Cathepsin G Research Articles

Structural and functional properties of protein hydrolysates from myofibrillar protein of crocodile (Crocodylus siamensis) meat

In this study, three proteases (papain, neutral protease, bromelain) were employed to hydrolyze crocodile (Crocodylus siamensis) myofibrillar protein (MP), effects of hydrolysis duration on functional and structural properties of MP hydrolysates was investigated. The findings revealed that solubility of all hydrolysates exceeded 60%, with the highest reaching 96.7%. The emulsifying activity of neutral protease hydrolysates exhibited a significant superiority over the other two hydrolysates (P < 0.05). The Fe2+ chelating and DPPH radical scavenging activity of hydrolysates ranged from 20% to 60%, and 60%–90%, respectively. Based on the experiment results, the hydrolysates of papain at 1h, neutral protease at 1h, and bromelain at 2h were used for structural analysis. Compared to MP, the secondary structure of hydrolysates showed a reduction of 9.6%, 15.2%, and 4.7% in α-helix and the increase of 42.4%, 29.6%, and 44.2% in β-fold content of papain, neutral protease and bromelain hydrolysates respectively, suggesting the transition from spiral to folding structure and the exposure of hydrophobic group, which was proved by free amino acid analysis. SEM results confirmed the alterations in MP structure. The molecular weight distributions of three hydrolysates were predominantly below 1000 Da, demonstrating that crocodile MP was effectively hydrolyzed into bioactive peptides.

Effect of Glucose Concentration on the Production of Proteolytic Extract by Different Strains of Aspergillus under Solid-State Fermentation

Proteases are important enzymes because of their extended uses in several industries, such as food, beverages, pharmacy, detergents, and many others. Aspergillus is one of the most used fungi strains for enzyme production by solid-state fermentation (SSF). Disponibility of the carbon source is a key factor for protease production. In addition, the selection of solid support has great importance, as it must provide suitable airflow through the packed bed and nutrient diffusion inside the fermentable mass. Six Aspergillus strains and two inert supports (Agrolite (AL) and Polyurethane (PUF)) were tested for protease production from fish flour (FF) at different glucose concentrations (0, 5, 10, 15%) by SSF. The FF/PUF mixture at 70/30 (w/w) ratio, with 75.39% moisture, and a critical moisture point of 0.11 gH2O/g, presented a texture that allowed heat and mass transfer and provided enough moisture to make free water available as required for microorganism growth during the fermentation process. Aspergillus oryzae 2095 produced higher amounts of neutral and alkaline proteases with the addition of 5% glucose to the growth medium. Kinetics studies reveal that protease production is partially associated with growth. The extracts obtained can be used in different industries, and especially to prepare fish high-value by-product hydrolysates.

Integrated Genomics and Transcriptomics Provide Insights into Salt Stress Response in Bacillus subtilis ACP81 from Moso Bamboo Shoot (Phyllostachys praecox) Processing Waste.

Salt stress is detrimental to the survival of microorganisms, and only a few bacterial species produce hydrolytic enzymes. In this study, we investigated the expression of salt stress-related genes in the salt-tolerant bacterial strain Bacillus subtilis ACP81, isolated from bamboo shoot processing waste, at the transcription level. The results indicate that the strain could grow in 20% NaCl, and the sub-lethal concentration was 6% NaCl. Less neutral protease and higher cellulase and β-amylase activities were observed for B. subtilis ACP81 under sub-lethal concentrations than under the control concentration (0% NaCl). Transcriptome analysis showed that the strain adapted to high-salt conditions by upregulating the expression of genes involved in cellular processes (membrane synthesis) and defense systems (flagellar assembly, compatible solute transport, glucose metabolism, and the phosphotransferase system). Interestingly, genes encoding cellulase and β-amylase-related (malL, celB, and celC) were significantly upregulated and were involved in starch and sucrose metabolic pathways, and the accumulated glucose was effective in mitigating salt stress. RT-qPCR was performed to confirm the sequencing data. This study emphasizes that, under salt stress conditions, ACP81 exhibits enhanced cellulase and β-amylase activities, providing an important germplasm resource for saline soil reclamation and enzyme development.

