Proteinase Activity Research Articles

Sars-Cov2 Induced Biochemical Mechanisms in Liver Damage and Intestinal Lesions.

The online version contains supplementary material available at 10.1007/s12291-022-01089-x.

Dietary calcium requirements of bighead carp (Aristichthys nobilis)

To investigate dietary calcium requirement of bighead carp (Aristichthys nobilis), six purified diets were formulated to contain different concentrations of calcium (0.09% (control), 0.43%, 0.76%, 1.12%, 1.44%, and 1.79% of dry diets). Each diet was hand-fed to triplicate 30 fish with an average initial body weight (3.31 ± 0.09 g) for 60 days. The results showed that weight gain (WG) and specific growth rate (SGR) significantly increased when dietary calcium level was from 0.09% to 0.76% (P < 0.05). The phosphorus and calcium contents of whole fish body were highest in the 0.76% and 1.12% group, respectively (P < 0.05). The serum phosphorus content in the 1.79% group was significantly lower than those in other groups (P < 0.05). As dietary calcium content was from 0.09% to 0.76%, the activities of lipase and proteinase in the intestine had a significant increase (P < 0.05), while the glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities were significantly decreased (P < 0.05). Based on quadratic curve model analysis with WG and WGR as the appraising criteria, the appropriate dietary requirement of calcium for the bighead carp larvae (3.31 ± 0.09 g) was 1.01% - 1.02%.

Investigation of the Thermal Stability of Proteinase K for the Melt Processing of Poly(l-lactide).

The enzymatic degradation of aliphatic polyesters offers unique opportunities for various use cases in materials science. Although evidently desirable, the implementation of enzymes in technical applications of polyesters is generally challenging due to the thermal lability of enzymes. To prospectively overcome this intrinsic limitation, we here explored the thermal stability of proteinase K at conditions applicable for polymer melt processing, given that this hydrolytic enzyme is well established for its ability to degrade poly(l-lactide) (PLLA). Using assorted spectroscopic methods and enzymatic assays, we investigated the effects of high temperatures on the structure and specific activity of proteinase K. Whereas in solution, irreversible unfolding occurred at temperatures above 75-80 °C, in the dry, bulk state, proteinase K withstood prolonged incubation at elevated temperatures. Unexpectedly little activity loss occurred during incubation at up to 130 °C, and intermediate levels of catalytic activity were preserved at up to 150 °C. The resistance of bulk proteinase K to thermal treatment was slightly enhanced by absorption into polyacrylamide (PAM) particles. Under these conditions, after 5 min at a temperature of 200 °C, which is required for the melt processing of PLLA, proteinase K was not completely denatured but retained around 2% enzymatic activity. Our findings reveal that the thermal processing of proteinase K in the dry state is principally feasible, but equally, they also identify needs and prospects for improvement. The experimental pipeline we establish for proteinase K analysis stands to benefit efforts directed to this end. More broadly, our work sheds light on enzymatically degradable polymers and the thermal processing of enzymes, which are of increasing economical and societal relevance.

Analysis of Volatile Secondary Metabolites in Ocimum basilicum Cell Suspensions: Inhibition, In Silico Molecular Docking, and an ADMET Analysis against Proteolytic Enzymes of Rhynchophorus ferrugineus.

Our study's overarching goal was to determine which O. basilicum cell suspensions approach yielded the most insecticidal and R. ferrugineus-inhibitory volatile secondary metabolites. After inoculation with Verticillium dahliae as an activator, the growth kinetics were measured, and the extract was identified using GC-MS. Validation was achieved for the insecticidal efficacy of a volatile extract, the pure phenolic content against larva and adult R. ferrugineus, and the inhibitory effect on proteases (in vivo and in vitro). The volatile extract achieved an LC50 of 1229 µg/mL and an LD50 of 13.8 µg/larva. The LC50 values for β-bergamotene, α-eudesmol, β-farnesene, linalool, 1,8-cineole, eugenol, α-guaiene, and β-caryophyllene were 1294, 1312, 1356, 1398, 1426, 1459, 1491, and 1523 g/mL, respectively. The LD50 activities of α-eudesmol, linalool, 1,8-cineole, eugenol, and nerol were 12.4, 13.7, 13.9, 14.2, and 15.6 g/larva, respectively. Active volatile extract of O. basilicum inhibited trypsin proteinase, elastase, cysteine, overall protease, and metalloprotease activity with IC50 values of 89.4, 101.7, 394.7, 112.4, and 535.2 µg/mL and 178.5, 192.4, 547.3, 208.3, and 924.8 µg/mL, in vitro and in vivo, respectively. There was evidence of action against total proteases (in vitro) with IC50 values of 78.9, 81.2, 88.6, 90.7, 91.5, 97.6, 107.4, and 176.3 µg/mL for β-bergamotene, α-eudesmol, β-farnesene, linalool, 1,8-cineole, eugenol, α-guaiene, and β-caryophyllene, respectively. Total proteases (in vivo) are inhibited by the α-eudesmol, linalool, 1,8-cineole, eugenol, nerol, and (E)-β-ocimene, with IC50 values of 162.3, 192.7, 193.1, 201.4, 248.6, and 273.2 µg/mL, respectively. ADMET and molecular docking modeling were the only two methods used to conduct in-depth computational analyses of compounds. The study recommended using an efficient cell suspension method to produce a volatile extract rich in useful secondary metabolites that may be utilized as a bio-insecticide.

