Search Results (13,473)

Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide. Full article

The use of animal models to study mental illnesses, such as depression, requires proper standardization and extensive expertise. Achieving good construct, face, and predictive validity in depression models is quite challenging. Currently, only a few environmental models, mostly based on chronic stress, and a limited number of genetic models fulfill these criteria. In the quest for new antidepressants, initial screening tests are employed as a preliminary step in research. While these tests do not always meet the requirements of a disease model, they are useful for the early identification of substances that may have antidepressant potential, paving the way for further studies based on established models. This approach to discovering antidepressants was originally designed for traditional medications, which typically act by modulating serotonergic, noradrenergic, and dopaminergic systems, and require multi-week administration to produce a therapeutic effect. In contrast, the new antidepressant ketamine offers rapid therapeutic effects following a single dose and exhibits distinctive behavioral outcomes in both screening tests and animal depression models. These effects have inspired a new model for the search for rapid-acting ketamine-like antidepressants. This review presents the behavioral effects of ketamine and discusses the methodologies used in the search for novel rapid-acting antidepressant drugs (RAADs). Full article

Photodynamic therapy (PDT) exploits photoactivated materials that generate reactive oxygen species (ROS) to induce selective cancer cell death. Nitrogen-doped carbon dots (N-CDs) have emerged as promising photosensitizers owing to their favorable optical properties, while hybridization with titanium dioxide (TiO₂) may further enhance photoinduced ROS generation through improved charge separation. Here, we synthesized a series of N-CD/TiO₂ hybrid composites with varying TiO₂ content using a hydrothermal approach and systematically investigated the relationship between their physicochemical characteristics and biological activity. The hybrid materials were characterized by Fourier-transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, dynamic light scattering, and UV–visible spectroscopy. Among the formulations investigated, the composite containing 90% N-CDs and 10% TiO₂ (N-CDs10T) exhibited the smallest hydrodynamic diameter, a relatively narrow particle size distribution, favorable optical properties, and the strongest irradiation-dependent biological responses. Biological activity was evaluated in A549 lung adenocarcinoma and Kelly neuroblastoma cells. Under dark conditions, all formulations displayed relatively low intrinsic cytotoxicity. Following irradiation with 365 nm UVA light, however, N-CDs10T induced a marked increase in intracellular ROS production, activation of antioxidant response element (ARE)-dependent signaling, disruption of cell-cycle progression, apoptosis-associated cell death, and inhibition of cell proliferation and migration. Kelly cells exhibited greater sensitivity than A549 cells, with IC₅₀ values decreasing from 0.98 mg/mL under dark conditions to 0.52 mg/mL following irradiation. Collectively, these findings demonstrate that N-CD/TiO₂ hybrid composites function as photoresponsive materials that enhance ROS-mediated cytotoxicity upon light activation. Beyond demonstrating phototoxicity, this study systematically links hybrid composition with oxidative stress signaling and multiple cellular responses, providing a comprehensive biological evaluation of N-CD/TiO₂ hybrid materials. While additional studies are required to identify the predominant ROS, evaluate selectivity in non-malignant cells, and optimize activation at clinically relevant wavelengths, the present work establishes a proof of concept for the development of N-CD/TiO₂ hybrid composites for photodynamic applications. Full article

The cellular immune response underlying post-injury multiple organ dysfunction syndrome (MODS) remains incompletely described. We hypothesized that early perturbations in innate lymphocyte behavior are critical to the development of MODS in trauma patients. To address this, we examined lymphocyte subsets in a prospective cohort of major trauma patients recruited at a single major trauma hospital. Circulating lymphocytes were profiled with flow cytometry in serial samples drawn within the hyperacute (≤2 h) and acute (24 h, 72 h) post-injury periods. Plasma levels of specific mediators derived from innate lymphocytes were also measured in a larger cohort. We observed a marked hyperacute increase in circulating NK (particularly the CD56^dim subset) and Vδ1 cells that were associated with MODS or early mortality. Absolute counts of NK activation markers CD69 and NKG2D were also higher in patients with adverse outcomes, although the proportion of NK cells expressing NKG2D was reduced. Exploratory cluster analyses of NK activating and inhibiting receptors identified patient groups with differing injury characteristics and outcomes. In plasma, patients who developed MODS had significantly higher levels of NK-associated cytotoxic mediators and cytokines. Collectively, these data indicate a specific pattern of hyperacute NK cell activation after major trauma that is characterized by a pattern consistent with cytotoxic lymphocyte activation and is associated with clinical outcome. Full article

