Search Results (1,234)

Identification of protease cleavage sites is essential for understanding biological regulation and disease mechanisms, yet many predictive approaches rely on annotated substrates and curated databases, limiting performance for poorly characterized proteases. We present PhageScout, a framework for database-independent generation of protease-specific features to predict cleavage sites using de novo experimental substrate phage display screening. We screened a randomized 5-mer phage display library against two neutrophil serine proteases (cathepsin G, elastase). Cleaved peptides generated position weight matrices (PWMs) and peptide enrichment scores to evaluate cleavage-site likelihood across substrate sequences. Sequence-derived scores were integrated with structural features, including accessibility and flexibility, using XGBoost classification models. Performance was benchmarked against annotated cleavage sites from the MEROPS peptidase database as reference data. Phage-derived PWM scores alone captured protease preferences and discriminated cleavage sites from background sites. Without model fitting, PWM scores achieved an area under the curve (AUC) of 0.756 (95%CI: 0.714–0.797) (cathepsin G) and 0.787 (95%CI: 0.753–0.821) (elastase). Combining broad and specific phage-derived scores improved cathepsin G prediction (AUC = 0.783), whereas this improvement was not observed for elastase. Compared to only phage-derived features, XGBoost models integrating phage sequence and structural features provided modest gains for elastase (AUC = 0.775 to 0.806), with phage-derived features ranking among the strongest predictors, but not cathepsin G (AUC = 0.702 to 0.710). Our findings demonstrate that PhageScout can use experimentally derived cleavage signatures to generate protease-specific predictive features and prioritize protease cleavage sites, providing a framework that warrants further validation across diverse proteases and biological contexts. Full article

Endothelial dysfunction is a key mechanism linking obesity, metabolic disturbances, and cardiovascular disease, contributing to the development and progression of atherosclerosis and other vascular complications. This review provides a comprehensive overview of the molecular mechanisms underlying endothelial dysfunction in obesity and discusses current diagnostic approaches and therapeutic strategies aimed at restoring vascular homeostasis. The available evidence indicates that chronic inflammation, oxidative stress, insulin resistance, reduced nitric oxide bioavailability, increased reactive oxygen species production, and dysregulated adipokine signaling play central roles in endothelial impairment. Recent advances in functional vascular assessment, circulating biomarkers, and imaging techniques have improved the early identification of endothelial dysfunction and cardiovascular risk. Current therapeutic strategies include pharmacological agents, such as glucagon-like peptide-1 receptor agonists, sodium-glucose co-transporter 2 inhibitors, metformin, and dipeptidyl peptidase-4 inhibitors, together with lifestyle interventions based on healthy dietary patterns and regular aerobic and resistance exercise. These approaches improve glycemic control, reduce inflammation and oxidative stress, enhance endothelial function, and contribute to cardiovascular protection. Overall, the evidence supports an integrated and personalized management strategy targeting both metabolic and vascular abnormalities to reduce cardiovascular risk and improve long-term clinical outcomes in individuals with obesity. Full article

Dual-protein systems combining whey protein isolate (WPI) and soy protein isolate (SPI) offer complementary nutritional benefits but are limited by the presence of major allergens. Lactic acid bacteria (LAB) fermentation provides a promising strategy to mitigate this limitation. In this study, Lacticaseibacillus paracasei JM053, selected from 13 LAB strains based on phenotypic screening, significantly reduced the in vitro allergenicity of the dual-protein system, increasing the IgE-binding inhibition rate to 48.75%. Whole-genome sequencing and characterization of JM053 revealed a comprehensive proteolytic system, including the proline-specific peptidase genes pepX and pepQ, which may contribute to the degradation of allergenic peptide sequences. Combined with in silico bioinformatic analysis, potential cleavage sites within the linear epitopes of the dual-protein system were predicted based on the substrate specificity of the identified proteases, offering a testable hypothesis for the strain’s mechanism of action. In addition, in vitro safety assessment and genomic analysis supported the safety potential, stress tolerance, and probiotic characteristics of JM053. Collectively, this study provides a valuable candidate strain for the development of hypoallergenic dual-protein products and offers preliminary genomic insights into LAB-mediated allergenicity reduction. Full article