Successful Pre-Clinical Management of Irinotecan-Debilitated Animals: A Protein- Based Accessory Phytomedicine.

Calotropis procera is a laticiferous plant (Apocynaceae) found in tropical regions all over the world. The ultrastructural characteristics of laticifers, their restricted distribution among different taxonomic groups, and in some species in each clade, as peptidases from latex, make them very attractive for biological analysis. The study aims to investigate the effects of LP-PII-IAA (laticifer protein (LP) sub-fraction II (PII) of C. procera presenting an iodoacetamide-inhibited cysteine proteinase activity) on irinotecan-induced intestinal mucositis, a serious adverse effect of this medicine for the treatment of cancer. LP-PII-IAA is composed of closely related isoforms (90%) of peptidases derived from catalysis and an osmotin protein (5%). Animals receiving co-administration of LP-PII-IAA presented a significant decrease in mortality, absence of diarrhea, histological preservation, and normalization of intestinal functions. Clinical homeostasis was accompanied by a reduction in MPO activity and declined levels of IL-1β, IL-6 and KC, while the IL-10 level increased in LP-PII-IAA-treated animals. COX-2 and NF-kB immunostaining was reduced and the levels of oxidative markers (GSH, MDA) were normalized in animals that received LP-PII-IAA. We suggest that peptidases from the latex of Calotropis procera were instrumental in the suppression of the adverse clinical and physiological effects of irinotecan.

Effects of Allium mongolicum regel essential oil supplementation on growth performance, nutrient digestibility, rumen fermentation, and bacterial communities in sheep.

The objectives of this research were to investigate the effects of Allium mongolicum Regel essential oil on growth performance, nutrient digestibility, rumen fermentation, and bacterial communities in sheep Twenty sheep were randomly divided into two dietary groups with 10 replicates each: (1) a basal diet without AMO as the control group (n = 10) and (2) a basal diet supplemented with 40 mg/kg AMO as the AMO group (n = 10). The average daily gain (ADG) was increased (P < 0.05), and the feed conversion ratio (FCR) was reduced (P < 0.05) in the AMO group compared with the control. The ruminal acetate, propionate, total volatile fatty acids (TVFA), and microbial protein (MCP) were higher (P < 0.05) in the AMO group than in the control. Moreover, ruminal pH and ammonia nitrogen (NH3-N) were lower (P < 0.05) in the AMO group than in the control. The relative abundances of the phylum levels of Firmicutes, Actinobacteriota, and Verrucomicrobiota were higher (P < 0.05) in the AMO group than in the control, and the relative abundances of Bacteroidetes and Spirochaetota were lower (P < 0.05) in the AMO group than in the control. The relative abundance of Prevotella and Prevotellaceae_UCG-003 at the genus level was increased (P < 0.05) in the AMO group compared with the control; however, the relative abundance of Succiniclasticum, Norank_f__F082, Christensenellaceae_R-7_group, and Norank_f__Muribaculaceae was decreased (P < 0.05) in the AMO group compared with the control. The activities of cellulase, α-amylase, and proteinase were higher (P < 0.05) in the AMO group than in the control. The apparent digestibility of dry matter (DM) and crude protein (CP) was increased (P < 0.05) in the AMO group compared with the control. In conclusion, AMO supplementation has the potential to improve growth performance. Moreover, supplementation with AMO improved nutrient digestibility, rumen fermentation, and bacterial communities in the rumen of sheep.

