Search Results (310)

The intestine is considered a habitat for both bacteria and parasites. In this study, many fecal bacterial isolates and the protozoan Blastocystis sp. were recovered from stool samples of individuals with gastrointestinal conditions. Isolated bacteria were tested for extracellular protease production, and the most potent producer was identified by 16SrDNA gene sequencing as P. aeruginosa FZM498. The enzyme was extracted and purified to electrophoretic homogeneity by the DEAE-Sepharose ion-exchanger and SDS-PAGE revealed a major band at 42.15 KDa. It exhibited maximal activity at 35 °C with thermostability at 60 °C (T_1/2 = 200.04 min). It was most active at pH 8.0 and stable at 5.0–9.5. Enzymatic activity was greatly stimulated in the presence of Fe²⁺ ions, but was repressed by Zn²⁺ and Hg²⁺ ions. Inhibition by PMSF, TLCK, aprotinin, benzamidine, and SBTI protease reagents suggests a serine protease family. The V_max and K_m dynamic constants against azocasein were 36.232 U/mL and 0.0072 mM, respectively. It exhibited the lowest K_m value against the synthetic substrate D-Val-Leu-Lys-pNA among all substrates, indicating a plasmin-like activity. Interestingly, when tested against Blastocystis sp., cysts appeared progressively shrunken, ruptured, and mycelial-like, indicating complete structural collapse with leakage of intracellular contents. The importance of this research is that it is the first study to test the anti-Blastocystis activity of an extracted bacterial serine protease from the gut. This could be a promising, eco-friendly, natural alternative as an anti-Blastocystis agent. The objective of this study was to isolate, purify, and biochemically characterize an extracellular serine protease produced by gut-associated bacteria, as well as to assess its in vitro anti-Blastocystis efficacy as a potential natural and ecologically friendly antiparasitic therapy. Full article

In this study, new improved inhibitors of the viral enzyme 3-chymotrypsin-like protease (3CL^pro) were designed using structure-based drug design techniques in an effort to discover more effective treatment of coronavirus disease 2019 (COVID-19). Three-dimensional models of 3CL^pro–inhibitor complexes were prepared by in situ modification of the crystal structure of the submicromolar covalent inhibitor IPCL6 for a set of 25 known inhibitors with published inhibitory potencies (${IC}_{50}^{\mathrm{e}\mathrm{x}\mathrm{p}}$). The QSAR model was prepared with a reasonable correlation between the calculated free energies of formation of the 3CL^pro-IPCL complex (∆∆G_com) and the experimentally determined activities ${IC}_{50}^{\mathrm{e}\mathrm{x}\mathrm{p}}$, which explained approximately 92% of the variation in the 3CL^pro inhibition data. A similar agreement was achieved for the QSAR pharmacophore model (PH4) built on the basis of the active conformations of the IPCL inhibitors bound at the active site of the 3CL^pro. The virtual combinatorial library of more than 567,000 IPCL analogues was screened in silico using the PH4 model and resulted in the identification of 39 promising analogues. The best inhibitors designed in this study show high predicted affinity for the 3CL^pro protease, as well as favourable predicted ADME properties. For the best new virtual inhibitor candidate IPCL 80-27-74-4, the inhibitory concentration ${IC}_{50}^{\mathrm{p}\mathrm{r}\mathrm{e}}$ was predicted equal to 0.8 nM, which represents a significant improvement in the inhibitory potency of known IPCLs. Ultimately, molecular dynamics simulations of the 12 newly designed top-scoring IPCL inhibitors demonstrated that the 3CL^pro–inhibitor complexes exhibited good structural stability, confirming the potential for further development of the designed IPCL analogues. Full article