A secreted protein toxin encoded by a satellite dsRNA-enabled dissection of the genetic control of replication and expression of a single-segment dsRNA virus, the first Totivirus, L-A. Among the then-novel findings were i. “head-full replication”, ii. a supposedly forbidden “T = 2” capsid symmetry based on an asymmetric dimer, iii. a host N-acetyltransferase whose modification of the coat protein is necessary for packaging, iv. Kex1 and Kex2 pro-toxin peptidases leading to discovery of the pre-pro-insulin processing enzymes, and v. specific viral (+) strand sites/structures needed for RNA packaging and (-) strand synthesis. L-A viral (+) strands made in the particle are extruded to the cytoplasm. Those destined for translation are 5′ 7meGMP-capped by a coat protein activity that steals the cap from cellular mRNAs. (+) strands destined for encapsidation in new coats are not capped. Three host-encoded anti-viral systems were found, one based on blocking translation of the viral non-polyA mRNAs (Ski2,3,8 complex), another a 5′->3′ exoribonuclease specific for uncapped molecules (such as the viral (+) strands)(Ski1/Xrn1), and the third a mitochondrial nuclease released in cells undergoing meiosis/sporulation (Nuc1). All of these systems protect cells from virus-induced pathology and have clear animal homologs. The 3′ polyA of yeast mRNAs is dispensable for translation in ski2Δ slh1Δ cells, and such cells are healthy unless the L-A and M dsRNAs are present, suggesting that this polyA is primarily a device allowing cells to distinguish viral and cellular mRNAs. We suggest that the ribosome-associated Ski2,3,8 proteins block 60S subunit joining on polyA⁻ mRNAs. Recent evidence of roles for other cellular components controlling viral expression and replication suggests that yeast viruses will continue to be a fertile area for study of viral pathogenesis and host anti-viral systems. Full article

The peptidoglycan (PG) sacculus of Gram-negative bacteria is continuously reorganized by a diverse enzymatic repertoire, including lytic transglycosylases, endopeptidases, carboxypeptidases, amidases and LD-transpeptidases, that operate alongside PG synthases to maintain envelope integrity throughout the cell cycle. This remodeling machinery has been extensively characterized in the context of growth and division and it is now emerging also as a key determinant of bacterial survival under non-growing and stress conditions. This review summarizes current knowledge on PG remodeling in Gram-negative bacteria, with emphasis on its regulation during stationary phase, environmental stress, and outer membrane perturbation. How these remodeling pathways, characterized by increased 3–3 cross-linking and enhanced PG–outer membrane coupling, contribute to survival under β-lactam exposure and how related enzymatic configurations can give rise to antibiotic tolerance and resistance is also discussed. Finally, recent progress in targeting PG remodeling enzymes, particularly lytic transglycosylases and peptidases, as adjuvant strategies to potentiate existing antibiotics are reviewed. In summary, PG remodeling represents a mechanistically validated but still underexploited target for addressing antibiotic tolerance and resistance in Gram-negative pathogens. Full article

Hymenopteran venoms contain diverse proteins and peptides that shape envenomation, defense, predation, and allergic responses. Honeybee venom from Apis mellifera is well characterized, while molecular resources for the Asian giant hornet Vespa mandarinia remain less curated. We compared protein- and peptide-fraction LC-MS/MS identification datasets from V. mandarinia venom (VM-V) and A. mellifera venom (AM-V), supported by a de novo V. mandarinia transcriptome-derived database. Protein-level identification yielded 197 protein groups in VM-V PRO and 164 protein groups in AM-V PRO. AM-V contained well-recognized honeybee venom components, including phospholipase A2, hyaluronidase, venom acid phosphatase, venom dipeptidyl peptidase 4, melittin precursor, mast cell degranulating peptide precursor, secapin, and allergen Api m 6. VM-V PRO contained transcriptome-supported candidate venom-associated proteins, including venom dipeptidyl peptidase 4-like, hyaluronidase-like, venom allergen 5-like, serine protease-like, apolipophorin-like, and hexamerin-like entries. Peptide-fraction annotation was strongest in AM-V PEP, led by melittin precursor, whereas VM-V PEP remained largely unannotated. The study focuses on identification, annotation, and hypothesis-generating functional summaries, not replicate-level differential abundance. GO, KEGG, and STRING analyses organized functional annotation patterns and prioritized candidate protein groups. These data provide an identification-based comparative venomomics resource for VM-V and AM-V and a foundation for targeted validation of candidate V. mandarinia venom-associated components. Full article

