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In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing., (© 2024 Wiley Periodicals LLC.)

The inflamed mucosa contains a complex assortment of proteases that may participate in wound healing or the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies and of the T84 colon cancer cell line were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing was visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound-healing response in T84 cells but not primary cells. The PAR2-enhanced wound-healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent on Src kinase activity and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase, and epidermal growth factor receptor, which typically mediate wound-healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines TNF and IFNγ revealed a synergistically enhanced PAR2 wound-healing response in T84s but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of inflammatory bowel disease (IBD) and inflammation-associated colon cancer. NEW & NOTEWORTHY Protease-activated receptor 2 enhances wound healing in the T84 colon cancer cell line, but not in primary cells derived from patient biopsies, an effect that is synergistically enhanced in the presence of the inflammatory cytokines TNF and IFNγ.

Remarkable resistance of bacterial biofilms to high doses of antimicrobials and antibiotics is one of their main challenges. Encapsulation of proteolytic enzymes is one of the suggested strategies to tackle this problem. In this regard, the antibacterial and anti-biofilm activity of biocompatible hyaluronic acid- Lysine nanogels containing serratiopeptidase (SRP-loaded HA-Lys nanogel) was assessed against P. aeruginosa and S. aureus strains. SRP-loaded HA-Lys nanogel was prepared using dropping method and optimized by Box-Behnken experimental design. These formulations were studied for physical characterization, release profile, stability, bioactivity, and anti-biofilm effects. The particle size, polydispersity index (PDI), and surface charge were measured by Zetasizer Nano ZS. The average particle size and zeta potential of the optimum sample were 156 nm and -14.1 mV, respectively. SRP release showed an initial burst followed by sustained release and the highest release was around 77%. Enzyme biological activity data revealed the higher efficiency of free SRP compared to SRP-loaded HA-Lys nanogel. The time-kill assay showed that both forms of SRP-loaded HA-Lys nanogel and blank HA-Lys nanogel showed significant antimicrobial activity against examined bacteria in comparison to the free enzyme. The obtained results demonstrated improved anti-biofilm efficacy and down regulation of tested biofilm genes for both SRP-loaded HA-Lys nanogel 100% and blank HA-Lys nanogel 100% compared to SRP 100%., (© 2024. The Author(s).)

Purpose: The PRESTIGIO Registry was established in 2017 to collect clinical, virological and immunological monitoring data from people living with HIV (PLWH) with documented four-class drug resistance (4DR). Key research purposes include the evaluation of residual susceptibility to specific antiretrovirals and the validation of treatment and monitoring strategies in this population., Participants: The PRESTIGIO Registry collects annual plasma and peripheral blood mononuclear cell samples and demographic, clinical, virological, treatment and laboratory data from PLWH followed at 39 Italian clinical centres and characterised by intermediate-to-high genotypic resistance to ≥1 nucleoside reverse transcriptase inhibitors, ≥1 non-nucleoside reverse transcriptase inhibitors, ≥1 protease inhibitors, plus either intermediate-to-high genotypic resistance to ≥1 integrase strand transfer inhibitors (INSTIs) or history of virological failure to an INSTI-containing regimen. To date, 229 people have been recorded in the cohort. Most of the data are collected from the date of the first evidence of 4DR (baseline), with some prebaseline information obtained retrospectively. Samples are collected from the date of enrollment in the registry., Findings to Date: The open-ended cohort has been used to assess (1) prognosis in terms of survival or development of AIDS-related or non-AIDS-related clinical events; (2) long-term efficacy and safety of different antiretroviral regimens and (3) virological and immunological factors predictive of clinical outcome and treatment efficacy, especially through analysis of plasma and cell samples., Future Plans: The registry can provide new knowledge on how to implement an integrated approach to study PLWH with documented resistance to the four main antiretroviral classes, a population with a limited number of individuals characterised by a high degree of frailty and complexity in therapeutic management. Given the scheduled annual updates of PLWH data, the researchers who collaborate in the registry can send study proposals at any time to the steering committee of the registry, which evaluates every 3 months whether the research studies can be conducted on data and biosamples from the registry and whether they are aimed at a better understanding of a specific health condition, the emergence of comorbidities or the effect of potential treatments or experimental drugs that may have an impact on disease progression and quality of life. Finally, the research studies should aim to be inclusive, innovative and in touch with the communities and society as a whole., Trial Registration Number: NCT04098315., Competing Interests: Competing interests: RG reports payments to her institution from Gilead Sciences; personal fees for speaker panels and educational material from ViiV Healthcare, Merck Sharp and Dohme and Gilead Sciences and advisory boards from Theratechnologies, Janssen-Cilag and Gilead Sciences. FL reports personal fees for speaker panels from ViiV Healthcare, Janssen-Cilag and Merck Sharp and Dohme; travel grants from Gilead Sciences, ViiV Healthcare and Janssen-Cilag and advisory boards from ViiV Healthcare and Janssen-Cilag. EF reports personal fees for consultancy from Merck Sharp and Dohme, ViiV Healthcare, Gilead Sciences and Swedish Orphan Biovitrum; speaker panels and educational material from ViiV Healthcare and Gilead Sciences and advisory boards from ViiV Healthcare, Gilead Sciences and Merck Sharp and Dohme. ADB reports personal fees for speaker panels and educational material from ViiV Healthcare and Gilead Sciences and travel grants from ViiV Healthcare. AdCe reports personal fees for speaker panels from ViiV Healthcare. FM reports personal fees for consultancy and advisory boards from Merck Sharp and Dohme, ViiV Healthcare and Gilead Sciences. GM reports personal fees for speaker panels and educational material from Gilead Sciences; travel grants from Janssen-Cilag, Gilead Sciences and ViiV Healthcare and advisory boards from Gilead Sciences, ViiV Healthcare and Angelini Pharma. GC reports personal fees for speaker panels and educational material from Gilead Sciences, ViiV Healthcare and AbbVie and travel grants from Gilead Sciences. SR reports payments to his institution from Gilead Sciences, Janssen-Cilag and ViiV Healthcare; personal fees for travel grants from Gilead Sciences, Janssen-Cilag and ViiV Healthcare and advisory boards from Gilead Sciences, Janssen-Cilag, ViiV Healthcare and Merck Sharp and Dohme. MZ reports personal fees for consultancy and speaker panels and educational material from Gilead Sciences, ViiV Healthcare and Merck Sharp and Dohme. MMS reports personal fees for speaker panels and educational material from ViiV Healthcare, Merck Sharp and Dohme and Janssen-Cilag and advisory boards from ViiV Healthcare and Theratechnlogies. VS reports grants from Gilead Sciences and personal fees for speaker panels from Gilead Sciences, ViiV Healthcare and Merck Sharp & Dohme. AnCa reports personal fees for advisory boards, speaker panels and educational materials from Gilead Sciences, ViiV Healthcare, Janssen-Cilag, Merck Sharp & Dohme and Theratechnlogies. PRESTIGIO Registry received funding from Gilead Sciences, ViiV Healthcare, Merck Sharp & Dohme, and Theratechnologies.All other authors: no potential conflicts., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human. Since there are still no effective treatment options against the new emerging variants of SARS-CoV-2, it is necessary to devote a continuous endeavor for more targeted drugs and the preparation for the next pandemic. Salvia miltiorrhiza and its active ingredients possess wide antiviral activities, including against SARS-CoV-2. Danshensu, as one of the most important active ingredients in Salvia miltiorrhiza, has been reported to inhibit the entry of SARS-CoV-2 into ACE2 (angiotensin-converting enzyme 2)-overexpressed HEK-293T cells and Vero-E6 cells. However, there is a paucity of information regarding its detailed target and mechanism against SARS-CoV-2. Here, we present Danshensu as a covalent inhibitor of 3-chymotrypsin-like protease (3CL pro ) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. Further molecular docking, site-directed mutagenesis, circular dichroism (CD) and fluorescence spectra revealed that Danshensu covalently binds to C145 of SARS-CoV-2 3CL pro , meanwhile forming the hydrogen bonds with S144, H163 and E166 in the S1 site. Structure-based optimization of Danshensu led to the discovery of the promising compounds with good inhibitory activity and microsomal stability in vitro. Due to Danshensu inhibiting lung inflammation in the mouse model, we found that Danshensu derivatives also showed better anti-inflammatory activity than Danshensu in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Thus, our study provides not only the clue of the efficacy of Salvia miltiorrhiza against SARS-CoV-2, but also a detailed mechanistic insight into the covalent mode of action of Danshensu for design of covalent inhibitors against SARS-CoV-2 3CL pro , highlighting its potential as a bifunctional molecule with antivirus and anti-inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Objective: The COVID-19 pandemic has been recognized as severe acute respiratory syndrome, one of the worst and disastrous infectious diseases in human history. Until now, there is no cure to this contagious infection although some multinational pharmaceutical companies have synthesized the vaccines and injecting them into humans, but a drug treatment regimen is yet to come., Aim: Among the multiple areas of SARS-CoV-2 that can be targeted, protease protein has significant values due to its essential role in viral replication and life. The repurposing of FDA-approved drugs for the treatment of COVID-19 has been a critical strategy during the pandemic due to the urgency of effective therapies. The novelty in this work refers to the innovative use of existing drugs with greater safety, speed, cost-effectiveness, broad availability, and diversity in the mechanism of action that have been approved and developed for other medical conditions., Methods: In this research work, we have engaged drug reprofiling or drug repurposing to recognize possible inhibitors of protease protein 6M03 in an instantaneous approach through computational docking studies., Results: We screened 16 FDA-approved anti-viral drugs that were known for different viral infections to be tested against this contagious novel strain. Through these reprofiling studies, we come up with 5 drugs, namely, Delavirdine, Fosamprenavir, Imiquimod, Stavudine, and Zanamivir, showing excellent results with the negative binding energies in Kcal/mol as - 8.5, - 7.0, - 6.8, - 6.8, and - 6.6, respectively, in the best binding posture. In silico studies allowed us to demonstrate the potential role of these drugs against COVID-19., Conclusion: In our study, we also observed the nucleotide sequence of protease protein consisting of 316 amino acid residues and the influence of these pronouncing drugs over these sequences. The outcome of this research work provides researchers with a track record for carrying out further investigational procedures by applying docking simulations and in vitro and in vivo experimentation with these reprofile drugs so that a better drug can be formulated against coronavirus., (© 2023. The Author(s), under exclusive licence to Royal Academy of Medicine in Ireland.)