Palatability and Bio-Functionality of Chalky Grains Generated by High-Temperature Ripening and Development of Formulae for Estimating the Degree of Damage Using a Rapid Visco Analyzer of Japonica Unpolished Rice.

Global warming inhibits grain filling in rice and leads to chalky grains, which are damaged in physical and cooking qualities. In the present paper, we evaluated 54 Japonica unpolished rice grains harvested in Japan in 2020, and these samples (original grains) were divided to two groups (whole grains and chalky grains). Using rice grains of 100% whole grains or those blended with 30% of chalky grains, we measured contents of sugars and amino acids, and textural properties of boiled rice grains. It was shown that the α-amylase activity and proteinase activity of raw chalky rice were significantly higher than those of whole rice grains, which led to the significant increase of low-molecular-weight sugars and free amino acids after boiling. Furthermore, hardness and toughness of the boiled rice grains were decreased markedly by blending chalky grains. The ratio of α-amylase activity of chalky grains to that of whole grains was shown to be a useful indicator for damage degree by high-temperature ripening. It became possible to estimate the degree of high-temperature damage of rice grains based on only the pasting properties of unpolished rice.

Detection of ERG11 gene in fluconazole resistant urinary candida isolates

Candida species resistant to fluconazole and voriconazole were screened for the presence of ERG11gene by polymerase chain reaction (PCR). Also, the association of this gene with the demonstration of Candida virulence factors; biofilm formation, phospholipase and proteinases activities were studied. A total of 61 Candida isolates were collected from urine specimens. Candida species were identified by API 20 C Aux test. Extracellular phospholipase, secretory aspartyl proteinase and biofilm formation were determined. ERG11 gene was detected by PCR. C. albicans was identified in 34.5%, C. glabrata in 29.5% and C. tropicalis and C. krusei in 18% each. Candida species was resistant to fluconazole and voriconazole in 55.7% and 27.9%, respectively. Seventeen (50%) of fluconazole resistant Candida isolates were sensitive to voriconazole. The most frequently Candida species revealed fluconazole resistance were C. glabrata (47.1%), C. krusei (29.4%), and C. tropicalis and C. albicans (11.8% each). Biofilm formation, phospholipase and proteinase activity were determined in 41.2%, 67.6% and 35.3% of fluconazole resistant Candida isolates, respectively. Erg 11 gene was determined in 82.4% of fluconazole resistant Candida isolates and prominent in C. glabrata (93.75%), followed by C. krusei (90%), C. tropicalis (75%) and C. albicans (25%). Erg 11 gene was detected in 64.7% (11/17) of fluconazole resistant-voriconazole sensitive Candida isolates. Regarding, correlation of Erg11 gene positivity and virulence factors among fluconazole resistant Candida isolates, 34.5% exhibited biofilm formation and 62.1% and 31% showed phospholipase and proteinase activities, respectively. There were statistically significant difference concerning the association of proteinase activities and Erg 11 gene expression among fluconazole resistance Candida isolates (P=0.04). The study emphasizes the high prevalence of Erg11 gene among fluconazole resistant Candida species. There was association between the proteinase activity, fluconazole resistance and the presence of Erg11 among Candida species. Voriconazole maintains better activity towards Candida species and represent an alternative therapy.

Dynamics of indicators of the proteinase-inhibitory system of the body in destructive forms of chronic apical periodontitis during treatment

BACKGROUND: Destructive forms of chronic apical periodontitis (CAP) remain one of the most difficult problems not only in dentistry and endodontics but also in medicine. In addition to the progressive local process in the alveolar process of the jaw, they are a source of autoinfection and auto-intoxication, causing or exacerbating pathologies in other organs.&#x0D; AIM: This study aimed to analyze the dynamics of the activity of nonspecific proteinases and their inhibitors in patients with destructive forms of CAP when performing a standard endodontic treatment.&#x0D; MATERIALS AND METHODS: Some indicators of the proteinase-inhibitory system of the oral fluid and blood plasma were studied in 28 somatically healthy patients aged 2555 years with destructive forms of CAP in multi-root teeth. The canals were treated according to the protocol of the European Society of Endodontics. After instrumental and medical treatments of the root canal, it was dried and temporarily sealed with calcium hydroxide-based preparation. The tooth was closed with temporary fillers for 1014 days.&#x0D; RESULTS: The comparative analysis of the indicators of the proteinase-inhibitory system showed that destructive forms of CAP and rehabilitation of the pathological focus, to a greater or lesser extent, affect its condition.&#x0D; CONCLUSIONS: Changes in the parameters of the proteinase-inhibitory system in the development of CAP during endodontic treatment were observed both locally (oral fluid) and systemically (blood plasma).

