Nucleoside analogs are successful in treating viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when embedded in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM structures demonstrate that SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) preferentially incorporates remdesivir triphosphate (RTP), outcompeting 10-fold excess ATP; however, successive RTP incorporations are disfavored when ATP is present. The RdRp structures demonstrate that 1'-cyano-imposed conformational restriction of the remdesivir:UMP base-pair is resistant to translocation, reducing successive RTP incorporations. The S759A mutant confers RTP resistance. We show that the mutation switches the RdRp preference to ATP; RTP is incorporated only at 10-fold excess to ATP. The structures of S759A RdRp reveal that the primer 3'-end nucleotide repositioning and its altered ribose-ring conformation contribute to RTP resistance. These findings have implications for designing non-obligate nucleoside analogs with different inhibition mechanisms.

Cyphenothrin, a type II synthetic pyrethroid, is widely recommended by the World Health Organization (WHO) for control programs for mosquitoes such as Culex quinquefasciatus. This cosmopolitan mosquito species plays a pivotal role in transmitting several neglected tropical diseases, including lymphatic filariasis, West Nile virus, Japanese encephalitis, and so on. Besides treatment, vector control programs heavily rely on insecticides, leading to varying resistance due to prolonged exposure. The present study assessed the susceptibility status of field-collected Cx.quinquefasciatus larvae from two filarial endemic districts of sub-Himalayan West Bengal against cyphenothrin. All the wild populations have been exposed to their respective lethal concentration doses. Monooxygenase levels and carboxylesterase activities were quantified both in the wild and cyphenothrin-exposed populations. The expression profiles of selected carboxylesterase (esterase A and esterase B) and CYP450 (CYP6AA7, CYP9J40, and CYP9J45) genes were also assessed. Molecular docking analyses were performed to evaluate the binding affinities and interaction mechanisms of cyphenothrin with the selected proteins. Most of the populations showed elevated resistance status to cyphenothrin. In enzymatic assays, elevated levels of monooxygenases and carboxylesterases are found both in the wild and cyphenothrin-exposed populations. The expression profiles of selected CYP450s and carboxylesterase genes indicated an upregulation in the studied wild and exposed populations. Furthermore, molecular docking simulations corroborated the strong binding affinities of cyphenothrin to these detoxification enzymes. These findings suggest the involvement of a few detoxifying genes in resistance development against cyphenothrin in Cx.quinquefasciatus mosquitoes and advocates the urgent need for regular resistance monitoring, molecular surveillance, gene knockdown studies, and incorporation of synergists into integrated vector management frameworks to sustain effectiveness of mosquito control programs.

Thyroid hormone (TH) homeostasis depends on the coordination of several key events to maintain proper local TH signaling, including iodide uptake, hormone synthesis, metabolism, and elimination. Three selenoprotein isoenzymes, deiodinases 1-3 (DIO1-3), are essential components of TH metabolism, and their activities have been identified as relevant endpoints regarding the screening of compounds influencing the TH system. Given the importance of DIO2 as the key enzyme for local activation of the prohormone T4 to the active T3 in various tissues, and limited data on selective biochemical DIO2 inhibition, there is a clear need to identify potent and selective DIO2-inhibiting compounds. Human-recombinant DIO2 enzyme pools were prepared from HEK293 cells overexpressing DIO2 and used as a robust enzyme source for the development, optimization, and semiautomated miniaturization of a nonradioactive DIO2 high-throughput screening (HTS) enzyme assay for the identification of DIO2-selective small molecule inhibitors. LT4 was used as substrate, and enzymatic release of iodide was colorimetrically quantified by the iodide-catalyzed Sandell-Kolthoff reaction. Eight comprehensive small molecule libraries were screened, covering ∼1/5 of the synthetic chemicals currently registered, natural products, as well as FDA-approved drugs. A total of 59,928 compounds were first screened at a single 10 µM concentration, followed by a validation screen to confirm the primary hits. Subsequently, DIO isoenzyme selectivity and cytotoxicity were evaluated. Utilizing this highly reproducible and robust HTS test system with a determined median Z'-factor of 0.70 identified 356 primary inhibitory hits. Concentration-response experiments verified 17 potent inhibitors, further characterized regarding their DIO isoenzyme selectivity and cytotoxicity. Six potent DIO2-selective inhibitors, including two FDA-approved drugs and various novel pan-DIO inhibitors, for example, the fungicide fluazinam, were identified. Specific DIO2 inhibitors, such as the FDA-approved drugs racecadotril and ibrutinib and the tyrosine kinase inhibitor rociletinib, might serve as a future toolbox for reversible pharmacological interference with the local provision of DIO2-generated T3 from T4 during development, tissue regeneration, and various DIO2-dependent metabolic processes. Furthermore, they can serve as reference compounds for the development and validation of regulatory in vitro tests. Identified FDA-approved drugs warrant a closer look at potential disturbances of local TH availability.