Neurogenesis is known to be closely associated with depression. We aimed to investigate whether a polypeptide monomer derived from pilose antler (polypeptide sequence LSALEGVFYP, PAP) exerts an antidepressant effect by influencing neurogenesis, and to elucidate the mechanism of its antidepressant action. Behavioral tests were performed to observe the antidepressant effect of PAP. Neurogenesis in the dentate gyrus (DG) region of hippocampus was observed by immunofluorescence. The expression of key proteins of Sentrin/SUMO-specific proteases 2 (SENP2)- Phosphoinositide-specific phospholipase C beta 4 (PLCβ4) pathway was accessed by co-immunoprecipitation (Co-IP), and the calcium homeostasis associated proteins were observed via Western blot (WB). Subsequently, temozolomide (TMZ) pharmacologically blocked neurogenesis to verify the antidepressant effect of PAP on neurogenesis. The mechanism of PAP antidepressant effect was verified by constructing a sh-SENP2 virus vector to silence SENP2 protein. Finally, corticosterone (CORT)-induced PC12 cell model was used to verify whether PAP was involved in the process of deconjugated PLCβ4 SUMOylated. The results showed that PAP improved depression-like behavior and neurogenesis induced by chronic unpredictable mild stimulation (CUMS). In addition, PAP acted on SENP2-PLCβ4 pathway to deconjugate the SUMOylation of PLCβ4 and affect calcium homeostasis. Pharmacological blockade of neurogenesis by TMZ treatment impaired the antidepressant efficacy of PAP. Knockout of SENP2 in the CUMS model attenuated the antidepressant response of PAP, and the impaired neurogenesis was not ameliorated by PAP treatment. In summary, PAP acted on the SENP2-PLCβ4 signaling pathway to inhibit the SUMOylation of PLCβ4 and maintain calcium homeostasis, thereby protecting neurogenesis and playing an antidepressant role., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

How different dietary fibers including pectin, cellulose and lignin affect casein digestibility was studied using in vitro static protocols. Peptides' profile, free amino acids (AAs) content, casein-DF interactions and their influences on enzymatic activities of proteolytic enzymes were studied using combined techniques. Under gastric and intestinal digestive conditions, while pectin could reduce casein digestibility (with an averaged decrease of 12.15% and 7.83, respectively) through both depletion flocculation and hydrogen-binding interactions, lignin inhibited the digestion of casein straightly through reducing the enzymatic activity of proteolytic enzymes, thereby altering the production of free AAs. Although cellulose showed the least detrimental effects, it still significantly reduced the content of Thr, Glu, Val, Leu, Phe, Lys, and no Arg was released. Deeper insight into casein-DF interactions and their influences on casein digestibility improves the development of more effective forms of DF for improving AA homeostasis in individuals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Enzyme combinations, particularly phytase (PHY) with various carbohydrases and proteases, are utilized in commercial broiler production to enhance nutrient and energy bioavailability., Objective: A feeding study was undertaken to determine whether the efficiency of an Escherichia coli-derived PHY and a feed enzyme complex (FEC) derived from Bacillus spp. containing carbohydrase and protease as main activities in broiler chickens is dependent on diet quality. A total of 900 male one-day-old broiler chickens (Ross 308) were assigned to a 2 × 3 factorial arrangement of the treatments with 2 different nutrient density diets, standard nutrient diet (SN diet) and a low-nutrient diet (LN diet; -100 kcal/kg for AMEn and -5% for crude protein [CP] and limiting amino acids), and 3 enzyme treatments (control [no enzymes], PHY and PHY + FEC). Each treatment group was composed of 6 replicates of 25 birds each., Results: The LN diet caused a decrease in performance index, tibia length and diameter, tibia calcium content and jejunal villus surface area (VSA). The interaction effects between diet and enzyme supplementation were observed (p < 0.05) on overall average daily gain (ADG), performance index, tibia ash content and jejunal villus height (VH) and VSA, with the favourable benefits of PHY + FEC treatment being more pronounced in the LN diets. Regardless of dietary nutrient density, supplementation with PHY alone or combined with FEC enhanced (p < 0.05) final body weight, overall ADG and jejunal villus height (VH)/crypt depth, with the highest values observed in the PHY + FEC group. The PHY + FEC treatment also improved (p < 0.05) overall feed conversion ratio, apparent ileal digestibility of dry matter, organic matter, CP, and energy, and tibia phosphorus content compared to the control treatment., Conclusions: The results indicate that the simultaneous addition of PHY and FEC to the LN diets improved the growth rate, bone mineralization and gut morphology., (© 2024 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Flavonoids are vital candidates to fight against a wide range of pathogenic microbial infections. Due to their therapeutic potential, many flavonoids from the herbs of traditional medicine systems are now being evaluated as lead compounds to develop potential antimicrobial hits. The emergence of SARS-CoV-2 caused one of the deadliest pandemics that has ever been known to mankind. To date, more than 600 million confirmed cases of SARS-CoV2 infection have been reported worldwide. Situations are worse due to the unavailability of therapeutics to combat the viral disease. Thus, there is an urgent need to develop drugs against SARS-CoV2 and its emerging variants. Here, we have carried out a detailed mechanistic analysis of the antiviral efficacy of flavonoids in terms of their potential targets and structural feature required for exerting their antiviral activity. A catalog of various promising flavonoid compounds has been shown to elicit inhibitory effects against SARS-CoV and MERS-CoV proteases. However, they act in the high-micromolar regime. Thus a proper leadoptimization against the various proteases of SARS-CoV2 can lead to high-affinity SARS-CoV2 protease inhibitors. To enable lead optimization, a quantitative structure-activity relationship (QSAR) analysis has been developed for the flavonoids that have shown antiviral activity against viral proteases of SARS-CoV and MERS-CoV. High sequence similarities between coronavirus proteases enable the applicability of the developed QSAR to SARS-CoV2 proteases inhibitor screening. The detailed mechanistic analysis of the antiviral flavonoids and the developed QSAR models is a step forward toward the development of flavonoid-based therapeutics or supplements to fight against COVID-19., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Our previous report established that RUNX family transcription factor 1 (RUNX1) promotes proliferation of mouse retinal microvascular endothelial cells (mRMECs) and exacerbates diabetic retinopathy (DR). However, the mechanism behind the upregulation of RUNX1 remains unclear. This study aims to investigate the possible correlation between histone SUMOylation and RUNX1 in DR, as well as the involved molecules. A mouse model of diabetes was induced by streptozotocin (STZ). These mice had increased retinal thickness and elevated production of inflammatory cytokines. Additionally, they showed elevated levels of SUMO1 and SUMO2/3, but reduced levels of SUMO specific peptidase 1 (SENP1) in retinal tissues. Co-immunoprecipitation and Western blot assays revealed that the RUNX1 protein was primarily modified by SUMO2/3, and SENP1 inhibited SUMO2/3 modification, thereby reducing RUNX1 expression. Overexpression of SENP1 alleviated symptoms in mice and alleviated inflammation. In vitro experiments demonstrated that the SENP1 overexpression suppressed the proliferation, migration, and angiogenesis of high-glucose-induced mRMECs. However, further overexpression of RUNX1 counteracted the alleviating effects of SENP1 both in vivo and in vitro. In conclusion, this study demonstrates that the downregulation of SENP1 in DR leads to SUMO2/3-dependent activation of RUNX1. This activation promotes proliferation of mRMECs and exacerbates DR symptoms in mice., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

ABSTRACT The deposition of β-amyloid plaques, either due to their over-production or insufficient clearance, is an important pathological process in cognitive impairment and dementia. Icariin (ICA), a flavonoid compound extracted from Epimedium, has recently gained attention for numerous age-related diseases, such as neurodegenerative diseases. We aimed to explore the possible neuro-protective effect of ICA supplementation in colchicine-induced cognitive deficit rat model and exploring its effect on the β-amyloid proteolytic enzymes. The study included four groups (10 rats each): normal control, untreated colchicine, colchicine + 10 mg/kg ICA, and colchicine + 30 mg/ kg ICA. Results revealed that intra-cerebro-ventricular colchicine injection produced neuronal morphological damage, β amyloid deposition, and evident cognitive impairment in the behavioral assessment. Icariin supplementation in the two doses for 21 days attenuated neuronal death, reduced the β amyloid levels, and improved memory consolidation. This was associated with modulation of the proteolytic enzymes (Neprilysin, Matrix Metalloproteinase-2, and insulin-degrading enzyme) concluding that β-amyloid enzymatic degradation may be the possible therapeutic target for ICA.