Type 2 diabetes mellitus (T2DM) is often linked to metabolic dysfunction-associated steatotic liver disease (MASLD), a metabolic liver condition defined by excessive lipid deposition within hepatocytes and is frequently accompanied by increased circulating liver enzyme levels. Dipeptidyl peptidase-4 (DPP-4) inhibitors, such as linagliptin, may exert pleiotropic effects on hepatic metabolism; however, their effects on liver enzyme profiles remain uncertain. This systematic review and meta-analysis aimed to evaluate the impact of linagliptin on liver enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT), in patients with T2DM. A systematic literature search was performed across five electronic databases up to 11 May 2026 following the PRISMA 2020 guidelines. Randomized controlled trials and cohort studies were included, and pooled mean differences (MDs) were calculated using random-effects models. Eight studies involving 1262 participants were analyzed. Linagliptin was associated with a modest reduction in AST (MD −1.58 U/L, 95% CI −2.85 to −0.31) with low heterogeneity, whereas the change in ALT was not statistically significant (MD −1.86 U/L, 95% CI −4.14 to 0.42), and substantial heterogeneity was observed. No significant effects were observed for GGT, while evidence for ALP was limited to a single study and was insufficient to determine the effect of linagliptin. Overall, linagliptin demonstrated limited and inconsistent effects on liver enzyme profiles. Full article

The kallikrein–kinin system (KKS) and the kallikrein-related peptidase (KLK) family are interconnected proteolytic networks that regulate inflammatory signaling, vascular permeability, extracellular matrix remodeling, and tissue barrier dynamics. Beyond their classical vasoactive and inflammatory functions, accumulating evidence indicates that kinin peptides, including bradykinin, Lys-bradykinin, and their des-Arg⁹ metabolites, together with selected KLKs, modulate cell–cell and cell–extracellular matrix adhesion. Through B1 and B2 kinin receptor activation, the KKS influences endothelial adhesion molecule expression, leukocyte integrin activation, neutrophil trafficking, focal adhesion kinase/Src signaling, cytoskeletal remodeling, and matrix metalloproteinase activity. In parallel, KLKs directly reshape the adhesive microenvironment by cleaving junctional proteins, including E-cadherin and desmosomal components, and extracellular matrix substrates such as fibronectin, laminin, vitronectin, fibrinogen, and collagens. These coordinated actions affect epithelial and endothelial barrier integrity, leukocyte transmigration, angiogenesis, fibrosis, epithelial–mesenchymal transition, tumor cell migration, invasion, and metastatic dissemination. This review critically summarizes current evidence linking KKS and KLK activity to adhesion-dependent processes in inflammation and cancer, emphasizing how proteolytic signaling may either preserve tissue homeostasis or promote pathological barrier disruption depending on cellular context, receptor expression, protease activity, and microenvironmental cues. Understanding these mechanisms may refine the identification of adhesion-related biomarkers and support the development of targeted therapeutic strategies for inflammatory disorders, fibrotic remodeling, and cancer progression. Full article

As the prevalence of type 2 diabetes increases rapidly, the demand for natural origin dipeptidyl peptidase-IV (DPP-IV) inhibitors with fewer side effects is increasing. In this study, protein-rich Limnospira platensis was investigated as a source of bioactive peptides to enhance its value as a functional food ingredient. Hydrolysates were prepared using three food-processing proteases, individually and in two-step combinations, followed by in silico analysis and peptide identification via liquid chromatography–tandem mass spectrometry. Subsequently, the thermal stability, gastrointestinal resistance, and intestinal permeability (using Caco-2 cells) of the identified peptides were evaluated. It was revealed that the Orientase 22BF digest exhibited high DPP-IV inhibitory activity. From the digest, three novel peptides—SPSPN (IC₅₀ = 144.1 ± 2.2 μM), VPSV (IC₅₀ = 93.6 ± 5.8 μM), and IPIGG (IC₅₀ = 13.4 ± 2.3 μM)—were identified, exhibiting DPP-IV inhibitory potencies comparable to or higher than previously reported Limnospira-derived peptides. Although VPSV exhibited low epithelial permeability (P_app = 4.02 ± 0.69 × 10⁻⁸ cm/s), it remained stable under simulated gastrointestinal digestion conditions, suggesting potential local luminal inhibitory activity within the small intestine. Overall, these findings highlight Limnospira-derived VPSV as a promising functional ingredient candidate with high bioactivity and digestive stability. Full article

Endosymbiosis is a necessary process in eukaryotic evolution, yet the steps leading to full integration remain poorly understood. Trypanosomatids provide a comparative framework to study this transition through two independent systems representing distinct levels of integration. Strigomonadinae comprises a subfamily derived from a single ancestral acquisition, which maintains a single cytosolic bacterium closely synchronized with the host cell cycle. In contrast, Novymonas esmeraldas harbors multiple bacteria within symbiontophorous vacuoles, presenting a more limited coordination of division. The generation of aposymbiotic strains enables direct functional assessment of endosymbiont contributions to host biology. Comparative analyses reveal strong metabolic interdependence, with the endosymbiont supplying essential amino acids, heme, vitamins, and purines, while relying on the host for key metabolic resources including non-essential amino acids, cofactors, energy metabolism, and lipid biosynthesis. Beyond metabolism, the endosymbiont impacts protein homeostasis and cellular regulation, modulating proteolytic pathways such as GP63, cysteine peptidases, and the ubiquitin–proteasome system, as well as host networks involving kinetoplast-associated proteins (KAPs). Host-encoded endosymbiont-targeted proteins (ETPs) further mediate endosymbiont positioning, organization, and coordinated maintenance and division. Here, we critically synthesize current knowledge on mEHTs using a comparative framework based on interconnected dimensions of host–endosymbiont integration. Full article