P470 Determination of the virulence potential of yeast fungi isolated from cattle milk

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMIt is reported about the activity of proteolytic enzymes and phospholipases of yeast fungi of the genus Candida isolated from raw milk of clinically healthy cattle in some regions of Russia. The most common species were C. kefyr (16%), C. membranifaciens (15%), C. catenulata (13%), C. rugosa (9%), C. lambica (8%), C. pararugosa (7%), C. krusei (7%), and Candida albicans (6%). Candida albicans isolates had the highest enzyme activity, C. membranifaciens had the lowest.ObjectivesProteolytic enzymes and phospholipases are known to be pathogenic factors in yeast fungi of the genus Candida. We examined raw milk taken from clinically healthy cows to determine the diversity of yeast fungi of the genus Candida that are found in milk, and also determined the activity of proteolytic enzymes and phospholipases of isolated isolates.MethodsRaw milk samples (50 ml) were taken after the treatment of the udder with antiseptic agents, manually into sterile plastic tubes of the Falcon type, immediately before the planned milking. Then the samples were cooled to + 4°C and transported to the laboratory in this form.Isolation of yeast fungi: 1 ml of milk was mixed with 20 ml of melted and cooled to 45°C Sabouraud medium with the addition of an antibiotic (M063 + FL033, HiMedia), poured into Petri dishes, and cultivated for 48 h. The isolated yeast fungi were inoculated onto a tube-slant wort-agar medium (M129, HiMedia).Identification: Yeasts were identified using MALDI-TOF MS according to the standard procedure.Determination of enzyme activity: filter paper discs (diameter 5.0 mm) were impregnated with 10 μL of pure culture with a density of 0.5 McF and laid out on the surface of the nutrient medium, cultivated at + 37°C for 7 days.Enzyme activity was determined as the ratio of the diameter of the substrate hydrolysis zone to the disk diameter.The composition of the nutrient medium for determining the activity of proteolytic enzymes: YCB (HiMedia) 11.7 g, Yeast extract 1.0 g, bacteriological agar 15.0 g, 1.0 g BSA after autoclaving. To assess the zone of hydrolysis, the surface of the nutrient medium was stained after cultivation with a 0.1% solution of Amido Black.The composition of the nutrient medium for determining the activity of phospholipases: glucose 30.0 g, bacteriological peptone 10 g, 1 M NaCl, 5 mM CaCl2, bacteriological agar 15.0 g, 80 ml egg yolk emulsion after autoclaving.ResultsAs a result of the research, 257 isolates of yeast fungi of the genus Candida were segregated. The most common were tested for proteinase and phospholipase activity, the results are presented in Table 1.ConclusionYeast fungi known to be pathogenic for animals, such as C. albicans and C. krusei, have been isolated from the milk of healthy cows. Isolates of C. catenulata, C. rugosa, and C. pararugosa have a high activity of proteinases and phospholipases. Based on the results obtained, it was concluded that these types of yeast fungi have the potential to infect the host.

Anti-Helicobacter pylori, anti-Inflammatory, and Antioxidant Activities of Trunk Bark of Alstonia boonei (Apocynaceae).