Background: Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss and is strongly driven by oxidative stress and inflammation. This study investigated the protective effects of cynarin against sodium iodate (NaIO₃)-induced retinal pigment epithelium (RPE) injury, focusing on the MAPK and NF-κB signaling pathways. Materials and Methods: Human ARPE-19 cells were exposed to NaIO₃, and cell viability was assessed by the MTT assay. Protein expression of MAPK components (p38, JNK, ERK) and the NF-κB pathway was analyzed by Western blotting, and pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) mRNA expression was measured by RT-qPCR. In vivo, NaIO₃-induced retinal degeneration in C57BL/6 mice was treated with cynarin (3 or 10 mg/kg) for seven days, and retinal changes were evaluated by fundus photography, fluorescein angiography, and OCT. Results: Cynarin preserved ARPE-19 cell viability without cytotoxicity. It significantly attenuated NaIO₃-induced p38 and JNK phosphorylation, IκB degradation, and NF-κB activation while downregulating IL-1β, IL-6, and TNF-α expression. In vivo, cynarin reduced drusen-like lesions, hyperfluorescent abnormalities, and retinal thinning, and dose-dependently suppressed ocular pro-inflammatory cytokines. Conclusions: Cynarin protects against oxidative stress-induced retinal degeneration by suppressing MAPK and NF-κB inflammatory signaling, representing a promising therapeutic candidate for preventing or delaying NaIO₃-induced dry AMD-like retinal injury. Full article

Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are heterogeneous intestinal disorders that pose significant threats to human health and share common pathological features, including intestinal mucosal barrier disruption and gut microbiota dysbiosis, in which fucosylation acts as a critical regulatory mediator. Fucosylation is a highly conserved post-translational glycosylation modification involving the enzymatic transfer of fucose residues to glycoproteins and glycolipids. This tightly regulated process plays essential roles in maintaining intestinal homeostasis, mediating host–microbiota interactions and regulating immune responses. This review adopts a physiology-to-pathology framework, delineating fucosylation’s operational principles in healthy intestines and its dysregulation in IBD and CRC. It summarizes the spatial distribution of fucosylation, its regulatory mechanisms, and its roles in disease pathogenesis, and also discusses its potential as a diagnostic biomarker and therapeutic target. Finally, this review highlights future research directions to bridge mechanistic insights with clinical translation, emphasizing the promise of fucosylation in the precision diagnosis and treatment of intestinal disorders. Full article

Depression (major depressive disorder, MDD) is a globally prevalent, highly disabling, and complex mental disorder whose pathogenesis has not been fully elucidated. In recent years, the role of the gut microbiota in depression via the “microbiota–gut–brain axis” (MGB axis) has attracted increasing attention. A large body of evidence indicates that the gut microbiota and its metabolites can engage in bidirectional communication with the central nervous system through three core pathways—neural, immune, and metabolic—thereby profoundly influencing the onset and progression of depression. This article reviews the specific mechanisms by which the gut microbiota affects depression through the aforementioned pathways, including regulating the balance of neurotransmitters (e.g., GABA and 5-HT), mediating neuroinflammatory responses, and adjusting the levels of metabolites such as short-chain fatty acids. This study aims to provide a theoretical basis for an in-depth understanding of the pathophysiological mechanisms of depression and the development of novel microbiota-based intervention therapeutic strategies. Full article

Bone remodeling is a dynamic and tightly regulated process that maintains skeletal homeostasis through a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Disruption of this balance contributes to the development of bone-related disorders, particularly osteopenia, osteoporosis and osteogenesis imperfecta, which weaken, deform, or cause fractures. Increasing evidence indicates that oxidative stress and chronic inflammation impair osteoblast functions while promoting osteoclast differentiation and activity. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with cytoprotective, antioxidant, and anti-inflammatory properties. Besides its primary role in cellular defense against oxidative stress and inflammatory damage, HO-1 has been shown to be involved in both osteoblast differentiation and osteoclastogenesis. Through its interaction with key regulatory systems, including the receptor activator of nuclear factor κB (RANK)–receptor activator of nuclear factor κB ligand (RANKL)–osteoprotegerin axis and redox-sensitive signaling pathways, HO-1 contributes to maintenance of optimal bone remodeling. The enzyme also plays a role in modulating metabolic processes in the bone. This review highlights the role of HO-1 in bone formation, bone resorption, and related pathophysiologic conditions. Furthermore, the therapeutic potential of HO-1 as a target for bone disorders is discussed. Full article