We investigated the effects of senescence and hypoxia on the transcriptome and secretome of the colon cancer cell, HCT-116, in an in vitro model. Senescence was confirmed using SA-β Gal staining and the expression of p53 and p21 proteins, and hypoxia using HIF-1α protein. Control (CN) and senescent (SN) cells were exposed to normoxia or hypoxia, control hypoxia (CH), and senescent hypoxia (SH). Senescence (SN, SH) enhanced the expression of kallikrein-related peptidases, TPp53, p21, optineurin, lipocalin, ADH-1, and stratifin by several folds. Stratifin, with tumor suppressive functions, was upregulated in senescent cells under normoxia but not in hypoxia. Hypoxia (CH and SH) upregulated the expression of many glycolysis genes, especially HK, PFK, aldolase, PDH kinase, and LDH-A. Mitochondrial RNAs (tRNA and rRNA) were increased in SH compared to CH. Significant increases in the secretion of IL-1α, endothelin, bFGF, HB-EGF, PDGF-AB, CCL-5, 7, 22, and CXCL-1 and 8 were observed in SN and SH. VEGF-A, VEGF-C, and TNF-β secretion increased, while PLGF, TGF-α, IL-27, GM-CSF, and M-CSF decreased under hypoxic (CH and SH) conditions. Thus, senescence and hypoxia contribute to cancer cell senescence pathophysiology by regulating the cellular transcriptome and secretome and by both positive and negative feedback mechanisms. Full article

Background and Objectives: Sodium–glucose cotransporter-2 (SGLT2) inhibitors provide cardio-renal protection in type 2 diabetes, but tolerability-related discontinuation is common, creating a need for effective replacement therapy. Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used as follow-on agents, yet the clinical course after such a switch has not been systematically characterized, and whether individual DPP-4 inhibitors differ in efficacy in this setting is unknown. Materials and Methods: We conducted a single-center retrospective cohort study at Chungnam National University Hospital (Daejeon, Republic of Korea) between January 2013 and January 2025, including 117 adults with type 2 diabetes who switched from an SGLT2 inhibitor to a DPP-4 inhibitor and met pre-specified criteria for medication stability and follow-up. The primary outcome was the change in glycated hemoglobin (HbA1c) from baseline to 3 months; secondary outcomes were changes in body weight, body mass index, blood pressure, and renal parameters. A pre-specified subgroup analysis compared teneligliptin (n = 60) with other DPP-4 inhibitors as a class (n = 57). Results: In the overall cohort, body weight rose by 0.7 kg and systolic blood pressure by 4.3 mmHg at 3 months (both p < 0.05), whereas HbA1c was unchanged (p = 0.157). In the subgroup analysis, HbA1c fell significantly with teneligliptin (−0.36%; 95% confidence interval, −0.62 to −0.10; p = 0.009) but not with other DPP-4 inhibitors (+0.13%). The between-group difference was −0.49% (95% confidence interval, −0.83 to −0.15; p = 0.005) and persisted after adjustment for baseline HbA1c and estimated glomerular filtration rate (p = 0.007). Conclusions: Switching preserved overall glycemic control at 3 months but produced modest, anticipated increases in body weight and blood pressure. Teneligliptin was associated with a greater HbA1c reduction than the pooled group of other DPP-4 inhibitors; this association persisted after adjustment for the two available baseline covariates but could not be adjusted for diabetes duration, medication adherence, diabetic complications, or other unmeasured factors. Because confounding by indication and other residual confounding cannot be excluded in this short-term, single-center analysis, this finding is hypothesis-generating only and requires confirmation in adequately powered prospective head-to-head trials. Importantly, this study evaluated only the glucose-lowering effect of the switch; because the cardio-renal protection of SGLT2 inhibition is not reproduced by DPP-4 inhibitors, a DPP-4 inhibitor should be regarded as an unavoidable substitute when an SGLT2 inhibitor cannot be maintained rather than a therapeutically equivalent replacement, and preserved HbA1c at 3 months does not establish clinical equivalence between the two strategies. Full article