Haemonchus contortus is a blood-feeding gastrointestinal nematode that significantly impacts the health and productivity of small ruminants. The burden of parasitism and the escalating incidence of anthelmintic resistance necessitate alternative control methods. Here, we characterize the enzymatic activities of five mammalian cell-expressed recombinant H. contortus cysteine proteases (HcCPs), which include two cathepsin B-like proteins (HcCBP1 and HcCBP2) and three cysteine protease 1 proteins (HcCP1a, HcCP1b, and HcCP1c). We hypothesize that these enzymes degrade host blood proteins, thereby facilitating the parasite’s nutrient acquisition and survival. Using synthetic cathepsin (cat) substrates, we show that HcCBP2 was the only protein that exhibited high catB/L but low catB or catK activity, which was inhibited by the cysteine protease inhibitor E-64. All mHcCPs degraded fibrinogen (Fg), which led to delayed plasma clotting, reduced clot density, and lysed plasma clots. All HcCPs partially degraded hemoglobin (Hb), except for mHcCBP2, which degraded Hb and bovine serum albumin completely and bovine IgG partially in the presence of a reducing agent. In conclusion, by sustaining blood feeding and facilitating immune evasion and nutrient acquisition, the HcCPs may play an essential role in the parasite’s survival. Thus, vaccines or cysteine protease inhibitors targeting these parasitic enzymes may mitigate or prevent infections. Full article

Aeromonas hydrophila is a typical pathogen that causes fish diseases and can easily infect different fish species. This study investigated the antibacterial activity, physicochemical properties and antibacterial mechanism of the BLIS UI-11 produced by Lactobacillus plantarum HYH-11, isolated from traditional kimchi in Hebei, China. It was found that BLIS UI-11 showed excellent inhibitory effect on the growth of A. hydrophila, and it also had a good antibacterial effect on various pathogens such as Vagococcus fluvialis, Listeria monocytogenes, Aeromonas dhakensis, Aeromonas salmonicida, Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus. By measuring growth kinetics, it was found that the maximum antibacterial activity was reached after 30 h of culture, and both the optical density value at 600 nm (OD₆₀₀₎ and pH basically entered the stable phase after 20 h. Whole-genome analysis and gene cluster prediction identified a RiPP-like biosynthetic gene cluster, which comprises genes encoding precursor peptides, modification enzymes, and transport/immunity components. The molecular weight of the antimicrobial active substance was detected by dialysis and Tricine-SDS-PAGE, and it was shown to be an ultra-small molecular substance (<1 kDa). BLIS UI-11 was sensitive to protease K, but its antibacterial activity remained stable after treatment with acidic environment (pH 3.0–6.0), high-temperature treatment (121 °C for 30 min), and ultraviolet irradiation (4 h). After the sub-live cell assay (PI/SYTO9) and scanning electron microscopy (SEM), BLIS UI-11 inhibited the growth of bacteria by destroying the cell membrane of A. hydrophila to deform, collapse, and form holes that lead to accounting leakage. The hemolysis assay indicated that BLIS UI-11 exhibited incomplete hemolysis, suggesting its safety for application. The results showed that BLIS UI-11 produced by strain HYH-11 has great potential as an antimicrobial agent against A. hydrophila infection. Full article

Plant protease inhibitors (PPI) play a significant role against microbes, insects, and, to a considerable extent, human pathogens. PPIs inactivate hydrolase enzymes or depolarize the plasma membrane of the pathogens, thereby inhibiting their growth, replication, and invasion. Here, an active serine protease inhibitor was isolated and purified from the seeds of Cleome viscosa. The purified inhibitor was homogenous and exhibited a molecular weight of around 12 kDa as a monomer. The secondary structure analysis indicated that the inhibitor was predominantly composed of α-helical content. The kinetics experiments demonstrated a noncompetitive mode of inhibition towards serine protease when casein was used as the enzyme substrate. The inhibitor formed a stable complex with serine protease, having a likely 1:1 stoichiometry, as inferred from ITC, and the dissociation constant was examined to be K_d = 1.9 × 10⁻⁶ M with a Gibbs free energy of ΔG = −8.079 (kcal/mol). The inhibitor exhibits stable protease inhibition up to 90 °C. Further, in vitro preliminary studies revealed its inhibitory effects against HSV-2 function, evidence that it may have a role in the treatment of viral infections. Full article

