Search Results (179)

Candidozyma auris is a multidrug-resistant, thermo- and osmotolerant yeast capable of persisting on biotic and abiotic surfaces, attributes likely linked to its cell wall composition. Here, seven putative genes encoding yapsins, aspartyl proteases GPI-anchored to the membrane or cell wall, were identified in the genomes of C. auris CJ97 and 20-1498, from clades III and IV, respectively. The C. auris YPS1 gene is orthologous to the SAP9 of C. albicans. The YPS7 gene is orthologous to YPS7 in C. glabrata and S. cerevisiae, so that they may share similar roles. An in silico analysis suggested an interaction between pepstatin and the catalytic domain of Yps1 and Yps7. Although this inhibitor, when combined with caffeine, had a subtle effect on the growth of C. auris, it induced alterations in the cell wall. CauYPS1 and CauYPS7 expression increased under nutrient starvation and NaCl, and at 42 °C. The transcriptome of the 20-1498 strain suggests that autophagy may play a role in thermal stress, probably degrading deleterious proteins or maintaining cell wall and vacuolar homeostasis. Therefore, CauYps1 and CauYps7 may play a role in the cell wall integrity of C. auris in stress conditions, and they could be a target of new antifungal or antivirulence agents. Full article

Background/Objectives: Maternal exposures are known to influence the risk of isolated cleft lip with or without cleft palate (CL/P)—a common and highly heritable birth defect with a multifactorial etiology. Methods: To identify new risk loci, we conducted a genome-wide gene–environment interaction (GEI) analysis of CL/P with maternal smoking and vitamin use in Filipinos (N_cases = 540, N_controls = 260). Since GEI analyses are typically low in power and the results can be difficult to interpret, we applied multiple testing frameworks to evaluate potential GEI effects: a one degree-of-freedom (1df) GxE test, the 3df joint test, and the two-step EDGE approach. Results: While no genome-wide significant interactions were detected, we identified 11 suggestive GEIs with smoking and 24 with vitamin use. Several implicated loci contain biologically plausible genes. Notable interactions with smoking include loci near FEZF1, TWIST2, and NET1. While FEZF1 is involved in early neuronal development, TWIST2 and NET1 regulate epithelial–mesenchymal transition, which is required for proper lip and palate fusion. Interactions with vitamins encompass CECR2—a chromatin remodeling protein required for neural tube closure—and FURIN, a critical protease during early embryogenesis that activates various growth factors and extracellular matrix proteins. The activity of both proteins is influenced by folic acid. Conclusions: Our findings highlight the critical role of maternal exposures in identifying genes associated with structural birth defects such as CL/P and provide new paths to explore for CL/P genetics. Full article

Human noroviruses (HuNoVs) are the primary cause of acute viral gastroenteritis. There are no antivirals or vaccines available to treat and/or prevent HuNoV. Norovirus 3C-like protease (3CLpro) is essential for viral replication; consequently, the inhibition of this enzyme is a fruitful avenue for antinorovirus therapeutics. To discover novel 3CLpro inhibitors with diverse scaffolds, a multi-stage virtual screening approach was performed by docking >10 million compounds into the 3CLpro catalytic site. An initial subset of 18 compounds was selected, and compounds YY-1029 and YY-4204 were identified as the best two molecules. Molecular dynamics (MD) simulations and binding free energy calculations (MM/GBSA) of YY-1029 and YY-4204 were performed to elucidate the binding mechanisms. The ADMET properties were also estimated to predict the potential druggability of representative molecules. All 18 compounds were evaluated for their antinorovirus activity and cytotoxicity in a cell-based replicon system. This work could provide information for the development of 3CL pro inhibitors. Full article