Middle East Respiratory Syndrome (MERS) is a viral respiratory disease caused b a special type of coronavirus called MERS-CoV. In the search for effective substances against the MERS-CoV main protease, we looked into compounds from brown algae, known for their medicinal benefits. From a set of 1212 such compounds, our computer-based screening highlighted four-CMNPD27819, CMNPD1843, CMNPD4184, and CMNPD3156. These showed good potential in how they might attach to the MERS-CoV protease, comparable to a known inhibitor. We confirmed these results with multiple computer tests. Studies on the dynamics and steadiness of these compounds with the MERS-CoV protease were performed using molecular dynamics (MD) simulations. Metrics like RMSD and RMSF showed their stability. We also studied how these compounds and the protease interact in detail. An analysis technique, PCA, showed changes in atomic positions over time. Overall, our computer studies suggest brown algae compounds could be valuable in fighting MERS. However, experimental validation is needed to prove their real-world effectiveness.

Antimicrobial resistance by pathogenic bacteria has become a global risk to human health in recent years. The most promising approach to combating antimicrobial resistance is to target virulent traits of bacteria. In the present study, a biosurfactant derived from the probiotic strain Lactobacillus acidophilus was tested against three Gram-negative bacteria to evaluate its inhibitory potential on their biofilms, and whether it affected the virulence factors controlled by quorum sensing (QS). A reduction in the virulence factors of Chromobacterium violaceum (violacein production), Serratia marcescens (prodigiosin production) and Pseudomonas aeruginosa (pyocyanin, total protease, LasB elastase and LasA protease production) was observed at different sub-MIC concentrations in a dose-dependent manner. Biofilm development was reduced by 65.76%, 70.64% and 58.12% at the highest sub-MIC levels for C. violaceum, P. aeruginosa and S. marcescens, respectively. Biofilm formation on glass surfaces exhibited significant reduction, with less bacterial aggregation and reduced formation of extracellular polymeric materials. Additionally, swimming motility and exopolysaccharides (EPS) production were shown to be reduced in the presence of the L. acidophilus-derived biosurfactant. Furthermore, molecular docking analysis performed on compounds identified through gas chromatography-mass spectrometry (GC-MS) analysis of QS and biofilm proteins yielded further insights into the mechanism underlying the anti-QS activity. Therefore, the present study has clearly demonstrated that a biosurfactant derived from L. acidophilus can significantly inhibit virulence factors of Gram-negative pathogenic bacteria. This could provide an effective method to inhibit the formation of biofilms and QS in Gram-negative bacteria.

Mast cell granules are packed with proteases, which are released with other mediators by degranulating stimuli. Several of these proteases are targets of potentially therapeutic inhibitors based on hypothesized contributions to diseases, notably asthma and ulcerative colitis for β-tryptases, heart and kidney scarring for chymases, and airway infection for dipeptidyl peptidase-I. Small-molecule and antibody-based β-tryptase inhibitors showing preclinical promise were tested in early-phase human trials with some evidence of benefit. Chymase inhibitors were given safely in Phase II trials without demonstrating benefits, whereas dipeptidyl peptidase-I inhibitor improved bronchiectasis, in effects likely related to inactivation of the enzyme in neutrophils., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Antimicrobial resistance (AMR) is a global health threat. The dramatic increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections emphasizes the need to find new anti-infective agents with a novel mode of action. The Caseinolytic protease (ClpP) is a central virulence factor in stress survival, virulence, and antibiotic resistance of MRSA. Here, we found ayanin, a flavonoid isolated from Callicarpa nudiflora, was an inhibitor of MRSA ClpP with an IC 50 of 19.63 μM. Using quantitative real-time PCR, ayanin reduced the virulence of Staphylococcus aureus (S. aureus) by down-regulating the level of some important virulence factors, including agrA, RNAⅢ, hla, pvl, psmα and spa. The results of cellular thermal shift assay and thermal shift assay revealed a binding between ayanin and ClpP. Molecular docking showed that ASP-168, ASN-173 and ARG-171 were the potential binding sites for ClpP binding to ayanin. ClpP mutagenesis study further indicated that ARG-171 and ASN-173 were the main active sites of ClpP. The affinity constant (K D ) value of ayanin with ClpP was 3.15 × 10 -5 M measured by surface plasmon resonance. In addition, ayanin exhibited a significant therapeutic effect on pneumonia infection induced by S. aureus in mice in vivo, especially in combination with vancomycin. This is the first report of ayanin with in vivo and in vitro efficacy against S. aureus infection. In conclusion, ayanin is a promising therapeutic agent to combat MRSA infections by targeting ClpP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Feed additives that increase nutrient availability in feeds have gained a lot of interest., Aim: An experiment was conducted to determine whether amylase, protease, and their combined supplementation affected broiler performance., Methods: Two hundred eighty broiler chicks were selected and distributed randomly into 28 replicate pens with four treatment groups and seven replicates under a completely randomized design. A total of four diets were developed, having 0, 100, 100, and 100 + 100 g of control (AP0), amylase (A1), protease (P1), and amylase + protease (AP1)/ton of feed, respectively. Four replicates of each treatment were fed each diet. Each diet was randomly allotted to each group. Ad-libitum feeding was provided to the birds. The feeding program had starter and finisher diets. Upon completion of the experiment, three birds from each pen were slaughtered to analyze the carcass characteristics and organ weight., Results: Differences were insignificant between 100 g/ton of amylase supplementation and FI, body weight gain (BWG), or feed conversion ratio (FCR) ( p > 0.05). Supplementation with 10 0g/ton of protease did not significantly affect FI, BWG, and FCR ( p > 0.05). Similarly, 100 + 100 g/ton of amylase + protease addition had no significant effect on FI, BWG, and FCR ( p > 0.05). None of the treatments significantly affected carcass weight, abdominal fat percentage, dressing percentage, drumstick, wings, breast, and thigh weights ( p > 0.05). In addition, there were no significant effects ( p > 0.05) on the weight of the heart, liver, gizzard, and spleen., Conclusion: In conclusion, amylase, protease, and their combined supplementation at a rate of 100 g/ton of feed did not influence BWG, FI, FCR, carcass characteristics, or organ weight., Competing Interests: The authors declare that there is no conflict of interest.

The COVID-19 pandemic has spurred intense research efforts to identify effective treatments for SARS-CoV-2. In silico studies have emerged as a powerful tool in the drug discovery process, particularly in the search for drug candidates that interact with various SARS-CoV-2 receptors. These studies involve the use of computer simulations and computational algorithms to predict the potential interaction of drug candidates with target receptors. The primary receptors targeted by drug candidates include the RNA polymerase, main protease, spike protein, ACE2 receptor, and transmembrane protease serine 2 (TMPRSS2). In silico studies have identified several promising drug candidates, including Remdesivir, Favipiravir, Ribavirin, Ivermectin, Lopinavir/Ritonavir, and Camostat Mesylate, among others. The use of in silico studies offers several advantages, including the ability to screen a large number of drug candidates in a relatively short amount of time, thereby reducing the time and cost involved in traditional drug discovery methods. Additionally, in silico studies allow for the prediction of the binding affinity of the drug candidates to target receptors, providing insight into their potential efficacy. This study is aimed at assessing the useful contributions of the application of computational instruments in the discovery of receptors targeted in SARS-CoV-2. It further highlights some identified advantages and limitations of these studies, thereby revealing some complementary experimental validation to ensure the efficacy and safety of identified drug candidates.

The study aimed to evaluate the tissue-dissolving ability of papain and bromelain with respect to that of sodium hypochlorite (NaOCl) at the temperatures of 25°C and 60°C. The study also assessed the effects of these proteolytic agents on radicular dentine microhardness. Warming NaOCl, papain and bromelain solutions resulted in significant tissue dissolution at all time intervals (p < 0.001). At 60°C, bromelain showed significantly higher tissue weight loss at every time interval when compared to NaOCl (p < 0.001). All of the three organic tissue dissolvents reduced the microhardness at 1 hr when compared to their respective baseline values. The reduction in microhardness from the baseline reading was statistically significant only in the papain group at 30 min (p = 0.018) and at 60 min (p = 0.03) when compared to the control group. Hence it was concluded that bromelain exerted superior tissue dissolution action, especially when warmed, with minimal effect on dentine microhardness., (© 2022 Australian Society of Endodontology Inc.)