Flavor enhancement of strong fragrant rapeseed oils by enzymatic treatment

Fragrant rapeseed oils with the sensory characteristics attract increasing market in China. The unique flavor of strong fragrant rapeseed oil (SFRO) is the first choice to attract the consumers’ preference. Thus, enzymatic treatments of rapeseeds were used to enhance the flavor properties of SFROs in the present study. Results showed that SFRO produced from neutral protease and α-amylase-treated rapeseeds (NRO+ARO) had the highest percentages of unsaturated fatty acid (USFA), monounsaturated fatty acid (MUSFA) and oleic acid (C18:1) of 93.30%, 65.90% and 65.22%, respectively, with the highest tocopherol content of 577.35 mg/kg and total sterol content of 4523.46 mg/kg. The NRO+ARO showed a strong nutty and rich roasted fragrance and distinguishing sweet flavor with the highest overall sensory evaluation score. Majority of volatile compounds in NRO+ARO belonged to pyrazines and furans originated from Maillard reactions with the highest contents. The highest decrease trends of monosaccharide and amino acid contents from 8.42 g/kg to 2.04 g/kg and from 128.91 mg/kg to 47.39 mg/kg, respectively, were observed in the neutral protease and α-amylase-treated rapeseeds after roasting. Glucose/xylose-lysine reaction products contained the high contents of pyrazines, suggesting enhancement of supply of glucose/xylose and lysine in rapeseeds would significantly improve the fragrance of SFROs. The outputs of present study would provide a clue to understand the formation of unique flavor of SFROs and a reference to develop series of SFRO products.

Novel peptides from sea cucumber intestines hydrolyzed by neutral protease alleviate exercise-induced fatigue via upregulating the glutaminemediated Ca2+ /Calcineurin signaling pathway in mice.

Sea cucumber intestines are considered a valuable resource in the sea cucumber processing industry due to their balanced amino acid composition. Studies have reported that peptides rich in glutamate and branched-chain amino acids have anti-fatigue properties. However, the function of the sea cucumber intestine in reducing exercise-induced fatigue remains unclear. In this study, we enzymatically hydrolyzed low molecular weight peptides from sea cucumber intestines (SCIP) and administered SCIP orally to mice to examine its effects on exercise-induced fatigue using swimming and pole-climbing exhaustion experiments. The results revealed that supplementation with SCIP significantly prolonged the exhaustion time of swimming in mice, decreased blood lactate and urea nitrogen levels, and increased liver and muscle glycogen levels following a weight-loaded swimming test. Immunofluorescence analysis indicated a notable increase the proportion of slow-twitch muscle fiber and a significant decrease the proportion of fast-twitch muscle fiber following SCIP supplementation. Furthermore, SCIP upregulated mRNA expression levels of Ca2+ /Calcineurin upstream and downstream regulators, thereby contributing to the promotion of skeletal muscle fiber type conversion. This study presents the initial evidence establishing SCIP as a potential enhancer of skeletal muscle fatigue resistance, consequently providing a theoretical foundation for the valuable utilization of sea cucumber intestines.