Norovirus, a major cause of acute gastroenteritis, possesses a single-stranded positive-sense RNA genome. The viral 3C-like cysteine protease (3CL^pro) plays a critical role in processing the viral polyprotein into mature non-structural proteins, a step essential for viral replication. Targeting 3CL^pro has emerged as a promising strategy for developing small-molecule inhibitors against Norovirus. In this study, we employed a combination of virtual screening and the FlipGFP assay to identify potential inhibitors targeting the 3CL^pro of Norovirus genotype GII.4. A library of approximately 58,800 compounds was screened using AutoDock Vina tool, yielding 20 candidate compounds based on their Max Affinity scores. These compounds were subsequently evaluated using a cell-based FlipGFP assay. Among them, eight compounds demonstrated significant inhibitory activity against 3CL^pro, with Gedatolisib showing the most potent effect (IC₅₀ = 0.06 ± 0.01 μM). Molecular docking and molecular dynamics simulations were conducted to explore the binding mechanisms and structural stability of the inhibitor–3CL^pro complexes. Our findings provide valuable insights into the development of antiviral drugs targeting Norovirus 3CL^pro, offering potential therapeutic strategies to combat Norovirus infections. Full article

Porcine teschovirus (PTV) circulates in pig populations, causing clinical diseases such as poliomyelitis, reproductive disorders, and pneumonia. However, the molecular mechanisms underlying the pathogenesis of PTV infection have not been fully elucidated. Here, we found that PTV infection does not activate the promoters of NF-κB or IFN-β. The expression of PTV 3C^pro inhibits the promoter activity of NF-κB and IFN-β stimulated by SeV and inhibits the downstream transcription of NF-κB and IFN-β by blocking the phosphorylation and nuclear translocation of NF-κB. Coimmunoprecipiation (co-IP) experiments demonstrated that 3C^pro and NF-κB interact. The degradation of NF-κB was unaffected by inhibitors targeting lysosomes (NH4Cl), proteasomes (MG132), or caspases (Z-VAD-FMK). The protease activity of 3C^pro, which relies on its catalytic active site, is vital for NF-κB cleavage and degradation. Loss of proteolytic activity in mutants abolished NF-κB degradation, impairing the ability of 3C^pro to suppress SeV-induced innate immunity and restore VSV-GFP replication, thereby underscoring its critical role in immune evasion by targeting NF-κB. This study reveals novel mechanisms underlying PTV-mediated suppression of host innate immunity. Full article

Proteolytic enzymes, which play a crucial role in peptide bond cleavage, are widely applied in various industries. In this study, protease-producing bacteria were isolated and characterized from marine sediments collected from the Yellow Sea, China. Comprehensive screening and 16S rDNA sequencing identified a promising G4 strain as Bacillus atrophaeus. Following meticulous optimization of fermentation conditions and medium composition via response surface methodology, protease production using strain G4 was significantly enhanced by 64%, achieving a yield of 3258 U/mL. The G4 protease exhibited optimal activity at 50 °C and pH 7.5, demonstrating moderate thermal stability with 52% residual activity after 30-min incubation at 50 °C—characteristics typical of an alkaline protease. Notably, the enzyme retained over 79% activity across a broad pH range (6–11) and exhibited excellent salt tolerance, maintaining over 50% activity in a saturated NaCl solution. Inhibition by phenylmethylsulfonyl fluoride, a serine protease inhibitor, confirmed its classification as a serine protease. The enzyme’s potential in generating bioactive peptides was further demonstrated through hydrolysis of sheep (Ovis aries) placenta, resulting in a hydrolysate with notable antioxidant properties. The hydrolysate exhibited a 64% superoxide anion scavenging activity, surpassing that of reduced glutathione. These findings expand the current understanding of Bacillus atrophaeus G4 proteases and provide a foundation for innovative sheep placenta utilization with potential industrial applications. Full article