Structural and functional investigation of RD3-GCAP1 interaction in retinal photoreceptors under normal and disease conditions.

In its simplest representation, apoptosis is a two-step peptidase cascade in which initiator caspases (caspases-8, -9, and -10) activate executioner caspases (caspases-3, -6, and -7). Although many intricacies exist-such as the proteolysis of initiator caspases by executioner caspases, which further regulates their activity-apoptotic pathways ultimately converge on the activation of caspase-3, the most proteolytically proficient member of the family. This central role has led to the development of numerous enzymatic assays to detect caspase-3 activity, its activation, and the cleavage of hallmark substrates, such as poly(ADP-ribose) polymerase 1 (PARP1). Like other members of the caspase family, caspase-3 minimally recognizes a five-amino-acid motif, usually located in a well-exposed loop within its substrates. Caspase-3 cleavage-site motif preferences have been systematically studied using peptides but not proteins. Here, we use a simple recombinant protein-based double brilliance bioluminescence resonance energy transfer (BRET2) biosensor assay for caspase-3 that enables robust and quantitative kinetic measurements in vitro. We used the biosensor by assessing its ability to distinguish between optimal and suboptimal cleavage-site motifs using a panel of BRET2 biosensors incorporating all 20 amino acids at the critical P4 position (the fourth residue N-terminal to the scissile bond). Except for arginine and lysine, we successfully determined the catalytic specificity (kcat/KM) for all other residues at P4. Notably, the range of proteolytic efficacies observed with BRET biosensors was significantly narrower than that previously reported using peptide-based libraries. Finally, we confirmed the biosensor's utility in apoptotic cells, demonstrating its robustness and broad applicability.

Integrated UiO-66-bacterial composite for methylene blue removal: Mechanistic insights and detoxification.

PurposeCorneal scarring and fibrosis following injury remain the leading causes of visual impairment worldwide. This study aimed to evaluate the therapeutic effects of tannic acid (TA), a natural polyphenolic compound with antioxidant, anti-inflammatory, and metal-chelating properties, and to elucidate its underlying molecular mechanisms in corneal wound healing.MethodsIn vitro (human corneal epithelial and stromal cells) and in vivo (rabbit anterior lamellar keratectomy) models were established to evaluate TA’s efficacy. Epithelial migration was assessed by scratch and Transwell assays, whereas TGF-β-induced models evaluated anti-fibrotic activity. Histopathology was examined by hematoxylin and eosin, Masson’s trichrome staining, and α-SMA immunohistochemistry. Transcriptomic sequencing, Western blot, and RT-qPCR analyzed gene and protein expression. Copper ion concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS), and lysyl oxidase (LOX) enzymatic activity was determined using fluorometric activity assay kits.ResultsWithin biocompatible concentrations, TA dose-dependently promoted epithelial cell migration and suppressed stromal fibrosis in vitro. In vivo, TA treatment achieved rapid scar-free healing, outperforming rhEGF treatment. Mechanistic investigations revealed that TA exerts anti-fibrotic effects by downregulating LOX family gene expression and modulating collagen fibril assembly pathways. Protein and mRNA analyses confirmed that TA suppresses LOX expression and activity through chelation of excess copper ions, with functional validation via exogenous copper supplementation further supporting this mechanism.ConclusionsThese findings demonstrate that TA chelates excess copper ions accumulated in injured corneas, thereby mediating LOX inhibition and reducing fibrosis, representing an important mechanistic complement to TA’s established antioxidant and anti-inflammatory properties.