Whole-genome sequencing of a protease-producing strain isolated from fermented golden pompano and molecular docking of the protease to fish proteins

In fermented fish products, microorganisms catalyze protein degradation by secreting proteases, thereby altering the nutritional values of foods. In this study, we isolated a high proteinase-producing strain from fermented golden pompano (Trachinotus ovatus), and from the 16S rRNA, we identified the strain as Bacillus tropicus, which we named Bacillus tropicus MX-8. This strain could form a distinct proteolytic circle around the colony when plated on agar containing 3% skimmed milk powder and could grow under 0–5% salinity. Its protease significantly degraded fish proteins. Genome sequencing analysis revealed that the genome of B. tropicus MX-8 comprised one circular chromosome and three circular plasmids. A total of 27 genes encoding proteases, including a neutral protease and three serine proteases, were identified in the non-redundant protein database. Homology modeling showed that serine protease 2 had the best model quality and the highest sequence match. Molecular docking experiments revealed the presence of several amino acid active sites on serine protease 2. These active sites mainly rely on hydrogen bonding to interact with actin heavy chains and myosin to form complexes, which can promote further protein degradation. This study demonstrated that B. tropicus MX-8 has the potential to ferment fish, providing a theoretical basis for the production of rapidly fermented fish products.

Neutrophil extracellular traps induced by chemotherapy inhibit tumor growth in murine models of colorectal cancer.

Neutrophil extracellular traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and cause tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibited the growth of PIK3CA-mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors were associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of ROS in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, entered CRC cells through the RAGE cell surface protein. The internalized CTSG cleaved 14-3-3 proteins, released BAX, and triggered apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.

Regulatory role of VvsB protein on serine protease activity of VvsA in Vibrio vulnificus.

Vibrio vulnificus NCIMB2137, a Gram-negative, metalloprotease negative estuarine strain was isolated from a diseased eel. A 45kDa chymotrypsin-like alkaline serine protease known as VvsA has been recently reported as one of the major virulence factor responsible for the pathogenesis of this strain. The vvsA gene along with a downstream gene vvsB, whose function is still unknown constitute an operon designated as vvsAB. This study examines the contribution of VvsB to the functionality of VvsA. In this study, VvsB was individually expressed using Rapid Translation System (RTS system), followed by an analysis of its role in regulating the serine protease activity of VvsA. The proteolytic activity of VvsA increased upon the addition of purified VvsB to the culture supernatant of V. vulnificus. However, the attempts of protein expression using an E. coli system revealed a noteworthy observation that protein expression from the vvsA gene exhibited higher protease activity compared to that from the vvsAB gene within the cytoplasmic fraction. These findings suggest an intricate interplay between VvsB and VvsA, where VvsB potentially interacts with VvsA inside the bacterium and suppress the proteolytic activity. While outside the bacterial milieu, VvsB appears to stimulate the activation of inactive VvsA. The findings suggest that Vibrio vulnificus regulates VvsA activity through the action of VvsB, both intracellularly and extracellularly, to ensure its survival.

Maintenance of an Acidic Skin Surface with a Novel Zinc Lactobionate Emollient Preparation Improves Skin Barrier Function in Patients with Atopic Dermatitis.

The skin of patients with atopic dermatitis (AD) is characterised by elevated pH. As a central homeostatic regulator, an increasedpH accelerates desquamation and suppresses lipid processing, resulting in diminished skin barrier function. The aim of this study was to determine whether a novel zinc lactobionate emollient cream can strengthen the skin barrier by lowering skin surface pH. A double-blind, forearm-controlled cohort study was undertaken in patients with AD. Participants applied the test cream to one forearm and a vehicle cream to the other (randomised allocation) twice daily for 56days. Skin surface pH and barrier function (primary outcomes) were assessed at baseline and after 28days and 56days of treatment, amongst other tests. A total of 23 adults with AD completed the study. During and after treatment, a sustained difference in skin surface pH was observed between areas treated with the test cream and vehicle (4.50 ± 0.38 versus 5.25 ± 0.54, respectively, p < 0.0001). This was associated with significantly reduced transepidermal water loss (TEWL) on the test cream treated areas compared with control (9.71 ± 2.47 versus 11.20 ± 3.62g/m2/h, p = 0.0005). Improvements in skin barrier integrity, skin sensitivity to sodium lauryl sulphate, skin hydration, and chymotrypsin-like protease activity were all observed at sites treated with the test cream compared with the control. Maintenance of an acidic skin surface pH and delivery of physiologic lipids are beneficial for skin health and may help improve AD control by reducing sensitivity to irritants and allergens.