Chronic wound healing is often negatively impacted by infection. Efficient infection assessment is crucial for effective treatment, and biofilm inhibition could improve treatment efficacy. To that end, we developed a bacterial protease-responsive shape memory polymer based on a segmented polyurethane with incorporated poly(glutamic acid) peptide (PU-Pep). Poly(glutamic acid) degrades in response to bacterial proteases to trigger shape recovery of PU-Pep films that are programmed into a secondary shape. These materials have transition temperatures well above body temperature (~60°C), which enables stable storage in temporary shapes after implantation. Synthesized polymers have high shape fixity (~74%-88%), shape recovery (~93%-95%), and cytocompatibility (~100%). Strained PU-Pep samples underwent shape recovery within ≤24 h in response to the V8 enzyme from Staphylococcus aureus (S. aureus, ~50% recovery) and multiple bacteria strains (S. aureus [~40%], Staphylococcus epidermidis [~30%], and Escherichia coli [~25%]), and they had minimal shape change in response to media controls and mammalian cells. Shape recovery of strained PU-Pep samples prevented biofilm formation on the sample surfaces, and resulting attached planktonic bacteria were vulnerable to applied treatments. PU-Pep with physically incorporated antimicrobials simultaneously prevented biofilm formation and killed isolated bacteria. PU-Pep dressings displayed visible shape change and resistance to biofilm formation in in vitro and ex vivo models. In the in vitro model, PU-Pep shape change also disrupted pre-formed biofilm structures. This novel bacterial protease-responsive biomaterial could serve as a wound dressing that changes shape specifically during bacterial colonization to alert clinicians to infection and make biofilm-associated infections easier to treat., (© 2023 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Recently, much emphasis has been placed on solving the intrinsic defects of antimicrobial peptides (AMPs), especially their susceptibility to protease digestion for the systemic application of antibacterial biomaterials. Although many strategies have increased the protease stability of AMPs, antimicrobial activity was severely compromised, thereby substantially weakening their therapeutic effect. To address this issue, we introduced hydrophobic group modifications at the N-terminus of proteolysis-resistant AMPs D1 (AArIIlrWrFR) through end-tagging with stretches of natural amino acids (W and I), unnatural amino acid (Nal) and fatty acids. Of these peptides, N1 tagged with a Nal at N-terminus showed the highest selectivity index (GM SI =19.59), with a 6.73-fold improvement over D1. In addition to potent broad-spectrum antimicrobial activity, N1 also exhibited high antimicrobial stability toward salts, serum and proteases in vitro and ideal biocompatibility and therapeutic efficacy in vivo. Furthermore, N1 killed bacteria through multiple mechanisms, involving disruption of bacterial membranes and inhibition of bacterial energy metabolism. Indeed, appropriate terminal hydrophobicity modification opens up new avenues for developing and applying high-stability peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: To improve the potency and stability of proteolysis-resistant antimicrobial peptides (AMPs) without increasing toxicity, we constructed a convenient and tunable platform based on different compositions and lengths of hydrophobic end modifications. By tagging an Nal at the N-terminal, the obtained target compound N1 exhibited strong antimicrobial activity and desirable stability under multifarious environments in vitro (proteases, salts and serum), and also showed favorable biocompatibility and therapeutic efficacy in vivo. Notably, N1 exerted its bactericidal effect by damaging bacterial cell membranes and inhibiting bacterial energy metabolism in a dual mode. The findings provide a potential method for designing or optimizing proteolysis-resistant AMPs thus promoting the development and application of peptide-based antibacterial biomaterial., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Herein, we described for the first time, an efficient biogenic synthesis of APTs-AgNPs using acid protease from Melilotus indicus leaf extract. The acid protease (APTs) has an essential role in the stabilization, reduction, and capping of APTs-AgNPs. The crystalline nature, size, and surface morphology of APTs-AgNPs were examined using different techniques such as XRD, UV, FTIR, SEM, EDS, HRTEM, and DLS analysis. The generated APTs-AgNPs demonstrated notable performance as dual functionality (photocatalyst and antibacterial disinfection). By destroying 91 % of methylene blue (MB) in <90 min of exposure, APTs-AgNPs demonstrated remarkable photocatalytic activity. APTs-AgNPs also showed remarkable stability as a photocatalyst after five test cycles. Furthermore, the APTs-AgNPs was found to be a potent antibacterial agent with inhibition zones of 30(±0.5 mm), 27(±0.4 mm), 16(±0.1 mm), and 19(±0.7 mm) against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, respectively, under both light and dark conditions. Furthermore, APTs-AgNPs effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, demonstrating their potent antioxidant activity. The outcomes of this study thus demonstrates the dual functionality of APTs-AgNPs produced using the biogenic approach method as a photocatalyst and an antibacterial agent for effective microbial and environmental control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz. , the two-component signal transduction system CpxRA ( c onjugative p ilus e x pression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers., Competing Interests: The authors declare no conflict of interest.

Background: In low- and middle-income countries where most patients receive standardized third-line ART through national programmes, real-world data are scarce. This study was done to assess the long-term survival, and virological and mutational outcomes of people living with HIV receiving third-line ART between July 2016 and December 2019 in an ART centre in India., Methods: Eighty-five patients were started on third-line ART. Genotypic resistance testing to identify drug resistance mutations in the integrase, reverse transcriptase and protease genes was done at the start of third-line therapy, as well as in those who did not attain virological suppression after 12 months of therapy., Results: Survival was 85% (72/85) at 12 months and 72% (61/85) at the end of follow-up in March 2022. Virological suppression was present in 82% (59/72) and 88% (59/67) at 12 months and at the end of follow-up, respectively. Five out of 13 patients who had virological failure at 12 months showed virological suppression at the end of the study. At the start of third-line therapy, 35% (14/40) and 45% (17/38) of patients had major integrase- and protease-associated mutations, respectively, even though they had never been on integrase inhibitor-based regimens. At 1 year follow-up, among those failing third-line therapy, 33% (4/12) of patients had major integrase mutations, but none had major protease mutations., Conclusions: This study demonstrates good long-term outcome in patients on standardized third-line ART in programmatic conditions with very few mutations in those failing the therapy., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

COVID-19 has become a global public health crisis since its outbreak in China in December 2019. Currently there are few clinically effective drugs to combat SARS-CoV-2 infection. The main protein (M pro ), papain-like protease (PL pro ) and RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 are involved in the viral replication, and might be prospective targets for anti-coronavirus drug development. Here, we investigated the antiviral activity of oridonin, a natural small-molecule compound, against SARS-CoV-2 infection in vitro. The time-of-addition analysis showed that oridonin efficiently inhibited SARS-CoV-2 infection by interfering with the genome replication at the post-entry stage. Mechanistically, the inhibition of viral replication by oridonin depends on the oxidation activity of α, β-unsaturated carbonyl. Further experiments showed that oridonin not only effectively inhibited SARS-CoV-2 M pro activity, but also had some inhibitory effects on PL pro -mediated deubiquitinating and viral polymerase-catalyzed RNA elongation activities at high concentrations. In particular, oridonin could inhibit the bat SARS-like CoV and the newly emerged SARS-CoV-2 omicron variants (BA.1 and BA.2), which highlights its potential as a pan-coronavirus antiviral agent. Overall, our data provide strong evidence that oridonin is an efficient antiviral agent against SARS-CoV-2 infection., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). To precisely define the inflammatory mediators, we examined the transcriptomic profile of human retinal endothelial cells exposed to advanced glycation end products, which revealed the neutrophil chemoattractant chemokine CXCL1 as one of the top genes upregulated. The effect of neutrophils in the alteration of the blood-retinal barrier (BRB) was further assessed in wild-type C57BL/6J mice intravitreally injected with recombinant CXCL1 as well as in streptozotocin-induced diabetic mice. Both intravitreally CXCL1-injected and diabetic animals showed significantly increased retinal vascular permeability, with significant increase in infiltration of neutrophils and monocytes in retinas and increased expression of chemokines and their receptors, proteases, and adhesion molecules. Treatment with Ly6G antibody for neutrophil depletion in both diabetic mice as well as CXCL1-injected animals showed significantly decreased retinal vascular permeability accompanied by decreased infiltration of neutrophils and monocytes and decreased expression of cytokines and proteases. CXCL1 level was significantly increased in the serum samples of patients with DR compared with samples of those without diabetes. These data reveal a novel mechanism by which the chemokine CXCL1, through neutrophil recruitment, alters the BRB in DR and, thus, serves as a potential novel therapeutic target., Article Highlights: Intravitreal CXCL1 injection and diabetes result in increased retinal vascular permeability with neutrophil and monocyte recruitment. Ly6G antibody treatment for neutrophil depletion in both animal models showed decreased retinal permeability and decreased cytokine expression. CXCL1 is produced by retinal endothelial cells, pericytes, and astrocytes. CXCL1 level is significantly increased in serum samples of patients with diabetic retinopathy. CXCL1, through neutrophil recruitment, alters the blood-retinal barrier in diabetic retinopathy and, thus, may be used as a therapeutic target., (© 2023 by the American Diabetes Association.)