CD38 is a multifunctional enzyme that plays a pivotal role in NAD+ metabolism and calcium signaling, and its abnormal activity is closely associated with multiple myeloma, age-related metabolic decline, neurodegenerative diseases, and other disorders. Although monoclonal antibodies such as daratumumab have been approved for clinical application, their inherent limitations necessitate the development of novel small-molecule CD38 inhibitors. In this study, we employed DNA-encoded library (DEL) technology for the high-throughput screening of CD38 inhibitors, using a DEL library containing more than 100,000 unique compounds to screen against recombinant human CD38. A total of 1043 enriched compounds were initially identified, and after rigorous validation and screening to exclude non-specific binding and previously reported active compounds, eight hit compounds with diverse chemical scaffolds were obtained, among which Fenbendazole-a clinically approved antiparasitic drug-was included. Surface plasmon resonance (SPR) assays confirmed the direct binding of these hit compounds to CD38, with dissociation constants (KD) ranging from 7.74 × 10-5 M to 2.15 × 10-4 M. Fluorescence-based enzymatic activity assays demonstrated that these compounds exert dose-dependent inhibitory effects on both the hydrolase (with ε-NAD as substrate) and cyclase (with NGD as substrate) activities of CD38. Further structure-activity relationship (SAR) analysis of Fenbendazole analogues revealed the critical structural features that regulate CD38 inhibitory potency, and Flubendazole was found to exhibit excellent inhibitory activity, with an IC50 of 14.78 ± 4.21 μM against CD38 hydrolase and 26.31 ± 3.40 μM against cyclase. Molecular docking and 100 ns molecular dynamics (MD) simulations further elucidated the molecular mechanism of CD38 inhibition by lead compounds, confirming that van der Waals interactions are the main driving force for the binding of small-molecule ligands to CD38, with conserved aromatic residues in the active site mediating ligand recognition. This study validates DEL technology as an efficient and reliable platform for the discovery of CD38 inhibitors, and the identified lead compounds-especially Fenbendazole and its analog Flubendazole-provide valuable molecular scaffolds for the further structural optimization of CD38 inhibitors. These findings lay a solid foundation for the development of novel therapeutic agents for the treatment of CD38-associated diseases.

Background/Objectives: Collagen hydrolysates are widely used as nutritional ingredients for skin and joint health; however, growing evidence indicates that collagen may also exert beneficial effects on cardiometabolic pathways. Short peptides have been shown to modulate angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), key regulators of blood pressure and glucose homeostasis. This study aimed to assess the dual ACE- and DPP-IV inhibitory and GLP-1 stimulation activities, respectively of a tripeptide-enriched formulation (CH). The study was performed using a benchmark collagen hydrolysate (BCH) as reference. Methods: ACE and DPP-IV inhibitory activities were evaluated using in vitro enzymatic assays. Cellular compatibility and in situ DPP-IV inhibition were assessed in Caco-2 intestinal cells, while glucagon-like peptide-1 (GLP-1) secretion was measured in STC-1 enteroendocrine cells. The degree of hydrolysis was determined by OPA assay, and nanoLC–HRMS was used to characterize and compare the proteomic profiles of the samples. Results: Both hydrolysates exhibited dose-dependent ACE and DPP-IV inhibition; however, CH showed significantly higher inhibitory activity at comparable concentrations. CH also reduced cellular DPP-IV activity in Caco-2 cells and stimulated GLP-1 secretion in STC-1 cells, whereas BCH showed limited or non-significant cellular effects. Peptidomic analysis revealed an enrichment of short- and medium-length peptides in CH, while BCH contained a higher proportion of long peptides (>2000 Da). Consistently, CH exhibited a 1.7-fold higher degree of hydrolysis than BCH. Conclusions: The tripeptide-enriched collagen hydrolysate demonstrated superior enzymatic and cellular bioactivity compared with the benchmark formulation, supporting its potential as a multifunctional bioactive ingredient for health applications.