Lactobacillus plantarum 69-2 combined with α-lactalbumin hydrolysate alleviates DSS-induced ulcerative colitis through the TLR4/NF-κB inflammatory pathway and the gut microbiota in mice.

Ulcerative colitis (UC), an inflammatory bowel disease, seriously affects people's quality of life. Diet-derived active peptides and Lactobacillus plantarum have shown promise for mitigating symptoms of UC. This investigation explored the combined effects of α-lactalbumin (α-LA) hydrolysate, which boasts a high antioxidant capacity, and L. plantarum 69-2 (L69-2) on a colitis mouse model. The results showed that α-LA hydrolysate with a molecular weight <3 kDa obtained with neutral protease had excellent antioxidant activity and potential to enhance probiotic proliferation. Furthermore, the synergistic application of α-LA hydrolysate and L69-2 could alleviate the adverse impact of colon inflammation by reducing oxidative stress and regulating immune disorders. It maintains the intestinal epithelial barrier, thereby reducing immune system over-activation, promoting the colonization of beneficial bacteria, and regulating intestinal immune responses. Simultaneously, it remodels the structure of the disrupted intestinal flora. The increase in the richness and diversity of the flora leads to the production of beneficial metabolites, which in turn inhibits the activation of the TLR4/NF-κB inflammatory pathway. This study provides a novel perspective on milk-derived peptide synergism with probiotics in alleviating UC.

Discovery of a nasal spray steroid, tixocortol, as an inhibitor of SARS-CoV-2 main protease and viral replication.

Coronaviruses rely on the viral-encoded chymotrypsin-like main protease (Mpro or 3CLpro) for replication and assembly. Our previous research on Mpro of SARS-CoV-2 identified cysteine 300 (Cys300) as a potential allosteric site of Mpro inhibition. Here, we identified tixocortol (TX) as a covalent modifier of Cys300 which inhibits Mpro activity in vitro as well as in a cell-based Mpro expression assay. Most importantly TX inhibited SARS-CoV-2 replication in ACE2 expressing HeLa cells. Biochemical analysis and kinetic assays were consistent with TX acting as a non-competitive inhibitor. By contrast, TX was a weaker inhibitor and modifier of C300S Mpro, confirming a role for Cys300 in inhibition of WT Mpro but also providing evidence for an additional Cys target. TX pivalate (TP), a prodrug for TX that was previously marketed as a nasal spray, also inhibited SARS-CoV-2 replication in HeLa-ACE2 cells at low micromolar IC50s. These studies suggest that TX and/or TP could possibly be repurposed for the prevention and/or treatment of SARS-CoV-2 infection.

Production and biochemical and biophysical characterization of fibrinolytic protease of a Mucor subtilissimus strain isolated from the caatinga biome.

Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease's far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/K∙mol.

QSAR of SARS-CoV-2 Main Protease Inhibitors Utilizing Theoretical Molecular Descriptors