The application of plant essential oil liposomes to prevent and control food safety risks caused by Campylobacter jejuni (C. jejuni) still faces challenges such as lack of targeting and low release rate. Here, a bacteria-targeted and protease-activated antibacterial liposome (ACCLPs) was successfully synthesized through encapsulation of clove essential oil (CEO) by film dispersion method, embedding of casein by freeze-thaw method, and conjugation of C. jejuni antibody on the liposome membrane by post-insertion method. The average particle size, the essential oil encapsulation rate, the casein mosaic rate, and the antibody coupling efficiency of ACCLPs were determined as185.87 nm,16.9%,70.1% and 87.5%, respectively. The modification with C. jejuni antibody could significantly improve the targeting of ACCLPs to C. jejuni. Controlled release experiments showed that the exocrine protease from C. jejuni could hydrolyze the embedded casein and perforation on the ACCLPs, thus leading to a bacteria-dependent CEO release and significant prolonging the antibacterial effects of ACCLPs. Application results of ACCLPs on C. jejuni-contaminated foods showed that ACCLPs could effectively inhibit C. jejuni in a variety of meat products, fruits and vegetables and extend their shelf life without significantly affecting food quality. The results above in this work would provide a new view for the development of high efficient liposome-based antibacterial system of plant essential oil., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: To explore whether nuclear factor I C (NFIC) alleviated inflammatory response of synovial fibroblasts (SFs) caused by rheumatoid arthritis (RA) by regulating transcription levels of phosphatase and tension homolog deleted on chromosome 10 (PTEN) and sentrin-specific protease 8 (SENP8)., Methods: NFIC, PTEN, and SENP8 levels in RASFs and normal SFs (NSFs) were measured by qRT-PCR and western blotting. The levels of Bax, Bcl-2, MMP-3, and MMP-13, as well as the content of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined in RASFs and NSFs using western blotting and ELISA. The binding of NFIC to promoter sequences of PTEN and SENP8 was predicted and verified. A mouse model of collagen-induced arthritis (CIA) was established and evaluated according to the degree of joint swelling and arthritis index., Results: NFIC, PTEN, and SENP8 were downregulated in RASFs. RASFs had increased viability and MDA levels as well as decreased cell apoptosis and SOD content. NFIC was demonstrated to modulate the transcription of PTEN and SENP8 as their transcription factor. NFIC ameliorated the inflammatory response induced by RA in vivo by promoting the transcription of PTEN and SENP8., Conclusion: NFIC acted as a transcription factor to facilitate the transcription of PTEN and SENP8, thereby inducing apoptosis of RASFs and effectively attenuating inflammatory response in CIA mice., Competing Interests: Declaration of interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Given the limited options and often harmful side effects of current analgesics and the suffering caused by the opioid crisis, new classes of pain therapeutics are needed. Protease-activated receptors (PARs), particularly PAR2, are implicated in a variety of pathologies, including pain. Since the discovery of the role of PAR2 in pain, development of potent and specific antagonists has been slow. In this study, we describe the in vivo characterization of a novel small molecule/peptidomimetic hybrid compound, C781, as a β-arrestin-biased PAR2 antagonist. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. Pharmacokinetic studies were done to assess pharmacokinetic/pharmacodynamic relationship in vivo. We used both prevention and reversal paradigms with protease treatment to determine whether C781 could attenuate protease-evoked pain. C781 effectively prevented and reversed mechanical and spontaneous nociceptive behaviors in response to small molecule PAR2 agonists, mast cell activators, and neutrophil elastase. The ED 50 of C781 (intraperitoneal dosing) for inhibition of PAR2 agonist (20.9 ng 2-AT)-evoked nociception was 6.3 mg/kg. C781 was not efficacious in the carrageenan inflammation model. Pharmacokinetic studies indicated limited long-term systemic bioavailability for C781 suggesting that optimizing pharmacokinetic properties could improve in vivo efficacy. Our work demonstrates in vivo efficacy of a biased PAR2 antagonist that selectively inhibits β-arrestin/MAPK signaling downstream of PAR2. Given the importance of this signaling pathway in PAR2-evoked nociception, C781 exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development. PERSPECTIVE: Our work provides evidence that PAR2 antagonists that only block certain aspects of signaling by the receptor can be effective for blocking protease-evoked pain in mice. This is important because it creates a rationale for developing safer PAR2-targeting approaches for pain treatment., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteostasis, i.e., the homeostasis of proteins, responsible for ensuring protein turnover, is regulated by proteases, which also participate in the etiopathogenesis of multiple conditions. The magic of proteases is such that, in blood coagulation, one same molecule, such as coagulation factor V, for example, can perform both a procoagulant and an anticoagulant function as a result of the activity of proteases. However, this magic has an insidious side to it, as it may also prevent the completion of the clinical value chain of factor V deficiency. This value chain encompasses the discovery of knowledge, the transfer of this knowledge, and its translation to clinical practice. In the case of rare and ultra-rare diseases like factor V deficiency, this value chain has not been completed as the knowledge acquisition phase has dragged out over time, holding up the transfer of knowledge to clinical practice. The reason for this is related to the small number of patients afflicted with these conditions. As a result, new indications must be found to make the therapies cost-effective. In the case of factor V, significant research efforts have been directed at developing a recombinant factor V capable of resisting the action of the proteases capable of inactivating this factor. This is where bioethics and health equity considerations come into the equation.

The anti-cancer activities of the compounds were evaluated against KYSE-150, KYSE-30, and KYSE-270 cell lines and also on investigated esophageal line HET 1 A as a standard. Modified inhibitory impact on enzymes of collagenase and elastase were used Thring and Moon methods, respectively. Among both compounds, both of them recorded impact on cancer cells being neutral against the control, both had IC50 lower than 100 µM and acted as a potential anticancer drug. The chemical activities of Skullcapflavone I and Skullcapflavone II against elastase and collagenase were investigated utilizing the molecular modeling study. IC50 values of Skullcapflavone I and Skullcapflavone II on collagenase enzyme were obtained 106.74 and 92.04 µM and for elastase enzyme were 186.70 and 123.52 µM, respectively. Anticancer effects of these compounds on KYSE 150, KYSE 30, and KYSE 270 esophageal cancer cell lines studied in this work. For Skullcapflavone I, IC50 values for these cell lines were obtained 14.25, 19.03, 25.10 µM, respectively. Also, for Skullcapflavone II were recorded 20.42, 34.17, 22.40 µM, respectively. The chemical activities of Skullcapflavone I and Skullcapflavone II against some of the expressed surface receptor proteins (CD44, EGFR, and PPARγ) in the mentioned cell lines were assessed using the molecular docking calculations. The calculations showed the possible interactions and their characteristics at an atomic level.

Soil bacteria participate in self-immobilization processes for survival, persistence, and production of virulence factors in some niches or hosts through their capacities for autoaggregation, cell surface hydrophobicity, biofilm formation, and antibiotic and heavy metal resistance. This study investigated potential virulence, antibiotic and heavy metal resistance, solvent adhesion, and biofilm-forming capabilities of six cellulolytic bacteria isolated from soil samples: Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Strains were subjected to phenotypic methods, including heavy metal and antibiotic susceptibility and virulence factors (protease, lipase, capsule production, autoaggregation, hydrophobicity, and biofilm formation). The effect of ciprofloxacin was also investigated on bacterial susceptibility over time, cell membrane, and biofilm formation. Strains MKAL2, MKAL5, and MKAL6 exhibited protease and lipase activities, while only MKAL6 produced capsules. All strains were capable of aggregating, forming biofilm, and adhering to solvents. Strains tolerated high amounts of chromium, lead, zinc, nickel, and manganese and were resistant to lincomycin. Ciprofloxacin exhibited bactericidal activity against these strains. Although the phenotypic evaluation of virulence factors of bacteria can indicate their pathogenic nature, an in-depth genetic study of virulence, antibiotic and heavy metal resistance genes is required., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

The genomic reshuffling, mutagenicity, and high transmission rate of the SARS-CoV-2 pathogen highlights an urgent need for effective antiviral interventions for COVID-19 control. Targeting the highly conserved viral genes and/or gene-encoded viral proteins such as main proteinase (M pro ), RNA-dependent RNA polymerase (RdRp) and helicases are plausible antiviral approaches to prevent replication and propagation of the SARS-CoV-2 infection. Coronaviruses (CoVs) are prone to extensive mutagenesis; however, any genetic alteration to its highly conserved M pro enzyme is often detrimental to the viral pathogen. Therefore, inhibitors that target the M pro enzyme could reduce the risk of mutation-mediated drug resistance and provide effective antiviral protection. Several existing antiviral drugs and dietary bioactives are currently repurposed to treat COVID-19. Dietary bioactives from three ayurvedic medicinal herbs, 18 β-glycyrrhetinic acid (Δ G  = 8.86 kcal/mol), Solanocapsine (Δ G  = 8.59 kcal/mol), and Vasicoline (Δ G  = 7.34 kcal/mol), showed high-affinity binding to M pro enzyme than the native N3 inhibitor (Δ G  = 5.41 kcal/mol). Flavonoids strongly inhibited SARS-CoV-2 M pro with comparable or higher potency than the antiviral drug, remdesivir. Several tannin hydrolysates avidly bound to the receptor-binding domain and catalytic dyad (His 41 and Cys 145 ) of SARS-CoV-2 M pro through H-bonding forces. Quercetin binding to M pro altered the thermostability of the viral protein through redox-based mechanism and inhibited the viral enzymatic activity. Interaction of quercetin-derivatives with the M pro seem to be influenced by the 7-OH group and the acetoxylation of sugar moiety on the ligand molecule. Based on pharmacokinetic and ADMET profiles, several phytonutrients could serve as a promising redox nutraceutical for COVID-19 management.

