduce adverse events. Full article

Autolysis in sea cucumber has long been a threat to raw material storage and product processing. The involvement of endogenous cysteine protease in sea cucumber autolysis has been proved extendedly. However, as an essential part of the mechanism of autolysis, the role of its endogenous inhibitor has seldom been reported. To investigate the role of cysteine protease inhibitors in the early stage of hypoxic exposure-induced autolysis, a novel cystatin gene (SjCyt) belonging to the subfamily of cystatin C was cloned from Apostichopus japonicus by homology cloning and rapid amplification of cDNA ends. The affinity of SjCyt to cysteine protease (cathepsin L and cathepsin B) was investigated by molecular dynamics simulations. Pertinent metrics, including the root mean square deviation, radius of gyration, Gibbs free energy, binding free energy, and bond-forming frequency, showed that the conformation of SjCyt–SjCL was more stable and confirmed a stronger interaction of SjCyt with cathepsin L than with cathepsin B. Thus, cathepsin L (SjCL) was selected to further study its co-expression with SjCyt over a period of 9 h at an early stage of hypoxic exposure. Quantitative RT-qPCR revealed a ubiquitous transcriptional profile of SjCyt and SjCL in all the tested tissues, with the highest abundance in the dorsal epidermis, tube feet, and coelomocytes. Temporal transcription of them showed an overall up-regulated co-expression in the dorsal epidermis and tube feet. However, up-regulated SjCyt and down-regulated SjCL were observed at the protein level. Further immunofluorescence double labeling also found increased staining of SjCyt and SjCyt–SjCL complexes and decreased SjCL. Additionally, recombinant SjCyt was prepared and demonstrated an evident autolysis-inhibiting effect. The results of this study indicated that the anti-autolytic regulation of SjCyt functions at the very early stage of hypoxic exposure, exerting effects at both the transcriptional and translational levels. The above finding offers new insights into the mechanisms of sea cucumber autolysis. Full article