An ulcer is an erosion of the gastric mucosa that occurs following an imbalance between the aggression and protective factors and/or an infection with Helicobacter pylori (H. pylori). About 90-100% of duodenal ulcers and 70-80% of gastric ulcers are caused by H. pylori. The objective of this work was to evaluate in vitro the anti-H. pylori activity and then the anti-inflammatory and antioxidant properties of aqueous and methanol extracts of Alstonia boonei. The anti-H. pylori tests (CMI and antiureasic activity) were determined using the agar well diffusion method, the microbroth dilution method, and the measurement of ammonia production by the indophenol method; the anti-inflammatory properties were evaluated by inhibition of proteinases, denaturation of albumin, production of NO by macrophages, cell viability, and hemolysis of red blood cells by heat; then, the antioxidant properties were evaluated by the FRAP method (ferric reducing antioxidant power) and the DPPH (1,1-diphenyl-2-picrylhydrazyl) test. The results show that the best trapping of the DPPH radical was obtained with the methanol extract (EC50 = 8.91 μg/mL) compared to the aqueous extract (EC50 = 19.86 μg/mL). The methanol extract also showed greater iron-reducing activity than the aqueous extract and vitamin C. Furthermore, at the concentration of 200 μg/mL, the methanol extract showed a percentage (96.34%) strains of H. pylori higher than that of the aqueous extract (88.52%). The MIC90 of the methanol extract was lower than that of the aqueous extract. The methanol extract showed a higher percentage inhibition (85%) of urease than the aqueous extract (73%). The methanol extract at a concentration of 1000 μg/mL showed the greatest ability to inhibit proteinase activity, albumin denaturation, and red blood cell hemolysis; on the other hand, maximum cell viability and greater production of nitrite oxide by macrophages were obtained with the aqueous extract. Aqueous and methanol extracts of Alstonia boonei possess anti-H. pylori which would probably be linked to their antioxidant and anti-inflammatory properties.

Phospholipase and proteinase activities of isolates of colonizing Candida spp. causing vulvovaginitis in pregnant women

Proteases and phospholipases are virulence factors of Candida spp. that play an important role in tissue invasion. Among the factors related to the host some are associated with environmental characteristics and others with Candida colonization. To determine phospholipase and protease activities in colonizing and pathogenic strains, isolated from pregnant women in Cartagena de Indias. Phospholipase and protease activity was determined in 56 isolates, evaluating substrate degradation and calculating the enzyme activity coefficient. Phospholipase and protease activities were compared between colonizing and pathogenic strains. “Very high” (<0.69) phospholipase and protease activity was found in 34 and 14 isolates, respectively. There was no significant difference when comparing phospholipase and protease activities between colonizing and pathogenic isolates. Phospholipase activity predominated as a virulence factor in the studied strains, but no significant difference found between colonizing and pathogenic strains for phospholipase and protease activities.

An optimized method of extracting and quantifying active Neutrophil serine proteases from human whole blood cells.

PurposeNeutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study.MethodsTwo NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3.Results and discussionThe zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method.ConclusionThe NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.

Стимулювання системи протеїназа-інгібітор протеїназ у результаті імерсійної гіпотермії

The effect of immersion hypothermia (IН) on proteolysis activity in adult male rats, which was performed at 0 °C for 5 min in the “forced swimming” test, studied; the body temperature decreased to 27.5 ± 0.5°C. In blood serum and non-nuclear fractions of homogenates of cerebral cortex, hypothalamus, cerebellum, brainstem, lungs, heart, liver and kidneys tissues were determined the total proteinase activity (TPA), non-trypsin-like proteinases (NTPs), such as chymase, prostate-specific antigen, partially tonin, kallikrein rK9, as well as trypsin-inhibitory activity of α-1-proteinase inhibitor (α-1-PI), α-2-macroglobulin (α-2-MG) by highly sensitive (10-9–10-10 g) enzymatic methods. It is noted that IH led to the TPA increase in all studied tissues (4-84 times), which corresponds to the psycho-emotional effects and the phenomenon of hormesis. After 24 h, TPA decreased, remaining above the control level, and in the kidneys increased further (90 times). NTLP activity as a result of IH increased in blood serum and cerebellum, decreased in the hypothalamus, and after 24 h increased further in serum (7 times), cerebellum, and heart. The activity of α-1-PI also increased in the brainstem, lungs, heart and kidneys, and decreased after 24 h in serum below the control level, and in the brainstem remained high, protecting against proteolytic attack. The activity of α-2-MG increased in the hypothalamus, heart, and cerebral cortex, maximum in the brainstem (2-5 times), and after 24 h - more significantly (24 times), but there were no changes in the cerebellum, where its initial level is the biggest. Thus, the development of IG as a result of swimming in cold water for 5 min at 0oC has a stimulating effect on the body and leads to a significant activation of the reactions of limited proteolysis.

Luteolin-Rich Extract of Thespesia garckeana F. Hoffm. (Snot Apple) Contains Potential Drug-Like Candidates and Modulates Glycemic and Oxidoinflammatory Aberrations in Experimental Animals.