Alphaviruses pose a growing global health threat, with Chikungunya virus (CHIKV) epidemics ongoing. Although several CHIKV vaccine candidates have progressed to late-stage clinical evaluation, none have yet achieved licensure or widespread availability. The CHIKV nonstructural proteins nsP2 and nsP4 encode essential enzymatic activities that represent key targets for antiviral development, yet the biochemical basis of nsP4 RNA-dependent RNA polymerase (RdRp) activity remains poorly understood. Here, we identify a minimal, functional precursor form of nsP4 derived from the nsP3–nsP4 polyprotein (P34) that is active in a cell-based RNA replicon system. Using synthetic, capped mRNAs, we show that cleavage of P34 by the nsP2 protease is required for robust reporter expression, and that a truncated form retaining only the C-terminal 50 residues of nsP3 (CT50-P34) supports near-wild-type replication. Unexpectedly, ubiquitin–nsP4 fusions failed to substitute for P34, likely reflecting the transient expression supported by our RNA-based system. We propose that precursor forms of nsP4 interact with the nsP1 dodecamer at the site of genome replication, where cleavage activates the RdRp and localization within the nsP1 dodecamer maintains nsP4 in its active conformation. Dissociation from the nsP1 dodecamer triggers a conformational switch to an inactive state. Together, these findings establish a tractable framework for interrogation of the assembly, activation, and regulation of the alphavirus polymerase. Full article

Enterovirus A71 (EV-A71) is a critical global pathogen, primarily causing Hand-Foot-and-Mouth Disease (HFMD) but frequently leading to severe neurological complications, including fatal neurogenic pulmonary edema (PE). This review elucidates the complex interplay between viral pathogenesis and the host immune response. EV-A71 utilizes receptors like SCARB2 and PSGL-1 for entry, while its proteases (2A^pro, 3C^pro) efficiently evade innate immunity by cleaving key signaling adaptors (MAVS, TRIF), suppressing Type I IFN response. Critical to disease progression is the age-dependent vulnerability in infants and the subsequent shift toward immunopathology. Severe disease is driven by a systemic cytokine storm and T cell dysregulation, characterized by a loss of control from Treg cells and a profound Th17/Treg imbalance, resulting in high levels of pathogenic cytokines (e.g., IL-17A, IFN-γ). Clinical progression is predicted by specific biomarkers, including Treg depletion, monocyte exhaustion (PD-1/PD-L1), and suppressed regulatory signaling (low cAMP). These findings highlight that effective therapeutic strategies must target host-mediated damage through immunomodulation (e.g., by exploring interventions against key pathogenic axes like IL-6 and IL-1β) and call for the development of next-generation vaccines capable of eliciting balanced cellular immunity to prevent immunopathology. Full article

Norrin–Wnt signaling is essential for retinal vascular development and generation of the inner blood retinal barrier. Norrin itself is a potential therapeutic for retinal vascular repair. We explored the feasibility of producing a recombinant protein therapeutic based on human Norrin for intravitreal injection. Norrin^K86P production was tested using MBP fusion and non-tagged versions. FZD4 binding was evaluated by an ELISA, and the activation of AXIN2 gene expression in primary human retinal microvascular endothelial cells was measured by qPCR. Intravitreal injection was tested in the rat eye, evaluated by fluoresceine angiography, OCT, and ERG. MBP-tagged Norrin was resistant to HRV3C protease cleavage unless linker polypeptides were also incorporated. MBP–Norrin or cleaved MBP–Norrin also required refolding with disulfide reshuffling to generate FZD4-binding activity and to affect AXIN-2 gene expression. A production strategy based upon untagged Norrin^K86P refolded from bacterial inclusion bodies was selected. Intravitreal injection of Norrin^K86P did not affect retinal thickness nor retinal function, the latter monitored by the ERG A-wave and B-wave amplitudes. We concluded that MBP–Norrin, cleaved Norrin, and untagged Norrin from inclusion bodies display Norrin-like biological activity after refolding with disulfide reshuffling. The untagged, bacterial inclusion body process was selected for future large-scale bacterial fermentation. Norrin^K86P could be produced with Norrin-like biochemical and biological activities and was tolerated after intravitreal injection into the rat eye. Full article