Post-harvest decay of Carica papaya L. is the primary cause of deterioration in papaya quality and the low economic impact of this sector in Cameroon. Field surveys conducted by teams from the Ministry of Agriculture and Rural Development (MINADER) in Cameroon have primarily associated these decays with fungal attacks. However, to date, no methodological analysis has been conducted on the identification of these fungal agents. To reduce post-harvest losses, rapid detection of diseases is crucial for the application of effective management strategies. This study sought to identify the fungal agents associated with post-harvest decay of papaya cv Sunrise solo in Cameroon and to determine their physiological and biochemical growth characteristics. Isolation and pathogenicity tests were performed according to Koch’s postulate. Molecular identification of isolates was achieved by amplification and sequencing of the ITS1 and ITS4 regions. Phylogenetic analysis was based on the substitution models corresponding to each fungal genus determined by jModeltest, according to the Akaike information criterion (AIC). Fungal explants of each identified species were subjected to variations in temperature, pH, water activity, and NaCl concentration. The ability to secrete hydrolytic enzymes was determined on specific media such as skimmed milk agar for protease, peptone agar for lipase, and carboxymethylcellulose for cellulase. These experiments allowed the identification of three fungi responsible for papaya fruit decay, namely Colletotrichum gloeosporioides, Fusarium equiseti, and Lasiodiplodia theobromae. All three pathogens had maximum mycelial growth at a temperature of 25 ± 2 °C, pH 6.5, NaCl concentration of 100 µM, and water activity (aw) equal to 0.98. The three fungal agents demonstrated a strong potential for secreting cellulases, lipases, and proteases, which they use as lytic enzymes to degrade papaya tissues. The relative enzymatic activity varied depending on the fungal pathogen as well as the type of enzyme secreted. This study is the first report of F. equiseti as a causal agent of papaya fruit decay in Cameroon. Full article

Psychrotrophic Morganella spp. is a typical histamine producer commonly found in seafood, exhibiting a high histamine-producing capacity. In this study, two strains of Morganella (GWT 902 and GWT 904) isolated from yellowfin tuna were subjected to phenotypic and genotypic characterization. Phenotypic analysis reveals differences in growth temperature, NaCl tolerance, and D-galactose fermentation capacity between the two strains. Notably, the histamine production capacity of GWT 902 is significantly higher than that of GWT 904 at 4 °C. The complete genome sequences of strains GWT 902 and GWT 904 were sequenced, identifying GWT 902 as Morganella psychrotolerans and GWT 904 as Morganella morganii subsp. sibonii. Genomic analysis confirms the presence of histidine decarboxylase gene clusters (hdcT1, hdc, hdcT2, hisRS) in both strains, and sequence alignment shows that the amino acid sequence similarity of histidine decarboxylase encoded by the hdc gene was 95.24%. Gene function analysis further identified genes associated with putrescine biosynthesis, sulfur metabolism, lipase and protease secretion, and detected key genes in quorum sensing (QS), stress adaptation, and antibiotic resistance. This study provides valuable insights into the taxonomic analysis of psychrotrophic Morganella spp. and contributes to the development of efficient strategies for preventing histamine formation in seafood. Full article