Background: COVID-19 is caused by a novel strain of severe acute respiratory syndrome coronaviruses (SARS-CoV-2). It has claimed casualties around the world since the end of 2019 due to its high virulence and quick multiplicity in the human body. Hence, there has been a requirement to develop effective remedial measures to mitigate the mortality. Scientists have been able to develop corona vaccines to provide immunity, but there are no specific small-molecule chemotherapeutics to combat the novel coronavirus which has spread to the whole world due to its contagiousness. In the viral genome exploration, it has been found that the main protease, also known as chymotrypsin-like cysteine protease ([Mpro] or 3C-like protease [3CLpro]) is responsible for the novel coronavirus replication, transcription, and host immunity destruction. Objectives: Therefore, the main protease has been selected as one of the major targets for the design of new inhibitors. The protein crystallographic and molecular docking studies on SARS-CoV-2 Mpro inhibitors and some quantitative structure-activity relationship (QSAR) studies have been carried out on SARSCoV main protease inhibitors to get some lead molecules for SARS-CoV-2 inhibition. However, there is hardly any QSAR done on the diverse data of SARS-CoV-2 main protease inhibitors. In view of it, QSAR studies have been attempted on SARS-CoV-2 Mpro inhibitors utilizing theoretical molecular descriptors solely computed from the structures of novel corona viral main protease inhibitors. Methods: Methods: As the number of structural descriptors is more than the observations, a genetic algorithm coupled with multiple linear methods has been applied for the development of QSAR models taking diverse SARS-CoV-2 Mpro inhibitors. Results: The developed best QSAR model showing R2 , Q2 Loo, and R2 pred values of 0.7389, 0.6666, and 0.6453 respectively has been further validated on an external data set where a good correlation (r = 0.787) has been found. Conclusion: Therefore, this model may be useful for the design of new SARS-CoV-2 main protease inhibitors.

The potential contributions of matrix metalloproteinase 8,9 and 13 (MMP-8,9,13) to cerebral vasospasm and the role of doxycycline inhibitors on gene expression after experimental subarachnoid haemorrhage.

Vasospasm has been reported as the most important cause of mortality and morbidity in patients with subarachnoid haemorrhage (SAH) who can reach the hospital. Matrix metalloproteinases (MMPs) are a gene family, which are called neutral proteases in the central nervous system (CNS). In this experimental study we studied the upregulation of MMPs (MMP-8, MMP-9, and MMP-13) gene expression and the inhibitor effects of doxycycline after experimental SAH model in rats. After 24 Wistar Albino rats were divided into groups, a SAH model was created by transfusion of autologous blood from the cisterna magna, and then 30 mg/kg doxycycline treatment was applied. In order to observe the efficacy of the treatment, MMP-8, MMP-9 and MMP-13 gene expression levels were examined, and histopathological examinations were made in the sections taken. There was a statistically significant increase ( p < 0.05) in MMP-8, MMP-9 and MMP-13 gene expression within the first 6 hours after SAH. The Ct parameter specifies the number of cycles in which the detected fluorescence radiation threshold value is exceeded. The MMP-13 Ct difference in the SAH group was significantly higher ( p = 0.037) than the control group. The pathophysiology of cerebral vasospasm is complex and multifactorial. Many studies are conducted to solve the complex mechanism of cerebral vasospasm. It has been shown that the use of doxycycline causes a statistically significant ( p < 0.05) inhibition at gene expression levels (MMP-8, MMP-9 and MMP-13), even in a single dose of usage and also these results have been confirmed by histopathology examination.

Association study between SNP markers located in meat quality candidate genes with intramuscular fat content in an endangered dual-purpose cattle population.