The picornavirus 3CD protein is a precursor to the 3C main protease and the 3D RNA-dependent RNA polymerase. In addition to its functions in proteolytic processing of the virus polyprotein and cleavage of key host factors, the 3C domain interacts with cis-acting replication elements (CREs) within the viral genome to regulate replication and translation events. We investigated the molecular determinants of RNA binding to 3C using a wide range of biophysical and computational methods. These studies showed that 3C binds to a broad spectrum of RNA oligonucleotides, displaying minimal sequence and structure dependence, at least for these shorter RNAs. However, they also uncovered a novel aspect of these interactions, that is, 3C-RNA binding can induce liquid–liquid phase separation (LLPS), with 3CD–RNA interactions likewise leading to LLPS. This may be a general phenomenon for other 3C and 3C-like proteases and polyproteins incorporating 3C domains. These findings have potential implications in understanding virally induced apoptosis and the control of stress granules, which involve LLPS and include other proteins with known interactions with 3C/3CD. Full article

Nemopilema nomurai is a species of common large toxic jellyfish in China seas, and its tentacle tissues contain various types of nematocysts. However, the correlation between the morphology and function of nematocysts still remains unclear. In this study, we first obtained two monomorphic nematocysts with high-purity from N. nomurai, namely Anisorhizas and O-isorhizas, by density gradient centrifugation: the Anisorhizas is small and rod-shaped and the O-isorhizas is larger and spherical. Upon deionized water stimulation, O-isorhizas exhibited a stronger discharge capability than Anisorhizas. The nematocyst contents of Anisorhizas (AnC) and O-isorhizas (OnC) were extracted separately, and their composition and bioactivities were analyzed simultaneously. The protein bands by SDS-PAGE revealed similar distributions in AnC and OnC, except that the protein band distribution in OnC was more extensive. OnC showed stronger cytotoxicity, hemolytic activity, metalloprotease activity, and serine protease activity than AnC. In contrast, AnC exhibited a higher antioxidant activity and significant proinflammatory activity. Both AnC and OnC exhibited antimicrobial activities against certain marine pathogenic Vibrios. These results suggest that O-isorhizas, with the larger capsule capability, stronger discharge ability and toxicity, likely plays a major role in inducing toxic effects and tissue damage, while Anisorhizas, being smaller and less toxic, may undertake preferentially other functions, such as synergistic predation, environmental stress adaptation, and energy balance maintenance. This study provides insights into the morpho-functional relationship between various types of nematocysts, and also lays a foundation for further exploration of the functional diversity of nematocysts and the mechanisms underlying jellyfish envenomation. Full article