The objective of this study was to investigate the effects of feeding rice gluten meal (RGM) as an alternative protein source along with protease enzyme supplementation on growth performance, expression of nutrient transporter genes, nutrient digestibility, immune response and gut histomorphometry of broiler chicken. Proximate analysis of RGM revealed 923 g dry matter (DM), 500 g crude protein (CP), 69.2 g ether extract, 94.7 g crude fiber, 215.4 g nitrogen-free extract, 43.7 g ash, 6.20 g calcium, 7.80 g total phosphorus, 18.99 MJ gross energy and 12.68 MJ metabolizable energy per kg diet. Significant upregulation of nutrient transporter genes (PepT1, EAAT3 and mucin) and better growth performance was observed in the birds fed control diet which was statistically similar to the birds fed 150 g RGM compared to birds fed higher RGM levels. Histomorphometry of jejunum, nutrient digestibility, and immune response of birds did not reveal any significant effect of RGM or protease enzyme supplementation. However, the inclusion of RGM up to 150 g/kg diet resulted in significant decline of feed cost/kg live weight gain, dressed meat yield and eviscerated meat yield by 13.13%, 12.99% and 13.36%, respectively compared to control. Thus, it was concluded that the inclusion of 150 g RGM/kg diet in broiler chicken ration has no adverse effects on the growth pattern of birds and can be used for least-cost feed formulation for chicken.

Aphid (Aphis gossypii) is one of the important pests of papaya crop. In this work, applications of Trichoderma harzianum and Beauveria bassiana (biocontrol agents) and malathion (insecticide) were conducted in vitro and in agrifields for testing their anti-aphid efficacy and compared their efficacy. Furthermore, the enzymatic mechanism of T. harzianum with respect to biocontrolling the pest was unearthed. The LD 50 dose of T. harzianum and B. bassiana was 1.2 × 10 5 spores mL -1 and 1.0 × 10 6 spores mL -1 respectively after 48 h of administration. The LT 50 of T. harzianum also exhibited a lower effective time (47.70 h) than B. bassiana (57.53 h) for the same concentration of spores applied (1 × 10 5 spores mL -1 ). The pooled data analysis of two years (2019-2020) showed that the application of T. harzianum spores in agrifields exhibited 31.75 ± 13.00a percentage of reduction of aphid population whereas malathion exhibited 23.93 ± 1.30a%, in comparison to control. The statistical analysis indicated that the application of malathion exhibited the same efficacy as T. harzianum isolate and placed in the same category. In plate detection assay, T. harzianum produced a higher hydrolytic zone for chitinase (8.0 ± 0.4 cm diameter) and protease (7.0 ± 0.4 cm diameter) enzymes, than B. bassiana (1.3 ± 0.2 cm and 1.1 ± 0.2 cm respectively). Quantitative estimation of enzymes exhibited that T. harzianum produced 299 ± 11a μg mL -1 of chitinase, 519 ± 19a μg mL -1 of protease, and 65 ± 12a μg mL -1 of PR1, and on the other hand, B. bassiana yielded 124 ± 12b, 361 ± 23b, and 29 ± 18b μg mL -1 of chitinase, protease, and PR1 respectively. It indicated that T. harzianum was superior over the B. bassiana in terms of production capacity of all three enzymes. In conclusion, all the above experimental results suggested that T. harzianum showed better aphid-killing efficacy than B. bassiana. It also suggested that T. harzianum should replace hazardous chemical pesticide (malathion) for eco-friendly biocontrol of aphids., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objectives: To investigate whether the adhesive-dentin interface stability of primary teeth would be enhanced by epigallocatechin-3-gallate (EGCG) with ethanol wet-bonding., Methods: Non-caries primary molars were sliced to achieve a flat dentin surface and etched then randomly distributed into five groups in accordance with different treatments: group 1, no treatment; group 2, applying absolute ethanol wet-bonding for 60 s; groups 3-5, applying 0.1%, 0.5%, and 1% (w/v) EGCG-incorporating ethanol wet-bonding (0.1%, 0.5%, and 1% EGCG) for 60 s. Singlebond universal adhesive was then applied followed by resin composite construction. Microtensile bond strength, fracture mode, and nanoleakage at adhesive-dentin interface were evaluated after 24 h of water storage or 10,000 times of thermocycling. Zymography of hybrid layer, biofilm formation of Streptococcus mutans by CLSM, FESEM, and MTT test, and cytotoxicity by CCK-8 assay were respectively assessed., Results: Irrespective of thermocycling, the dentin bond strength was preserved with reduced nanoleakage in the 0.5% and 1% EGCG groups. Furthermore, the activity of endogenous proteases and the growth of Streptococcus mutans biofilm were inhibited after treatment with 0.5% and 1% EGCG/ethanol solutions (groups 4 and 5). CCK-8 results of the 0.1% and 0.5% EGCG groups showed acceptable biocompatibility., Conclusions: Treatment by EGCG/ethanol solutions effectively enhanced the bond stability of primary teeth at the adhesive-dentin interface., Clinical Significance: Synergistic application of EGCG and ethanol wet-bonding suggesting a promising strategy to improve dentin bonding durability with bacterial biofilm inhibition, thus increasing resin-based restorations' service life in primary dentition., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier Ltd.)

The biggest application bottleneck of antimicrobial peptides (AMPs) is the low oral bioavailability caused by the poor stability of digestive enzymes in the gastrointestinal tract. However, the research methods and evaluation criteria of available studies about anti-proteolytic strategies are not uniform and far from the actual environment in vivo. Here, we developed a research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving the protease stability of AMPs on the same platform for the first time. After a comprehensive analysis, Dab modification is identified as the most effective strategy to improve the trypsin stability of AMPs. By further modulating the proteolytic resistance optimization motif (DabW) n , U1-2WD is obtained with ideal stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which forms amorphous aggregates in the bacteria environment to trigger the agglutination of bacterial cells to prevent bacterial escape. It then kills bacteria by disrupting bacterial membranes and inhibiting bacterial energy metabolism. Overall, our work has led to a new understanding of the effectiveness of proteolytic resistance strategies and accelerated the development of anti-proteolytic AMPs to combat multidrug-resistant bacterial infections. STATEMENT OF SIGNIFICANCE: We developed research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving protease stability of AMPs on the same platform for the first time. we found effective strategies to resist trypsin hydrolysis: modification with backbone (β-Arg), D-enantiomer (D-Arg) and L-2,4-diaminobutanoic acid (Dab). Further, the proteolytic resistance optimization motif (DabW) n was designed. When n=3, derivative U1-2WD was obtained with desirable stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which can self-aggregate into amorphous aggregates in the bacteria environment to mediate the agglutination and sedimentation of bacterial cells to prevent bacterial escape, and then kill bacteria by destroying bacterial membranes and inhibiting bacterial energy metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Pseudomonas aeruginosa is an opportunistic pathogen that causes several serious health problems and numerous forms of virulence. During the treatment of P. aeruginosa infections, the development of multidrug-resistant isolates creates significant clinical problems. Using antivirulence compounds to disrupt pathogenicity rather than killing the bacterium may be an interesting strategy to overcome this problem, because less harsh conditions will exist for the development of resistance. To reduce pathogenicity and biofilm formation, newly synthesized analogs of imidazolyl ( 8n ) and previously synthesized analogs ( 8a-8m ) with a similar backbone [the 5-(imidazolyl-methyl) thiazolidinediones] were tested against pyoverdine and pyocyanin production, protease activity, and biofilm formation. Compared to the positive control group, the best compounds reduced the production of pyoverdine ( 8n ) by 89.57% and pyocyanin ( 8i ) by 22.68%, and protease activity ( 8n ) by 2.80% for PAO1 strain, at a concentration of 10 μM. Moreover, the biofilm formation assay showed a reduction of 87.94% ( 8i) for PAO1, as well as 30.53% ( 8d ) and 44.65% ( 8m ) for 1074 and 1707 strains, respectively. The compounds used in this study did not show any toxicity in the human dermal fibroblasts and 4T1 cells (viability higher than 90%). The in silico study of these compounds revealed that their antivirulence activity could be due to their interaction with the PqsR, PqsE, and LasR receptors.