Chalkbrood disease caused by Ascosphaera apis threatens honey bee brood, yet the transcriptional mechanisms that coordinate antifungal immune responses in Apis cerana remain unclear. Here, RNA interference (RNAi) was used to examine two Dorsal homologs and selected dorsal1 transcript isoforms during larval infection. A. apis inoculation increased dorsal1, dorsal2, and five antimicrobial peptide (AMP) transcripts in larval midguts, consistent with an infection-associated humoral response. Gene-level RNAi produced selective transcript responses: dorsal1 knockdown was accompanied by reduced apidaecin and defensin1 expression, whereas dorsal2 knockdown was accompanied principally by reduced defensin1 expression. abaecin, defensin2, and hymenoptaecin transcripts were not significantly altered after dorsal knockdown under the tested conditions. Isoform-targeted RNAi of RNA9886, RNA9888, and RNA9890 was likewise associated with distinct AMP transcript responses, with RNA9888 and RNA9890 more closely associated with defensin1. These transcript-level data support selective and partially overlapping Dorsal-associated regulation during the A. cerana larval response to A. apis, while direct differences in Dorsal protein abundance or activity remain to be established. Full article

In proteins, tracts compositionally biased by a subset of amino acids are often linked to intrinsic disorder. Such compositionally biased regions (CBRs) are sometimes analyzed for ‘sequence complexity’ (‘information entropy’) despite being an unlikely substrate for natural selection per se, and therefore not functionally implicated. Here, an algorithmic strategy applying compositional bias detection was designed to capture the wide diversity of CBRs in structured protein domains (termed ‘sCBRs’), ranging from trihomopeptides to >200 residues, with conservation and partner binding as functional lenses. sCBRs are common, with about 1/4th of domains harbouring them, but domains dominated by sCBRs over >50% of their lengths are rare (~1 in 200). Despite general assumptions, very short sCBRs are highly significantly sequence-conserved, and associated with ligand binding, even when common nucleotide/phosphate-binding or glycine-rich cases are disregarded. However, regardless of length, ~50% of cases are evolutionarily dynamic, undergoing clade-specific expansion/contraction. Protein-binding associations include aversions for short (≤16 residues) valine-rich regions in protein interfaces, and enrichments of longer alanine-rich cases (>16 residues). Only ~5% of cases are (at least partly) in intrinsically disordered loops, and are significantly shorter than sCBRs generally. Functional implications of sCBRs are discussed with many examples. The sCBR data might help with hypothesis generation and protein design/engineering. Full article

Human immunodeficiency virus (HIV) is an enveloped virus with a remarkable capacity for genetic diversification, enabling rapid escape from host immune responses and therapeutic interventions. Despite extensive global efforts, the development of an effective vaccine has remained elusive owing to the virus’s high genetic variability and antigenic diversity. Consequently, considerable effort has been directed toward the development of therapeutic agents targeting viral entry, reverse transcriptase, integrase, protease, and more recently, capsid. Although antiretroviral therapy (ART) remains the cornerstone of HIV treatment, it is associated with challenges including drug resistance, adverse side effects, and limitations in access and affordability. Targeting viral entry offers distinct advantages by blocking infection at the earliest stage of the viral life cycle and enabling the neutralization of free virions, as well as Fc-mediated elimination of HIV-infected cells in some cases. This review highlights promising protein-based HIV entry inhibitors that have demonstrated efficacy in preclinical studies, and discusses ongoing efforts to optimize their valency, avidity, specificity, serum half-life, effector functions, and production platforms to improve their therapeutic potential and economic feasibility. Full article

Olive leaves represent a metabolically active tissue that plays an important role in plant responses to biotic stress. The present study comprised two independent experiments investigating biochemical responses of olive leaves to bacterial and fungal challenge under controlled conditions. Changes in primary metabolites (sugars, organic acids and free amino acids), phenolic compounds and lipid peroxidation were analyzed using chromatographic and spectrophotometric methods. In the bacterial experiment, pronounced differences were observed in primary metabolism. Tryptophan concentrations ranged from approximately 50 mg kg⁻¹ DW to more than 360 mg kg⁻¹ DW in ‘Istarska bjelica’, while sucrose concentrations reached up to 87 g kg⁻¹ DW, demonstrating cultivar-dependent differences in carbohydrate metabolism. Phenolic profiling showed that secoiridoids were the dominant phenolic class, with oleuropein concentrations exceeding 27 g kg⁻¹ DW across bacterial treatments. In the fungal experiment, amino acids showed greater variability than sugars and phenolic compounds, whereas MDA concentrations ranged from approximately 190 to 300 nmol g⁻¹ DW but did not differ significantly among pathogen treatments. Overall, the two experiments showed distinct patterns of metabolite variation associated with bacterial and fungal challenge. These findings contribute to a better understanding of cultivar-dependent metabolic responses and provide a basis for future studies of olive–microbe interactions. Full article