In the study presented herein, an aerobic, Gram-stain-positive, spore-forming bacterium, designated as WLY-B-L8^T, was isolated from a qu-starter (baobaoqu) cultivation facility used for the production of Wuliangye baijiu in Yibin city (Sichuan province, China). The strain comprised short, rod-shape cells of 1.2–1.9 μm in width and 1.7–4.8 μm in length, arranged singly or in pairs. The isolate was able to grow at temperatures of 20–42 °C (optimum growth at 40 °C), pH 5.0–10.0 (optimum growth at pH 8.0), and in the presence of 0–2% (w/v) NaCl (optimum growth with 1% NaCl). Ribose, xylose, arabinose, mannose, glucose, and galactose constituted the major cell-wall sugars. Moreover, meso-diaminopimelic acid (meso-DAP) constituted the diagnostic amino acid. The main polar lipids of WLY-B-L8^T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unidentified aminolipids (UAL 1–2), an unidentified aminophospholipid (UAPL), an unidentified aminoglycolipid (UAGL), and an unidentified lipid (UL). MK-7 was the predominant menaquinone and iso-C_15:0 (23.00%) was the major fatty acid. Comparisons of the 16S rRNA gene sequence indicated that WLY-B-L8^T was most closely related to Bacillus rhizoplanae JJ-63 DSM 12442^T (98.71%), Bacillus pseudomycoides DSM 12442^T (98.21%), and Bacillus cytotoxicus NVH 391–98^T (98.14%). The average nucleotide identity (ANI) values of strain WLY-B-L8^T and the three type strains mentioned above were 88.24%, 80.57%, and 78.70%. The average amino identity (AAI) values between them were 89.84%, 79.51%, and 80.41%. In addition, the digital DNA–DNA hybridization (dDDH) values between them were 36.70%, 26.10%, and 23.90%. The genomic DNA G+C content was 35.97%. Based on the evidence presented herein, WLY-B-L8^T (CICC 25210^T = JCM 36284^T) exhibits promise as the type strain of a novel species, designated as Bacillus multifaciens sp. nov., that can produce protease (119.38 ± 7.44 U/mL) and volatile flavor components when cultured on raw wheat, such as 2-pipendinone (21.95 ± 1.56 mg/L), phenylethyl alcohol (19.08 ± 0.82 mg/L), hydrocinnamic acid (18.60 ± 0.53 mg/L), and acetoin (7.58 ± 0.11 mg/L). Full article

3CL protease (3CL^pro), a key enzyme of SARS-CoV-2 replication, is one of the most selective targets of antivirals, as no homologous protease has been recognized in the human body. As proteolysis-targeting chimeras (PROTACs) are superior to traditional inhibitors, based on the reported cereblon (CRBN) ligands thalidomide and lenalidomide, 3CL^pro ligands of peptidomimetic inhibitors, and suitable linkers, we aimed to develop novel PROTACs that may trigger efficient intracellular 3CL^pro degradation through a balance of hydrophilicity and lipophilicity. In brief, we designed and synthesized 5 PROTAC molecules. The 3CL^pro degradation efficiency of the PROTACs was assayed in stable SARS-CoV-2 3CL^pro expression HEK293 cell models and evaluated by Western blot. All compounds showed prominent 3CL^pro degradation activity with tolerable HEK293 cytotoxicity. The most prominent PROTAC compounds, 15 and 16, have DC₅₀ values of approximately 1 µM, and D_max of 89.3% and 75% respectively, indicating good potential for further application. Full article

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

This study shows an evaluation of the condition of organic matter against enzymatic activity in soil. Long-term static field experiments with fertilisation with manure (FYM), different minerals, and mineral–manure were used for the research. Assays were obtained of the content of total organic carbon (TOC), dissolved fraction (DOC), susceptibility to oxidation (CL1) and (CL), total nitrogen (TN), dissolved nitrogen fraction (DTNT), and available forms of potassium, phosphorus, and magnesium. The activity of enzymes dehydrogenases, catalase, β-glucosidase, proteases, alkaline, and acid phosphatase was determined. We calculated the enzymatic indices and those evaluating the labile organic carbon management (CMI and CPI) in soil. An increase in TOC, up to 8.85 g kg⁻¹ and 8.56 g kg⁻¹ (FYM, FYM + KN), respectively, as compared with the control (5.67 g kg⁻¹), did not have a significant effect on the content of labile carbon fraction CL for the fertilisation treatments. Only a higher CL content was found in the soil with the FYM + PN and FYM + NPK + Mg treatments (2.07 g kg⁻¹ and 2.05 g kg⁻¹). All the fertilisation treatments under study demonstrated a decrease in the value of the carbon management index (CMI). Similar DOC values (on average, 75.14 mg kg⁻¹) were noted. The average percentage share of the DOC fraction accounted for 1.163% TOC, and it was lower as compared with the control variant (1.33% TOC). The mineral fertilisation treatments decreased soil enzyme activities. Multiparametric enzymatic soil fertility indices differed due to soil properties, depending on the fertilisation applied. Full article