Study Question: Is 17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, a better spermicide in cervicovaginal fluid (CVF) than its parental peptides, LL-37 and GF-17?, Summary Answer: At the same mass concentration, 17BIPHE2 exhibited the highest spermicidal activity on human sperm resuspended in CVF-containing medium., What Is Known Already: LL-37 and its truncated peptide GF-17 exert both spermicidal and microbicidal activities, although they are prone to proteolytic degradation in body fluids., Study Design, Size, Duration: Spermicidal activities of 17BIPHE2 were evaluated in vitro in mouse and human sperm, both resuspended in medium, and then on human sperm incubated in CVF-containing medium; in the latter condition, the spermicidal activity and peptide stability in CVF of 17BIPHE2 were compared with that of LL-37 and GF-17. The in vivo contraceptive effects of 17BIPHE2 and the reversibility thereof were then assessed in mice. Finally, in vitro microbicidal effects of 17BIPHE2 on Neisseria gonorrhoeae were determined., Participants/materials, Setting, Methods: Sperm motility and plasma membrane integrity were assessed by videomicroscopy and exclusion of Sytox Green, a membrane-impermeable fluorescent dye, respectively. Successful in vitro fertilization (IVF) was determined by the presence of two pronuclei in oocytes following their coincubation with capacitated untreated or 17BIPHE2-treated sperm. Sperm alone or with 17BIPHE2 were transcervically injected into female mice and successful in vivo fertilization was indicated by the formation of two-cell embryos 42-h postinjection, and by pregnancy through pup delivery 21-25 days afterwards. Peptide intactness was assessed by immunoblotting and HPLC. Reversibility of the contraceptive effects of 17BIPHE2 was evaluated by resumption of pregnancy of the female mice, pretranscervically injected with 17BIPHE2, following natural mating with fertile males. Minimum inhibitory/bactericidal concentrations of 17BIPHE2 on N. gonorrhoeae were obtained through microdilution broth assay., Main Results and the Role of Chance: At the same mass concentration, 17BIPHE2 was a more effective spermicide than LL-37 or GF-17 on human sperm resuspended in CVF-containing medium, with the spermicidal concentration of 32.4 µM. This was mainly due to lower susceptibility of 17BIPHE2 to CVF proteases. Importantly, the reproductive tract of mouse females treated three times with 32.4 µM 17BIPHE2 remained normal and their fecundity resumed after stopping 17BIPHE2 treatment., Limitations, Reasons for Caution: For ethical reasons, the inhibitory effects of 17BIPHE2 on fertilization and pregnancy cannot presently be performed in women. Also, while our study has proven the effectiveness of 17BIPHE2 as a spermicide for mouse and human sperm in vitro, dosage formulation (e.g. in hydrogel) of 17BIPHE2 still needs to be developed to allow 17BIPHE2 to remain in the vagina/uterine cavity with controlled release for its spermicidal action., Wider Implications of the Findings: Since 17BIPHE2 also exerted bactericidal activity against N. gonorrhoeae at its spermicidal concentration, it is a promising candidate to be developed into a vaginal multipurpose prevention technology agent, thus empowering women against unplanned pregnancies and sexually transmitted infections., Study Funding/competing Interest(s): This work was supported by the Canadian Institutes of Health Research (PJT 173268 to N.T.). There are no competing interests to declare., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Serratia marcescens (S. marcescens) is an environmental bacterium that causes infections with high morbidity and mortality. Notably, infections caused by multidrug-resistant S. marcescens have become a global public health issue. Therefore, the discovery of promising compounds to reduce the virulence of pathogens and restore antibiotic activity against multidrug-resistant bacteria is critical. Quorum sensing (QS) regulates virulence factors and biofilm formation of microorganisms to increase their pathogenicity and is, therefore, an important factor in the formation of multidrug resistance. In this study, we found that 3-phenylpropan-1-amine (3-PPA) inhibited S. marcescens NJ01 biofilm formation and virulence factors, including prodigiosin, protease, lipase, hemolysin, and swimming. The combination of 3-PPA (50.0 μg/mL) and ofloxacin (0.2 μg/mL) enhanced S. marcescens NJ01 sensitivity to ofloxacin. Based on crystalline violet staining, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), 3-PPA (50.0 μg/mL) reduced S. marcescens NJ01 biofilm formation by 48%. Quantitative real-time PCR (qRT-PCR) showed that 3-PPA regulated the expression of virulence- and biofilm-related genes fimA , fimC , bsmB , pigP , flhC , flhD , and sodB . Liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that 3-PPA affected intracellular metabolites of S. marcescens NJ01, leading to reduce metabolic activity. These results suggested that 3-PPA inhibits the pathogenicity of S. marcescens NJ01 by occluding QS. Thus, 3-PPA is feasible as an ofloxacin adjuvant to overcome multidrug-resistant S. marcescens and improve the treatment of intractable infections. IMPORTANCE Multidrug-resistant bacteria have become a major threat to global public health, leading to increased morbidity, mortality, and health care costs. Bacterial virulence factors and biofilms, which are regulated by quorum sensing (QS), are the primary causes of multidrug resistance. In this study, 3-PPA reduced virulence factors and eliminated biofilm formation by inhibiting QS in S. marcescens NJ01 bacteria, without affecting bacterial growth, thus restoring sensitivity to ofloxacin. Thus, the discovery of compounds that can restore antibiotic activity against bacteria is a promising strategy to mitigate multidrug resistance in pathogens.

Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 μg/mL) as well as minimum fungicidal concentrations (2 to 32 μg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC 50 ) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 μg/mL and 32 μg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1 , and ALS1 , were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB , and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Indole signaling is viewed as a potential target for antivirulence therapy against antibiotic-resistant pathogens because of its link with the production of virulence factors. This study examined the antimicrobial and antivirulence properties of 44 indoles toward Vibrio campbellii. Based on the results, 17 halogenated indole analogues were selected, as they significantly improved the survival of brine shrimp larvae challenged with V. campbellii . Specifically, 6-bromoindole, 7-bromoindole, 4-fluoroindole, 5-iodoindole, and 7-iodoindole showed a high protective effect, improving the survival of brine shrimp to over 80% even at a low concentration of 10 μM. To explore the impact of selected indole analogues on bacterial virulence phenotypes, swimming motility, biofilm formation, protease activity, and hemolytic activity of V. campbellii were determined. The results showed that all of the 17 selected indole analogues decreased swimming motility at both 10 μM and 100 μM. Most of the indole analogues decreased biofilm formation at a concentration of 100 μM. In contrast, only a slightly decreased protease activity and no effect on hemolytic activity were observed at both concentrations. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system, and the results demonstrate the potential of these compounds in applications aiming at the protection of shrimp from vibriosis, a major disease in aquaculture. IMPORTANCE Bacterial diseases are a major problem in the aquaculture industry. In order to counter this problem, farmers have been using antibiotics, and this has led to the evolution and spread of antibiotic resistance. In order for the aquaculture industry to further grow in a sustainable way, novel and sustainable methods to control diseases are needed. We previously reported that indole signaling is a valid target for the development of novel therapies to control disease caused by Vibrio campbellii and related bacteria, which are among the major bacterial pathogens in aquaculture. In the present study, we identified indole analogues that are more potent in protecting brine shrimp (a model organism for shrimp) from V. campbellii . To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system.

Antibiotic-resistant bacteria have become a public health problem. Thus, antimicrobial peptides (AMPs) have been evaluated as substitutes for antibiotics. Herein, we investigated PN5 derived from Pinus densiflora (pine needle). PN5 exhibited antimicrobial activity without causing cytotoxic effects. Based on these results, we examined the mode of action of PN5 against Gram-negative and -positive bacteria. PN5 exhibited membrane permeabilization ability, had antimicrobial stability in the presence of elastase, a proteolytic enzyme, and did not induce resistance in bacteria. Bacterial lipopolysaccharide (LPS) induces an inflammatory response in RAW 264.7 macrophages. PN5 suppressed proinflammatory cytokines mediated by NF-κB and mitogen-activated protein kinase signaling. In C57BL/6J mice treated with LPS and d-galactosamine, PN5 exhibited anti-inflammatory activity in inflamed mouse livers. Our results indicate that PN5 has antimicrobial and anti-inflammatory activities and thus may be useful as an antimicrobial agent to treat septic shock caused by multidrug-resistant (MDR) Escherichia coli without causing further resistance. IMPORTANCE Antibiotic-resistant bacteria are a global health concern. There is no effective treatment for antibiotic-resistant bacteria, and new alternatives are being suggested. The present study found antibacterial and anti-inflammatory activities of PN5 derived from Pinus densiflora (pine needle), and further investigated the therapeutic effect in a mouse septic model. As a mechanism of antibacterial activity, PN5 exhibited the membrane permeabilization ability of the toroidal model, and treated strains did not develop drug resistance during serial passages. PN5 showed immunomodulatory properties of neutralizing LPS in a mouse septic model. These results indicate that PN5 could be a new and promising therapeutic agent for bacterial infectious disease caused by antibiotic-resistant strains.

Ilamycins/rufomycins are marine cycloheptapeptides containing unusual amino acids. Produced by Streptomyces sp., these compounds show potent activity against a range of mycobacteria, including multidrug-resistant strains of Mycobacterium tuberculosis . The cyclic peptides target the AAA+ protein ClpC1 that, together with the peptidases ClpP1/ClpP2, forms an essential ATP-driven protease. Derivatives of the ilamycins with a simplified tryptophane unit are synthesized in a straightforward manner. The ilamycin derivative 26 with a cyclic hemiaminal structure is active in the nM-range against several mycobacterial strains and shows no significant cytotoxicity. In contrast, derivative 27 , with a glutamic acid at this position, is significantly less active, with MICs in the mid µM-range. Detailed investigations of the mode of action of 26 indicate that 26 deregulates ClpC1 activity and strongly enhances ClpC1-WT ATPase activity. The consequences of 26 on ClpC1 proteolytic activities were substrate-specific, suggesting dual effects of 26 on ClpC1-WT function. The positive effect relates to ClpC1-WT ATPase activation, and the negative to competition with substrates for binding to the ClpC1 NTD.