The aim of this study was to associate single nucleotide polymorphisms (SNP) of the bovine calcium-activated neutral protease µ-calpain, calpastatin, diacylglycerol-O-acyltransferase, adipose fatty acid binding protein, retinoic acid receptor-related orphan receptor C (RORC), and thyroglobulin (TG) gene with intramuscular fat content (IMF). Therefore, 542 animals of the cattle breed "Rotes Höhenvieh" (RHV) were phenotyped for IMF. Genotyping of the animals was performed using polymerase chain reaction-restriction fragment length polymorphism tests for six SNP from candidate genes for meat quality traits. In addition, we calculated allele substitution and dominance effects on IMF. A subgroup of animals (n = 44, reduced dataset) with extraordinary high IMF was analyzed separately. The mean IMF content was 2.5% (SD: 2.8) but ranged from 0.02% to 23.9%, underlining the breeds' potential for quality meat production. Allele and genotype frequencies for all SNP were similar in the complete and reduced dataset. Association analyses in the complete dataset revealed the strongest effects of RORC on IMF (P = 0.075). The log-transformed least-squares mean for IMF of genotype g.3290GG was 0.45 ± 0.16, 0.26 ± 0.14 for genotype g.3290GT, and 0.32 ± 0.14 for genotype g.3290TT. In the reduced dataset, we found a significant effect (P < 0.05) of the g.422C>T-SNP of TG on IMF, with highest IMF for genotype CT (0.91 ± 0.17), lowest IMF for genotype TT (0.37 ± 0.25), and medium IMF for genotype CC (0.59 ± 0.16; log-transformed values). Compared to the complete dataset, allele substitution effects increased in the reduced dataset for most of the SNP, possibly due to the selective genotyping strategy, with focus on animals with highest IMF implying strong phenotypic IMF contrast. Dominance effects were small in both datasets, related to the high heritability of IMF. Results indicated RHV breed particularities regarding the effects of meat quality genes on IMF. An explanation might be the breeding history of RHV with focus on adaptation and resilience in harsh outdoor systems. Consequently, it is imperative to develop breed-specific selection strategies. Allele substitution and dominance effects were in a similar direction in both datasets, suggesting the same breeding approaches for different RHV strains in different regions. Nevertheless, a selective genotyping approach (reduced dataset), contributed to more pronounced genotype effect differences on IMF and dominance values.

An assessment of a GMP schistosomiasis vaccine (SchistoShield®).

Schistosomiasis is a neglected tropical disease that puts over 200 million people at risk, and prevention options are sparse with no approved vaccine. Our vaccine candidate, SchistoShield®, is based on an approximately 87 kDa large subunit of calcium activated neutral protease - termed Sm-p80 - combined with a potent TLR4 agonist-based adjuvant. SchistoShield® has been shown to prevent disease throughout the parasitic life cycle - including egg, juvenile, and adult worm stages - in numerous animal models up to and including baboons. SchistoShield® has been shown safe in both preclinical toxicology studies in rabbits and in a Phase 1 clinical trial in the USA. A Phase 1b trial was initiated in 2023 in endemic regions of Africa, and to date no serious safety signals have been reported. In preparation for large-scale Phase 2 clinical trials and eventual vaccine deployment, the Sm-p80 antigen production process has been transferred to a manufacturing organization, Quratis Corporation in South Korea, which specializes in preparation of vaccines for large-scale European and African trials. The process of scaling from our current production level of ~2000 vaccine doses, to a process that will generate more than 100 million doses has required multiple improvement steps in the process including fermentation, downstream purification of the protein antigen, lyophilization, and fill and finish. In this study, we detail the large-scale production process of the SchistoShield® protein product by Quratis. In addition, an effort was made to analyze and compare the Quratis-made lot of Sm-p80, referred to as QTP-105, to the cGMP lot of Sm-p80 which is in use in human trials in the USA and Africa, referred to as Sm-p80 DP (made in USA). We show that QTP-105 demonstrates excellent potency, purity, identity, and endotoxin levels compared to our Phase 1 Sm-p80 DP and is suitable for use in Phase 2 studies and beyond.

Enzymatic hydrolysis of oat core flour improves physiochemical and sensory behaviors for oat milk

The aim of this study was to develop a novel plant-based milk using oat core flour (OCF) as the raw material. OCF was firstly treated with deep liquefaction and saccharification. After that, multienzyme treatments, including protease, lipase, and cellulase hydrolyzations, were optimized and applied. The physicochemical properties of the oat milk were characterized. Particle size distribution and zeta potential determinations showed that neutral protease and lipase could effectively prevent the phase separation of oat milk. The highest absolute zeta potential value and narrower particle distribution were obtained at pH 7. Microstructure analysis showed that neutral protease treatment prevented protein aggregations, whereas the cellulase treatment destroyed the network structures formed by the fibers. Multienzyme treatments decreased the centrifugal sedimentation rate (SR) of OCF and increased the water absorption index (WSI) and the denaturation temperature. Furthermore, multienzyme treatments improved the sensory acceptance of color and apparent viscosity. Above all, this research was of specific significance to the development, application, and industrialization of OCF.