Pearl oyster aquaculture is severely constrained by biofouling organisms, particularly fouling oysters, which substantially impair pearl oyster growth and farming efficiency. This study investigated the selective oyster-feeding behavior of the predatory gastropod Thais luteostoma and evaluated its potential as an ecological biofouling control agent in pearl oyster culture. Field co-culture experiments showed that T. luteostoma did not adversely affect the survival of Pinctada fucata martensii, while effectively reducing biofouling loads and significantly improving pearl oyster growth performance. Laboratory behavioral assays and quantitative analyses revealed a pronounced feeding preference for oysters in T. luteostoma, as evidenced by a higher number of feeding individuals, longer total feeding duration, and greater spatial overlap between feeding hotspots and oyster locations. In addition, digestive enzyme assays indicated marked post-feeding physiological responses in T. luteostoma, with a stronger induction of digestive activity in the digestive gland than in the stomach. Collectively, these findings suggest that T. luteostoma represents a promising and sustainable biological option for managing biofouling in pearl oyster aquaculture, with potential applicability to other high-value bivalve farming systems.

Abstract Background Chronic inhalation of cellulosic thinner, which contains toluene and other volatile solvents, induces oxidative stress–mediated neurotoxicity, particularly within the nucleus accumbens (NAc). This study evaluated the neuroprotective effects of melatonin and cortistatin against thinner-induced NAc injury. Methods Ninety male Wistar rats were assigned to nine groups (n = 10): control, thinner-only, thinner + cortistatin, thinner + melatonin, thinner + melatonin + cortistatin, and four vehicle controls. Rats were exposed to thinner vapour twice daily (1 h/session) for six weeks. Melatonin (10 mg/kg) and/or cortistatin were administered intraperitoneally once daily. NAc tissue was analysed by light microscopy, transmission electron microscopy, and biochemical assays of lipid peroxidation (MDA) and antioxidant enzymes (GPx, SOD, CAT). Results Thinner inhalation induced behavioral changes, fur discoloration, and significant weight loss ( p < 0.05). Histology and ultrastructure showed neuronal loss and cellular damage in the nucleus accumbens, least evident with melatonin + cortistatin. Catalase activity showed no differences among groups ( p > 0.05). Glutathione peroxidase was reduced in the thinner group vs. control (0.36 vs. 0.56, p = 0.046). Superoxide dismutase activity was lower in thinner (1.93 vs. 3.67, p < 0.001) and thinner + cortistatin (2.82 vs. 3.67, p = 0.028) groups, with melatonin mitigating reductions. Lipid peroxidation was elevated in the thinner group (3.59 vs. 2.38, p = 0.042), though overall group differences were not significant ( p = 0.127). Conclusions Chronic thinner inhalation causes oxidative stress–related degeneration of the NAc. Melatonin and cortistatin exert neuroprotective effects, with their combination offering superior preservation of cellular and biochemical integrity. These findings highlight potential therapeutic avenues for solvent-induced neurotoxicity.