The assembly of inflammasomes drives caspase-1 activation, which further promotes proinflammatory cytokine secretion and downstream pyroptosis. The discovery of novel caspase-1 inhibitors is pivotal to developing new therapeutic means for inflammasome-involved diseases. In our present study, sennoside A (Sen A), a popular ingredient in multiple weight-loss medicines and dietary supplements, is found to potently inhibit the enzymatic activity of caspase-1 in vitro . Sen A considerably decreased IL-1β production in macrophages stimulated by LPS plus ATP, nigericin or MSU as well as poly(dA:dT) transfection, and remedied ROS-involved pyroptosis via caspase-1 inhibition. Mechanistically, Sen A not only suppressed the assembly of both NLRP3 and AIM2 inflammasome but also affected the priming process of NLRP3 inflammasome by blocking NF-κB signaling. Sen A significantly ameliorated the pathophysiological effect in LPS-, MSU- and carrageenan-challenged rodent models by suppressing inflammasome activation. Furthermore, P2X7 was indispensable for Sen A inhibiting NLRP3 inflammasome since it failed to further decrease IL-1β and IL-18 production in LPS plus ATP-stimulated BMDMs that were transfected with P2X7 siRNA. Sen A also restrained the large pore-forming functionalities of the P2X7R as verified by the YO-PRO-1 uptake assay. Taken together, Sen A inactivates caspase-1 to inhibit NLRP3 and AIM2 inflammasome-involved inflammation in a P2X7-dependent manner, making it an attractive candidate as a caspase-1 small-molecular inhibitor.

Biocontrol microbes are environment-friendly and safe for humans and animals. To seek biocontrol microbes effective in suppressing tomato gray mold is important for tomato production. Therefore, serial experiments were conducted to characterize the antagonism of Bacillus velezensis HY19, a novel self-isolated biocontrol bacterium, against Botrytis cinerea in vitro and the control on tomato gray mold in greenhouse. This bacterium produced extracellular phosphatase, protease, cellulose and siderophores, and considerably inhibited the growth of B. cinerea. A liquid chromatography-mass spectrometry (LC-MS) detected salicylic acid and numerous antifungal substances present in B. velezensis HY19 fermentation liquid (BVFL). When B. cinerea was grown on potato glucose agar, BVFL crude extract remarkably suppressed the fungal growth and reduced protein content and the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Transcriptome studies showed that BVFL crude extract significantly induced different expression of numerous genes in B. cinerea, most of which were down-regulated. Theses differently expressed genes were involved in the biological process, cell compartment, molecular functions, and metabolisms of glycine, serine, threonine, and sulfur in pathogen hyphae. Thus, this biocontrol bacterium antagonized B. cinerea in multiple ways due to the production of numerous antifungal substances that acted on multiple targets in the cells. BVFL significantly increased antioxidant enzyme activities in tomato leaves and decreased the incidence of tomato gray mold, with the control efficacies of 73.12-76.51%. Taken together, B. velezensis HY19 showed a promising use potential as a powerful bioagent against tomato gray mold., Competing Interests: Declaration of Competing Interest The work described here was not published previously, and is not under consideration for publication elsewhere. There is no conflict of interest in the submission of this manuscript. The manuscript has been approved for publication by all authors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs) are being developed as potent alternative treatments to conventional antibiotics which are unlikely to induce bacterial resistance. They can be designed and modified to possess several druggable properties. We report herein a novel hybrid peptide of modified aurein (A3) and cathelicidin (P7), or A3P7, by a flipping technique. It exhibited potent antibacterial activity against both Gram-negative and -positive pathogenic bacteria but had moderate hemolytic activity. To reduce the sequence length and toxicity, C-terminal truncation was serially performed and eight truncated derivatives (AP12-AP19) were obtained. They had significantly less hemolytic activity while preserving antibacterial activity. Secondary structures of the candidate peptides in environments simulating bacterial membranes (30 mM SDS and 50% TFE), determined by CD spectroscopy, showed α-helical structures consistent with predicted in silico 3D structural models. Among the peptides, AP19 demonstrated the best combination of broad-spectrum antibacterial activity (including toward Acinetobacter baumannii) and minimal hemolytic and cytotoxic activities. A D-form peptide (D-AP19), in which all L-enantiomers were substituted with the D-enantiomers, maintained antibacterial activity in the presence of pepsin, trypsin, proteinase K and human plasma. Both isomers exhibited potent antibacterial activity against multi-drug (MDR) and extensively-drug resistant (XDR) clinical isolates of A. baumannii comparable to the traditional antibiotic, meropenem. D-AP19 displayed rapid killing via membrane disruption and leakage of intracellular contents. Additionally, it showed a low tendency to induce bacterial resistance. Our work suggested that D-AP19 could be further optimized and developed as a novel compound potentially for fighting against MDR or XDR A. baumannii., (© 2022. The Author(s).)

Background: Interleukin-1 (IL-1) signaling has an established role as a cytokine signaling pathway important for progression of abdominal aortic aneurysms (AAAs). While the IL-1β ligand and IL-1R1 have been previously investigated, the role of the IL-1α ligand in AAAs remains unknown. In this study, we sought to examine the role of IL-1α in AAAs using genetic and pharmacologic approaches., Methods: Eight-week-old wild-type (WT) or IL-1α knock-out (KO) male and female mice (n = 10-16/group) underwent experimental AAA and were harvested 14 days following surgery to assess AAA size and characteristics. In separate studies, 8-week-old WT mice were treated with an inhibitor to IL-1α during AAA formation and harvested 14 days following surgery. Finally, WT and IL-1α KO mice were administered Anakinra, an IL-R1 inhibitor, during AAA formation to determine the effect of inhibiting IL-1R1 when IL-1α is knocked out., Results: Male and female IL-1α KO mice had larger AAAs compared to WT AAAs (male: 153% vs. 89.2%, P = 0.0001; female: 86.6% vs. 63.5%, P = 0.02). IL-1α KO mice had greater elastin breakage (P = 0.01), increased levels of macrophage staining (P = 0.0045), and greater pro-metallo proteinase 2 (P = 0.02). Pharmacologic inhibition of WT male mice with an IL-1α neutralizing antibody resulted in larger AAAs (133.1% vs. 77.0%, P < 0.001). Finally, treatment of IL-1α KO male mice with Anakinra decreased AAA formation compared with vehicle control AAAs (Anakinra + IL-1α KO: 47.7% vs. WT: 147.1%; P = 0.0001)., Conclusions: IL-1α disruption using either genetic or pharmacologic approaches worsens AAAs., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Drug-resistant bacteria was the third leading cause of death worldwide in 2019, which sounds like a cautionary note for global public health. Therefore, developing novel strategies to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections is the need of the hour. Caseinolytic protease P (ClpP) represents pivotal microbial degradation machinery in MRSA involved in bacterial homeostasis and pathogenicity, considered an ideal target for combating S. aureus infections. Herein, we identified a natural compound, hinokiflavone, that inhibited the activity of ClpP of MRSA strain USA300 with an IC 50 of 34.36 μg/mL. Further assays showed that hinokiflavone reduced the virulence of S. aureus by inhibiting multiple virulence factors expression. Results obtained from cellular thermal transfer assay (CETSA), thermal shift assay (TSA), local surface plasmon resonance (LSPR) and molecular docking (MD) assay enunciated that hinokiflavone directly bonded to ClpP with confirmed docking sites, including SER-22, LYS-26 and ARG-28. In vivo , the evaluation of anti-infective activity showed that hinokiflavone in combination with vancomycin effectively protected mice from MRSA-induced fatal pneumonia, which was more potent than vancomycin alone. As mentioned above, hinokiflavone, as an inhibitor of ClpP, could be further developed into a promising adjuvant against S. aureus infections.

The pharmacological effects of curcumin can be ascribed to its dose-dependent activity. Therapeutic application of curcumin is hindered by its poor solubility and low bioavailability. Carbon dots are gaining attention in biomedical applications in view of their unique photo-physical properties. Some carbon dots derived from bioactive molecules have shown superior activity than the parent compound. With an aim to address the limitations of curcumin, herein we compared the wound healing activity of curcumin-derived carbon dots (CurCD) with curcumin. The improved solubility and stability of CurCD, combined with its superior proliferative, proangiogenic and anti-bacterial activity suggested that CurCD would be more beneficial than curcumin in wound healing. To enable the sustained release of CurCD at the wound site, a protease-responsive hydrogel (GHCD) was prepared with CurCD acting as a cross-linker. A comparative study using a skin excision model revealed that GHCD supported faster wound closure with improved angiogenesis and complete restoration of the epithelium. Apart from the establishment of CurCD as a wound healing agent, the study provides a novel carbon dot based approach for molecules with limitations of solubility and bioavailability., (Copyright © 2022 Elsevier B.V. All rights reserved.)