Effects of dietary microbial protease on growth performance, nutrient apparent digestibility, hepatic antioxidant capacity, protease activities and intestinal microflora in juvenile genetically improved farmed tilapia, Oreochromis niloticus

The study was to evaluate the effects of four types of microbial protease on growth performance, nutritional composition, apparent digestibility coefficients (ADC), physiological parameters and microbiota of genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Six diets were prepared, including a positive control diet (protein 300 g/kg, high protein group, HP) and one negative control diet (protein 280 g/kg, low protein group, LP) and four proteases were added to the low protein diets, included HuPro protease (LPH), acid protease (LPAC), neutral protease (LPN) and alkaline protease (LPAK). Compared with the fish fed LP diet, the weight gain rate (WGR), specific growth rate (SGR), feed conversion rate (FCR), protein efficiency ratio (PER) and feed intake ratio (FIR) were improved in fish fed LPH diet. The ADC of dry matter in LPH diet was improved. The serum total protein (TP) in LPH diet was higher than the LP diets. The activities of total antioxidant capacity (T-AOC) and catalase (CAT) in LPH group was higher than the LP and HP group, respectively. The protease activities of stomach and gut in fish fed LPH diet was higher than LP group. Shannon and Simpson indices in LPH group were higher than the LP group. The abundance of Fusobacteria in the six groups reached more than 70 % at the phylum level, and the largest difference in the relative abundance at the genus level was Cetobacterium. In conclusion, this study demonstrates the positive effects of HuPro protease on tilapias and selection of HuPro protease to the low protein diet is suitable.

An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CLpro (also known as the main protease or Mpro) and PLpro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CLpro and PLpro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7-O-glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PLpro and 3CLpro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CLpro and PLpro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

Characterization of the Microbial Community Structures, Soil Chemical Properties, and Enzyme Activity of Stellera chamaejasme (Thymelaeaceae) and Its Associated Forages in Alpine Grassland of Northwestern China.

The invasion of toxic weeds was detrimental to the growth of original vegetation and speed up the degraded grasslands. The purpose of this study was to explore the difference in microbial community, soil physicochemical properties, and enzyme activity in the rhizosphere of Stellera chamaejasme and its associated forages (Stipa purpurea and Polygonum viviparum). The rhizosphere soil microbial communities of S. chamaejasme and its associated forages were determined by high-throughput sequencing technology, the physicochemical properties, and enzyme activities were also measured using soil chemical methods. We performed biological statistical analyses to explore the correlation of rhizosphere micro-ecological environment between the invading poisonous herb S. chamaejasme and its associated forages. The Ascomycota community in the rhizosphere soil of S. chamaejasme was significantly decreased when compared with its associated forages. S. chamaejasme and S. purpurea had a similar bacterial composition, while the rhizosphere of P. viviparum was associated with more Acidobacteria and Bacteroidetes. The RDA analysis showed S. chamaejasme had highly correlated with acid proteinase, invertase, polyphenol oxidase, cellulose, and neutral protease and S. purpurea had highly associated with N-acetyl-beta-D-glucosaminidase, β-D-Glucosidase, and the P. viviparum had highly associated with total phosphorus, total nitrogen, ammonium nitrogen, soil organic matter, pH, acid phosphatase, and catalase. Along with the invasion of S. chamaejasme, the microbial composition, soil physicochemical properties, and enzyme activity of the growing area changed considerably compared with the associated forages. Taken together, our results suggested that the composition and diversity of microbial communities associated with S. chamaejasme and its associated forages exhibited different patterns, and the rhizosphere soil microbial communities in different plants were regulated by different environmental factors in this alpine grassland ecosystem.