The present study evaluated the polyphenolic contents and hypoglycemic, antioxidant, and anti-inflammatory effects of the diethyl ether fraction of Thespesia garckeana using various in vitro and in vivo models. Total phenol and flavonoid contents of the extract were 613.65 ± 2.38 and 152.83 ± 1.56 mg/100 g dry weight, respectively. The extract exhibited in vitro antioxidant activities against DPPH, FRAP, LPO, and ABTS with respective half-maximal inhibitory concentration (IC50) values of 30.91 ± 0.23, 16.81 ± 0.51, 41.29 ± 1.82, and 42.39 ± 2.24 μg/mL. In vitro anti-inflammatory studies using membrane stabilization, protein denaturation, and proteinase activities revealed the effectiveness of the extract with respective IC50 values of 54.45 ± 2.89, 93.62 ± 3.04, and 56.60 ± 2.34 μg/mL, while in vitro hypoglycemic analysis of the extract revealed inhibition of α-amylase (IC5064.59 ± 3.29 μg/mL) and enhancement of glucose uptake by yeast cells. Interestingly, the extract demonstrated in vivo hypoglycemic and anti-inflammatory effects in streptozotocin- (STZ-) induced diabetic and xylene-induced ear swelling models, respectively. In addition, the extract improved insulin secretion, attenuated pancreatic tissue distortion and oxidative stress, and increased the activities of superoxide dismutase (SOD), catalase, and reduced glutathione (GSH), while reducing the concentration of LPO in the diabetic rats. A high-performance liquid chromatography (HPLC) analysis identified the presence of catechin (6.81e − 1 ppm), rutin (8.46 e − 1 ppm), myricetin, apigenin (4.019 e − 1 ppm), and luteolin (15.09 ppm) with respective retention times (RTs) of 13.64, 24.269, 27.781, 29.58, and 32.23 min, and these were subjected to a pharmacoinformatics analysis, which revealed their drug-likeness and good pharmacokinetic properties. A docking analysis hinted at the potential of luteolin, the most abundant compound in the extract, for targeting glucose-metabolizing enzymes. Thus, the present study provides preclinical insights into the bioactive constituents of T. garckeana, its antioxidant and anti-inflammatory effects, and its potential for the treatment of diabetes.

Multilocus Sequence Typing and Antifungal Susceptibility of Candida albicans Isolates From Milk and Genital Tract of Dromedary Camel.

Multilocus sequence typing (MLST) was used to study the genetic diversity and population structure of 48 Candida albicans (C. albicans) isolates from the udder or genital tract of apparently healthy or diseased camels. This study aimed also to determine the frequency of C. albicans isolates in the genital tract and udder of healthy or diseased female dromedary camels. A total of 240 mature dromedary camels (230 females and 10 males) were categorized based on the clinical examination of gentile tract and udder into five groups [fertile females (n = 70), infertile females (n = 115), healthy udder (n = 15), mastitis (n = 30), and fertile males (n = 10)]. Swabs were collected from male and female genital tracts of dromedary camels and milk samples were collected from healthy and diseased udders. C. albicans was isolated from 20% of the samples. The frequency of isolation was significantly higher (p < 0.00001) in disease camels (75%) compared with apparently healthy camels (25%). Most of C. albicans was isolated from infertile female genitalia (62.50%) which was significantly higher than that isolated from fertile female genitalia (16.67%). Multilocus sequence (MLS) analysis identified seven different diploid sequence types (DSTs) including DST2, DST50, DST62, DST69, DST124, DST142, and DST144. The most frequently identified DTS was DST69 (13/48) which significantly higher (p ≤ 0.05) than DST2, DST62, and DST124. The frequency of identification of DST50, DST142, and DST 144 was significantly higher (p ≤ 0.05) than DST62. DST62 and DST124 were isolated only from diseased camels. DST62 was isolated only from mastitic milk. DST124 was isolated only from infertile female genitalia. The percentage of DST50 and DST 142 was significantly higher in diseased camels (infertile females) than in the apparently healthy ones (fertile females). DST2 and DST50 were isolated only from female genitalia of apparent health and diseased camels. The C. albicans isolated from diseased camels had significantly higher biofilm formation, hydrophobicity, phospholipase, proteinase, and hemolysin activities compared with the isolates from apparent healthy camels. All isolates were sensitive to amphotericin B, itraconazole, micafungin, posaconazole and voriconazole. In conclusion, the present study represents the first molecular typing of C. albicans in samples isolated from milk and the genital tract of the dromedary camel. MLST is a useful tool for studying the epidemiology and evolution of C. albicans. Early identification of Candida species and attention to Candida virulence factors and their antifungal susceptibility patterns is very important for establishing strategies to control and/or prevent candidiasis by novel therapeutic management. Amphotericin B, itraconazole, micafungin, posaconazole, or voriconazole can be efficient in treatment of candidiasis.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1–69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

Biomarkers of Skin Graft Healing in Venous Leg Ulcers.