Microphthalmia, anophthalmia and ocular coloboma (MAC) are rare developmental eye disorders. Although over 100 causative genes have been identified, the molecular spectrum and genotype–phenotype correlations remain incompletely understood, particularly in genetically diverse populations. We set out to molecularly characterize MAC in the Israeli population. Forty-seven MAC-affected individuals from 43 unrelated families were enrolled. DNA of all probands was subjected to whole exome sequencing. The most common phenotype was microphthalmia (64% of patients). Definite or possible molecular diagnoses were achieved in 13/43 probands (30%) and involved 10 different genes (MFRP, SMO, GJA8, SOX2, RARB, TSPAN12, SHH, PTPN11, BEST1, and TP63). An in vitro splicing assay was used to explore the pathogenicity of a variant in the SMO gene. Following stringent filtering of exome data, 226 rare possibly pathogenic variants were identified in 218 genes not previously associated with MAC. The rate of molecular diagnosis achieved in this Israeli MAC cohort is similar to the reported range in other studies. The results further demonstrate the genetic heterogeneity of MAC, while supporting the involvement of complex inheritance and/or environmental factors in many of the cases. Further studies are required to reveal these underlying etiological factors, and to support the novel genotype–phenotype associations suggested here. Full article

Methyl-dependent restriction endonucleases are promising tools for analyzing eukaryotic DNA methylation patterns. However, quantitative assessment of their substrate specificity requires the determination of the kinetic parameters of enzymatic reactions. Here, we present a method for determining initial reaction rates based on fluorescent probes and real-time monitoring of changes in fluorescence intensity. Initial rates of methyl-dependent GlaI and BlsI restriction endonucleases were determined as the slope of the linear part of the kinetic curves, after which the Michaelis–Menten constants (K_M) and reaction rates (V_max) were calculated using nonlinear regression. For both enzymes, K_M values were determined for the first time, indicating a high affinity of the methyl-dependent restriction endonucleases for methylated sites. K_M values for fully methylated duplexes were in the range of (4.4–7.0)·10² nM for GlaI and 2.4–55 nM for BlsI. K_M values were significantly lower for the hemimethylated duplexes: (1.9–8.0)·10² nM for GlaI and (0.7–15.0)·10² nM for BlsI; and even lower for unmethylated duplexes: (27–46)·10² nM for GlaI and (0.28–23)·10² nM for BlsI. Maximum reaction rates varied within relatively narrow limits: V_max values were in range (4.5–19.5)·10⁻⁴ nM/s for GlaI and (1.4–18)·10⁻⁴ nM/s for BlsI, respectively. V_max values were depended weakly on the degree of methylation compared to the K_M. The proposed fluorescence method was applied to determine the kinetic parameters of methyl-dependent restriction endonucleases for the first time. It may serve as a simpler and more environmentally friendly alternative to traditional electrophoretic approaches that use radioactive labels. Full article

Skeletal muscle mitochondria possess the ability to autoregulate their health and functioning by the orchestration of mitochondrial quality control (MQC) pathways. This plasticity allows them to adapt to various stimuli, such as exercise. However, under pathological conditions, mitochondria can become dysfunctional, generating damage-associated molecular patterns (DAMPs), such as reactive oxygen species (ROS) and oxidized mitochondrial DNA (mtDNA). These DAMPs can launch an innate immune response, with consequences of widespread inflammation and atrophy. Integral to this is the NLRP3 inflammasome complex. Activation of the NLRP3 inflammasome results in maturation of caspase-1, which processes pro-inflammatory cytokines IL-1β and IL-18, as well as GSDMD. Consequently, the pore-forming GSDMD-N fragment induces pyroptosis, releasing mature IL-1β and IL-18. Exercise training is widely accepted as a potent mechanism to promote skeletal muscle health, particularly by remodeling the mitochondrial network and reducing the production of DAMPs. It has also been shown promote an anti-inflammatory milieu with the release of various myokines. Indeed, the potential of exercise to mitigate NLRP3 inflammasome-mediated inflammation and atrophy is promising. This review will examine the mechanisms underpinning inflammasome priming and activation, as well the effects of exercise, with an emphasis on the skeletal muscle. Full article