Human immunodeficiency virus (HIV-1) and SARS-CoV-2 continue to co-burden global health, motivating discovery of broad-spectrum small molecules. Conessine, a steroidal alkaloid, has reported membrane-active and antimicrobial properties but remains underexplored as a dual antiviral chemotype. To interrogate conessine’s multi-target antiviral potential against key enzymatic and entry determinants of HIV-1 and SARS-CoV-2 and to benchmark performance versus approved comparators. Eight targets were modeled: HIV-1 reverse transcriptase (RT, 3V81), protease (PR, 1HVR), integrase (IN, 3LPT), gp120–gp41 trimer (4NCO); and SARS-CoV-2 main protease (M^pro, 6LU7), papain-like protease (PL^pro, 6W9C), RNA-dependent RNA polymerase (RdRp, 7BV2), spike RBD (6M0J). Ligands (conessine; positive controls: dolutegravir for HIV-1, nirmatrelvir for SARS-CoV-2) were prepared with standard protonation, minimized, and docked using AutoDock Vina v 1.2.0exhaustiveness 4; 20 poses). Binding modes were profiled in 2D/3D. Protocol robustness was verified by re-docking co-crystallized ligands (RMSD ≤ 2.0 Å). Atomistic MD (explicit TIP3P, OPLS4, 300 K/1 atm, NPT; 50–100 ns) assessed pose stability (RMSD/RMSF), pocket compaction (Rg, volume), and interaction persistence; MM/GBSA provided qualitative energy decomposition. ADMET was predicted in silico. Conessine showed coherent, hydrophobically anchored binding across both viral panels. Best docking scores (kcal·mol⁻¹) were: HIV-1—PR −6.910, RT −6.672, IN −5.733; SARS-CoV-2—spike RBD −7.025, M^pro −5.745, RdRp −5.737, PL^pro −5.024. Interaction maps were dominated by alkyl/π-alkyl packing to catalytic corridors (e.g., PR Ile50/Val82, RT Tyr181/Val106; M^pro His41/Met49; RBD L455/F486/Y489) with occasional carbon-/water-mediated H-bonds guiding orientation. MD sustained low ligand RMSD (typically ≤1.6–2.2 Å) and damped RMSF at catalytic loops, indicating pocket rigidification; MM/GBSA trends (≈ −30 to −40 kcal·mol⁻¹, dispersion-driven) supported persistent nonpolar stabilization. Benchmarks behaved as expected: dolutegravir bound strongly to IN (−6.070) and PR (−7.319) with stable MD; nirmatrelvir was specific for M^pro and displayed weaker, discontinuous engagement at PL^pro/RdRp/RBD under identical settings. ADMET suggested conessine has excellent permeability/BBB access (high logP), but liabilities include poor aqueous solubility, predicted hERG risk, and CYP2D6 substrate dependence.Conessine operates as a hydrophobic, multi-target wedge with the most favorable computed engagement at HIV-1 PR/RT and the SARS-CoV-2 spike RBD, while maintaining stable poses at M^pro and RdRp. The scaffold merits medicinal-chemistry optimization to improve solubility and de-risk cardiotoxicity/CYP interactions, followed by biochemical and cell-based validation against prioritized targets. Full article

Multidrug resistance has also been accompanied by the prolonged use of antibiotics that makes complications in treatment. Biofilm in pathogenic bacteria is the most serious challenge linked with chronic illnesses and also contributes to virulence and drug resistance. Several bacterial pathogens employ the Quorum-sensing (QS) mechanism to coordinate their collective behaviors like bioluminescence, virulence, and biofilm formation. Therefore, agents that inhibit or interfere with bacterial QS and biofilm formation are emerging as a new class of next-generation antibacterial. Recently, nanoparticles have been employed to improve the efficacy of existing antibacterial agents. In the present study, gold nanocrystals were synthesized by using Koelreuteria paniculata (KP) leaf extract. Synthesized nanocrystals were characterized by a face-centered cubic structure of ~20 nm by XRD, FTIR, Zeta sizer, and TEM. Biogenic Gold nanocrystals (BGNCs) exhibited extended QS inhibition in bio-indicator strains Chromobacterium violaceum and Pseudomonas aeruginosa biosensor strains. BGNCs strongly suppressed QS-controlled violacein production in C. violaceum CV026, and elastase, protease, pyocyanin, alginate, and biofilm formation in P. aeruginosa (PA01). In addition, BGNCs notably suppressed the relative expression of PA01 quorum sensing, biofilm-forming, and virulence-regulating genes, as quantified by qRT-PCR. As a result of the broad-spectrum suppression of QS and biofilm by BGNCs, it is anticipated that these nontoxic bioactive nanocrystals can be employed as surface sterilization agents in nosocomial infections. Full article

Biliary tract carcinoma (BTC) is a group of highly heterogeneous malignancies arising from the biliary epithelium. Anatomically, BTC is categorized into gallbladder cancer (GBC) and cholangiocarcinoma (CCA), with the latter further subdivided into intrahepatic (iCCA), perihilar (pCCA), and distal cholangiocarcinoma (dCCA). Epidemiological studies reveal a dismal five-year survival rate of less than 20% for BTC patients, with limited responses to current chemotherapy regimens, underscoring the urgent need to unravel its complex molecular pathogenesis. Recent research has increasingly focused on the regulatory networks of post-translational modifications, particularly the ubiquitin-proteasome system (UPS), in tumorigenesis. As the largest subfamily of deubiquitinating enzymes (DUBs), ubiquitin-specific proteases (USPs) regulate the stability of key oncoproteins such as phosphatase and tensin homolog (PTEN) and c-Myc, playing pivotal roles in tumor cell proliferation, apoptosis evasion, invasion, and metastasis. This review systematically summarizes the differential expression profiles of USP family members (e.g., USP1, USP3, USP7, USP8, USP9X, USP21, and USP22) in BTC and their clinical significance, with a focus on elucidating how specific USPs regulate tumor progression through key substrates, including poly(ADP-ribose) polymerase 1 (PARP1), dynamin-1-like protein (DNM1L), and O-GlcNAc transferase (OGT). Furthermore, based on recent advances, we discuss the therapeutic potential of small-molecule USP inhibitors in BTC targeted therapy, providing a theoretical foundation for developing novel precision treatment strategies. Full article