Carcinine is an imidazole dipeptide with potent antioxidant and antiglycation properties, although its chemical synthesis currently relies on severely environmentally harmful processes. In this work, a multi-enzyme cascade biotransformation system comprising 4′-phosphopantetheinyl transferase and non-ribosomal peptide synthetase was constructed. To overcome the limitations arising from stochastic spatial distribution and suboptimal mass transfer associated with independent enzymes, a fusion protein strategy was adopted. The two enzymes were fused via a flexible genetic linker within plasmid pET28a-SFP-L-Ebony, which enabled robust soluble expression in Escherichia coli. Concurrently, the endogenous peptidase genes (pepA, pepB, pepD, and pepN) were systematically knocked out using CRISPR/Cas9-mediated gene editing. This quadruple protease-deficient strain (designated SFP-L-Ebony-ΔpepABDN) effectively suppressed product degradation. Subsequent optimization revealed that optimal catalytic performance occurred at 25 °C and pH 7.0. The highest biotransformation efficiency was achieved using 15 g/L crude enzymes, in the presence of 2 mM ATP and 10 mM MgCl₂. Through a fed-batch substrate feeding strategy in a 50 mL reaction system, the final carcinine titer reached 7.0 g/L after 48 h. This study, therefore, provides an efficient and sustainable technological pathway for the green biomanufacturing of carcinine as well as other high-value dipeptides. Full article

Background: Hereditary angioedema (HAE) is a rare, potentially life-threatening disease in which delayed recognition and inappropriate medication use may result in preventable morbidity and mortality. We aimed to assess physicians’ knowledge regarding HAE-related triggers, clinical features, and management strategies across multiple medical specialties. Methods: This single-center, cross-sectional survey was conducted among 350 physicians at a tertiary training and research hospital. A structured electronic questionnaire assessed knowledge of HAE pathophysiology, diagnosis, medication-related triggers, and management. A predefined composite knowledge score (0–14) was calculated. Results: Although self-reported familiarity with HAE was high, overall disease-specific knowledge was limited. Awareness of critical medication-related triggers—including angiotensin-converting enzyme inhibitors, dipeptidyl peptidase-4 inhibitors, and estrogen-containing therapies—was low across all specialties, with no significant between-group differences. Substantial knowledge gaps were identified in the recognition of clinical features, diagnostic evaluation, and acute management. In multivariable analysis, prior clinical exposure to HAE patients was the only independent predictor of higher knowledge scores (B = 1.097, p = 0.001), whereas specialty group, gender, and years of professional experience were not independently associated with knowledge scores. However, the regression model explained only a small proportion of the variance in knowledge scores. Conclusions: Significant gaps in clinically relevant HAE knowledge persist among physicians from multiple medical specialties. Prior clinical exposure was associated with higher knowledge scores, whereas specialty group, gender, and years of professional experience were not independently associated with physician knowledge. Targeted, practice-oriented educational interventions focusing on medication-related triggers and acute management may help bridge these knowledge gaps, complement experiential learning, and ultimately enhance patient safety. Full article

Most patients with solid tumors do not respond to immune checkpoint blockade, and inadequate T cell infiltration of the tumor parenchyma is the dominant mechanism of primary resistance. Oncolytic viruses address this problem by a distinct route: they replicate selectively within tumor cells, produce immunogenic cell death, and convert infected cells into local sources of any encoded transgene. Most armed designs to date have carried cytokine or checkpoint-antibody payloads, and chemokines have attracted comparatively little attention despite bearing directly on the trafficking bottleneck. This review synthesizes the preclinical literature on chemokine-armed oncolytic viruses across three receptor axes: CXCR3 (CXCL9, CXCL10, CXCL11), CCR5 (CCL5/RANTES), and CCR7 (CCL19). The accumulated evidence indicates that therapeutic outcome depends less on the chemokine payload itself than on the interaction between payload and viral backbone. CXCL11 outperforms its sister CXCR3 ligands not through intrinsic potency but because it is non-redundant with the endogenous chemokines induced by vesicular stomatitis virus and vaccinia, and because it largely escapes proteolytic cleavage by dipeptidyl peptidase 4 (DPP4). CCL5 has shown the most consistent activity in dual-payload designs that pair chemotaxis with a T cell survival cytokine such as IL-15. CCL19, which addresses lymphoid organization rather than effector recruitment, rests on a single published construct. One evidence gap is central: no head-to-head comparison of chemokine payloads within a single viral platform has been published. We therefore propose a translational decision framework that aligns chemokine selection with the immune contexture of the target tumor. Full article