In this study, we utilized machine learning techniques to identify potential inhibitors of the MERS-CoV 3CL protease. Among the models evaluated, the Random Forest (RF) algorithm exhibited the highest predictive performance, achieving an accuracy of 0.97, an ROC-AUC score of 0.98, and an F1-score of 0.98. Following model validation, we applied it to a dataset of 14,194 naturally occurring compounds from PubChem. The top-ranked compounds were subsequently subjected to molecular docking, which identified Perenniporide B, Phellifuropyranone A, and Terrestrol G as the most promising candidates, with binding energies of −9.17, −9.08, and −8.71 kcal/mol, respectively. These compounds formed strong interactions with key catalytic residues, suggesting significant inhibitory potential against the viral protease. Furthermore, molecular dynamics simulations confirmed their stability within the active site, reinforcing their viability as antiviral agents. This study demonstrates the effectiveness of integrating machine learning with molecular modeling to accelerate the discovery of therapeutic candidates against emerging viral threats. Full article

This study aims to explore the protective effects of Hong-bai-lan-shen (HBLS) extract, a traditional Chinese medicine compound, on myocardial injury based on metabolomics. H9C2 cells were cultured with HBLS extract for 12 h, and then the cells were cultured in a CoCl₂-containing medium, a model simulating the ischemic-hypoxic damage in myocardial cells, for an additional 12 h. The cell viability, cytotoxicity, intracellular metabolite and reactive oxygen species (ROS), mitochondrial membrane potential, apoptosis, and adenosine monophosphate-activated protein kinase (AMPK) signal pathway were determined. The results showed that HBLS extract significantly increased cell viability, stabilized cell morphology, reduced lactate dehydrogenase (LDH) release and ROS production, blocked cysteine-aspartic acid protease 3 (caspase-3) and bcl-2-associated X protein (Bax) expression and decreased apoptotic cell numbers. Meanwhile, HBLS increased membrane potential and the expression of B-cell lymphoma-2 (Bcl-2). Additionally, HBLS extract upregulated the expression of AMPK, PI3K, and protein kinase B (AKT) (p < 0.05, p < 0.01). These findings suggest that HBLS extract has a protective effect on myocardial cells by regulating the AMPK signal pathway and may be a promising therapeutic candidate for ischemic heart disease. Full article

The ongoing emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has led to resistance against multiple coronavirus disease 2019 (COVID-19) vaccines and therapeutic medications, making the development of effective therapeutics against SARS-CoV-2 a high priority. Studies have shown that bioactive polyphenols, particularly those with triphenol groups, can effectively inhibit the activity of SARS-CoV-2 3-chymotrypsin-like protease (3CL^pro). However, the structural instability of polyphenols necessitates further research. To address this, we conducted a literature review to identify triphenol compounds that are either approved or currently undergoing clinical trials, assessing their potential to inhibit SARS-CoV-2 3CL^pro. Exifone and benserazide hydrochloride were identified as the inhibitors of SARS-CoV-2 3CL^pro among these compounds, using a fluorescence resonance energy transfer (FRET)-based assay. Benserazide hydrochloride was confirmed as a covalent binder to SARS-CoV-2 3CL^pro through time-dependent inhibition and kinetic analysis, with its binding mode elucidated by molecular docking. Notably, exifone not only inhibited the protease activity but also blocked the interaction between the host cell receptor angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor binding domain (S-RBD), as identified by surface plasmon resonance (SPR) and flow cytometry. Additionally, exifone demonstrated antiviral activity against various SARS-CoV-2-S pseudovirus variants. In conclusion, the discovery of exifone and benserazide hydrochloride underscores the potential of polyphenols in developing conserved 3CL^pro inhibitors for coronaviruses, offering new strategies for the rapid development of effective drugs against both current and future coronavirus pandemics. Full article