There is a need for biomarkers that predict the success of transplantation of venous leg ulcers (with autologous split-thickness skin grafts). The primary objective of this exploratory study was to investigate the association between split-thickness skin graft healing in venous leg ulcers and candidate wound fluid biomarkers representing inflammatory cell and endogenous proteinase activities, and bioactivity. A secondary objective was to compare biomarker levels of the 17 venous leg ulcers with sterile split-thickness skin graft donor-site wounds in another 10 patients with venous leg ulcers. Wound fluids were collected for 24 h using a validated method. The concentration of pre-operative matrix metalloproteinase-9 in wound fluid was higher in venous leg ulcers showing good healing (n = 10) than in venous leg ulcers showing poor healing (n = 7) 12 weeks after transplantation with meshed split-thickness skin grafts. The diagnostic value of matrix metalloproteinase-9 was good according to receiver-operating characteristic curve analysis. Matrix metalloproteinase activity in wound fluids from split-thickness skin graft donor-site wounds increased as a function of time and healing, but was still lower than matrix metalloproteinase activity in venous leg ulcer wound fluids, which showed increased levels of most biomarkers except for matrix metalloproteinase-9 and matrix metalloproteinase-2. In conclusion, wound fluid matrix metalloproteinase-9 concentration is a potential predictive biomarker of split-thickness skin graft healing in venous leg ulcers.

Arctiin-reinforced antioxidant microcarrier antagonizes osteoarthritis progression

Loss of extracellular matrix (ECM) of cartilage due to oxidative stress injury is one of the main characteristics of osteoarthritis (OA). As a bioactive molecule derived from the traditional Chinese Burdock, arctiin exerts robust antioxidant properties to modulate redox balance. However, the potential therapeutic effects of arctiin on OA and the underlying mechanisms involved are still unknown. Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) tool, Burdock-extracted small molecule arctiin was identified as a potential anti-arthritic component. In vitro, treatment using arctiin rescued the interleukin (IL)-1β-induced activation of proteinases and promoted the cartilage ECM synthesis in human chondrocytes. In vivo, intraperitoneal injection of arctiin ameliorated cartilage erosion and encountered subchondral bone sclerosis in the post-traumatic OA mice. Transcriptome sequencing uncovered that arctiin-enhanced cartilage matrix deposition was associated with restricted oxidative stress. Mechanistically, inhibition of nuclear factor erythroid 2-related factor 2 (NRF2) abolished arctiin-mediated anti-oxidative and anti-arthritic functions. To further broaden the application prospects, a gellan gum (GG)-based bioactive gel (GG-CD@ARC) encapsulated with arctiin was made to achieve long-term and sustained drug release. Intra-articular injection of GG-CD@ARC counteracted cartilage degeneration in the severe (12 weeks) OA mice model. These findings indicate that arctiin may be a promising anti-arthritic agent. Furthermore, GG-modified bioactive glue loaded with arctiin provides a unique strategy for treating moderate to severe OA.Graphical

Effects of a Berry Polyphenolic Fraction on the Pathogenic Properties of Porphyromonas gingivalis.

Porphyromonas gingivalis expresses a broad array of virulence factors that enable it to play a central role in the etiopathogenesis of periodontitis. The objective of the present study was to assess the effects of a berry polyphenolic fraction (Orophenol®) composed of extracts from cranberry, wild blueberry, and strawberry on the main pathogenic determinants of P. gingivalis. Orophenol® attenuated the growth of P. gingivalis and decreased its hemolytic activity, its adherence to a basement membrane matrix model, and its proteinase activities. The berry polyphenolic fraction also impaired the production of reactive oxygen species (ROS) by oral keratinocytes stimulated with P. gingivalis. Lastly, using an in vitro model of oral keratinocyte barrier, the fraction exerted a protective effect against the damages mediated by P. gingivalis. In conclusion, the berry polyphenolic fraction investigated in the present study attenuated several pathogenic properties of P. gingivalis. Although future clinical investigations are required, our study provided evidence that the polyphenols contained in this fraction may represent bioactive molecules of high interest for the prevention and/or treatment of periodontal disease.