The continuous advancement of the food industry is accompanied by increased generation of animal waste, including poultry feathers. Composting presents a sustainable alternative to disposal methods such as incineration by converting waste into valuable fertilizer products. This study aimed to evaluate the impact of inoculation with the keratinolytic strain Bacillus subtilis P22 on the quality and maturity of compost produced from feathers combined with organic additives (wood shavings and lignite). The experiment involved evaluation of the keratinolytic potential of the tested strain, and characterization of its proteolytic enzymes, solid-state cultures and composting conducted at semi-technical scale. The B. subtilis P22 strain demonstrated the ability to solubilize 78% of feather material within 7 days of cultivation. The keratinolytic enzyme complex was likely dominated by polycatalytic alkaline serine proteases, i.e., subtilisins. The effectiveness of the inoculum was confirmed in laboratory solid-state cultures, where the dry mass loss in inoculated samples was twice that of the control containing only endogenous microflora. At the semi-technical scale, inoculation with B. subtilis P22 significantly accelerated compost maturation and mineralization (C/N = 10.2; N-NH₄⁺/N-NO₃⁻ = 0.4; Cw/Corg = 0.9) compared to the control. The final compost’s mineral composition indicates its potential for use as an organic soil amendment. Full article

Background/Objectives: Global warming and concomitant extreme weather events have markedly increased the incidence of heat stroke. Heat stroke (HS) poses a substantial threat to cerebral health by triggering neuroinflammation and accelerating neurodegenerative processes. The activation of the Nod-like receptor protein 3 (NLRP3) inflammasome for interleukin-1β (IL-1β) secretion has been implicated as a critical mechanism underlying HS-related fatalities. However, the potential role of specific dietary factors to counteract heat stroke-induced neurotoxicity remains largely underexplored. We previously reported that eicosapentaenoic acid (EPA) and urolithin A (UroA), a gut metabolite of ellagic acid, effectively suppress NLRP3 inflammasome activation against metabolic or pathogenic insults. This study aimed to assess the impact of eicosapentaenoic acid (EPA), urolithin A (UroA), and their combination on mitigating heatstroke-mediated NLRP3 inflammasome activation in microglial cells. Methods: In vitro heatstroke conditions were replicated by subjecting murine BV2 microglial cells to a high temperature (41 °C) under hypoxic conditions. To achieve nutrient loading, BV2 cells were preincubated with either EPA (50 µM) or UroA (10 µM). NLRP3 inflammasome activation was evaluated by proinflammatory gene expression, caspase-1 cleavage in cells, and IL-1β secretion to the medium. The caspase-1 activation was determined using a luciferase-based inflammasome and protease activity reporter (iGLuc) assay. Results: Exposure to high temperatures under hypoxia successfully mimicked HS conditions and promoted NLRP3 inflammasome activation in BV2 cells. Both EPA and UroA substantially attenuated the heat stroke-induced priming of proinflammatory genes. More importantly, EPA and UroA demonstrated a synergistic effect in mitigating HS-induced active caspase-1 production, leading to a dramatic decrease in IL-1β secretion. This synergistic effect between EPA and UroA was further confirmed by the iGLuc reporter assay. Conclusions: Dietary enrichment with EPA and UroA precursors may constitute an efficacious strategy for mitigating heat stroke-mediated neuroinflammation and neurodegenerative diseases. Full article