Introduction: In the context of chronic lymphocytic leukemia (CLL), the impact of baseline cytokine levels on biological features and clinical outcomes remains poorly understood.Methods. Plasma from 244 newly diagnosed CLL patients was extracted and then analyzed using a bead-based 27-plex enzymatic assay. Different optimal cut-offs for overall survival (OS) and probability of Richter transformation (RT) risk analyses were defined using maximally selected rank statistics for every specific cytokine/chemokine. The machine-learning algorithms self-organizing map (SOM) and K-means were applied to divide patients into clusters based on cytokine/chemokine levels.Results. Median age at diagnosis was 68.9 years; 73 patients (29.9%) had unmutated immunoglobulin heavy chain variable region (IGHV) genes, and 16 (6.6%) had TP53 disruption. After a median follow-up of 13.4 years, the median OS was 15.7 years. By correlating cytokines/chemokines with patient baseline clinical and molecular features, elevated MIP-1α levels were significantly associated with unmutated IGHV genes (p<0.0001), greater lymph node diameter (p<0.0001), and advanced Rai and Binet stages (both p<0.0001) (Figure 1A). Higher IP-10 levels significantly associated with advanced Rai (p=0.02) and Binet (p=0.01) stages (Figure 1A). Elevated GM-CSF levels were associated with del(17p) (p=0.03) (Figure 1A). At diagnosis, patients with IL-4 levels exceeding the cut-off of 5.78 pg/mL displayed shorter OS (p=0.02), also in multivariate analysis adjusted for age, IGHV, and TP53 status (HR 2.2, p=0.008). Regarding RT, high levels of GM-CSF (cut-off: 0.86 pg/mL) were significantly associated with increased risk of RT in univariate (p<0.0001) and multivariate analyses (HR 9.3, p=0.009). Unsupervised clustering identified 3 patient groups, namely clusters 1, 2, and 3, with distinct cytokine/chemokine profiles (Figure 1B). In the pathway gene enrichment analysis, cluster 1 displayed higher activation of inflammation-related pathways, while cluster 3 was characterized by the activation of autoimmunity and infection response pathways (Figure 1C). Survival analysis demonstrated that patients in cluster 1 had significantly worse OS (p=0.02), a finding confirmed in multivariate analysis (HR 2.9, p=0.02) (Figure 1D). Furthermore, Binet A and B patients in cluster 3 exhibited a significantly shorter time to first treatment (TTFT) compared to the other clusters (p=0.02), even when adjusted for the IPS-E staging items (HR 2, p=0.03) (Figure 1D).Conclusions: Plasma levels of immunomodulatory cytokines/chemokines are associated with unfavorable clinical and biological features at the time of CLL diagnosis and might predict lower OS and higher risk of RT and second malignancies. Moreover, patients can be divided into groups based on their plasma cytokine/chemokine profile to better stratify OS and TTFT. Consistently, if confirmed in other patient cohorts, cytokine/chemokine levels might integrate current prognostic biomarkers in CLL.

Could cannabigerol protect against neuroinflammation? Insights from an in vitro microglial study.

Targeting the antigen 85 complex of Mycobacterium tuberculosis: A three-fold validation of antimycobacterial activity of Centella asiatica.

Remodeling of phenolic compounds from celery pulp driven by Lactobacillus plantarum NCU116 fermentation: insights from omics, targeted quantification, and microstructural imaging.

Discovery of chalcone derivatives as antileishmanial agents: phenotypic screening, exploratory docking and enzymatic assays on trypanothione reductase and related flavoproteins.

Integrative ethnopharmacological and mechanistic evaluation of Gnidia glauca alkaloids: Multi-target anticancer potential correlated with traditional use.

Effects of micro(nano)plastics on nutrient removal and greenhouse gas production under heavy metals cooccurence in AMF-enhanced constructed wetlands.

Quercetin glucosides are important phytopharmaceutical metabolites in Descurainia sophia seeds, which are widely used in traditional herbal medicine. However, the key genes involved in quercetin glucoside biosynthesis in D. sophia have not been characterized. Herein, we present the telomere-to-telomere genomes of a tetraploid D. sophia, which accumulates high levels of quercetin glucoside, and a diploid D. sophia, which accumulates only trace amounts. Multiomics analyses and uridine diphosphate glucosyltransferase (UGT) enzyme assays revealed that the gene duplication and functional evolution of Dscd6AG01520, an UGT gene, led to high quercetin-3-O-β-d-glucoside and quercetin-3,7-O-β-d-diglucoside accumulation in tetraploid D. sophia seeds. Further UGT enzyme assays with the point mutations of Dscd6AG01520 showed that S213 was a critical amino acid for the enzymatic activity of Dscd6AG01520. In addition, we found that diploid D. sophia evolved from an ancestral crucifer karyotype through chromosome fusion and rearrangement. Collectively, our findings illuminate the mechanism of high quercetin glucoside accumulation in tetraploid D. sophia, clarify the origin of the diploid D. sophia genome, and provide valuable genomic resources for comparative genomics and research into polyploid evolution.

Paeoniflorin enhances tubular repair by promoting dihydroorotate dehydrogenase-dependent epithelial cell proliferation in cisplatin-induced chronic kidney disease.