Subtilase exhibits strong cytotoxicity that was first described in O113:H21 strain in Australia as a plasmid- encoded cytotoxin (subAB1). Subsequently, chromosomal variants including subAB2-1, subAB2-2, and subAB2-3 were described. We aimed to investigate the presence of subAB genes in a collection of Shiga toxin-producing Escherichia coli (STEC) strains (n=101) isolated from different sources in Iran. A collection of 101 archived STEC strains isolated from cattle (n=50), goats (n=25), sheep (n=15), wild captive animals (n=8: persian fallow deer, n=3; caspian pony, n=1; Macaca mulatta, n=4), and humans (n=3) during 2007-2016 were analyzed for the detection of different genes encoding the Subtilase variants, plasmidic and chromosomal virulence genes, phylogroups and serogroups. Overall, 57 isolates (56.4%) carried at least one variant of subAB. Most strains from small ruminants including 93% of sheep and 96% of caprine isolates carried at least one chromosomally encoded variant (subAB-2-1 and/or subAb2-2). In contrast, 12 cattle isolates (24%) only harbored the plasmid encoded variant (subAB1). STEC strains from other sources, including deer, pony and humans were positive for subAB-2-1 and/or subAb2-2. Our results reveal the presence of potentially pathogenic genotypes among locus of enterocyte effacement (LEE)-negative isolates, and some host specificity related to Subtilase variants and other virulence markers that may aid in source tracking of STEC during outbreak investigations.

Increasing resistance of dermatophytes against antifungals creates global public health problems, rendering essential a better understanding of virulence mechanisms and factors determining host-specificity of dermatophytes. Since dermatophytes switch from a saprophytic to a parasitic lifestyle by reprogramming gene expression, reliable experimental models are needed to investigate the pathogenesis of dermatophytosis. Here, a relevant mouse model of Trichophyton benhamiae dermatophytosis was assessed, together with a model based on reconstructed human epidermis (RHE), allowing their respective validation regarding fungal gene expressed during infection. The use of a standardized inoculum induced a natural-like superficial infection in mice. The severity and persistence of lesions enabled the assessment of infection markers, including mouse-specific pro-inflammatory molecules and fungal genes previously reported as potential virulence factors. Upregulated expression of fungal genes, including those encoding subtilisins, in infected RHE revealed that dermatophytes deploy similar processes as those observed during in vivo infection. The RHE model was then used to compare infections by anthropophilic Trichophyton rubrum and zoophilic T. benhamiae. Therefore, these two models represent complementary analytical tools to study the pathogenesis of acute dermatophytoses. In addition, we have identified certain fungal markers of infection and highlighted the existence of different mechanisms deployed by zoophilic versus anthropophilic dermatophytes.

The use of non-vitamin K antagonist oral anticoagulants (NOACs) has brought a significant progress in the management of cardiovascular diseases, considered clinically superior to vitamin K antagonists (VKAs) particularly in the prevention and treatment of thromboembolic events. In addition, numerous advantages such as fixed dosing, lack of laboratory monitoring, and fewer food and drug-to-drug interactions make the use of NOACs superior to VKAs. While NOACs are synthetic drugs prescribed for specific conditions, nattokinase (NK) is a natural enzyme derived from food that has potential health benefits. Various experimental and clinical studies reported the positive effects of NK on the circulatory system, including the thinning of blood and the dissolution of blood clots. This enzyme showed not only fibrinolytic activity due to its ability to degrade fibrin, but also an affinity as a substrate for plasmin. Recent studies have shown that NK has additional cardioprotective effects, such as antihypertensive and anti-atherosclerotic effects. In this narrative review, we presented the cardioprotective properties of two different approaches that go beyond anticoagulation: NOACs and NK. By combining evidence from basic research with clinical findings, we aim to elucidate the comparative cardioprotective efficacy of these interventions and highlight their respective roles in modern cardiovascular care.

The objective of this study was to investigate the nutrient composition of low-grade New Zealand commercial fish (Gemfish and Hoki) roe and to investigate the effects of delipidation and freeze-drying processes on roe hydrolysis and antioxidant activities of their protein hydrolysates. Enzymatic hydrolysis of the Hoki and Gemfish roe homogenates was carried out using three commercial proteases: Alcalase, bacterial protease HT, and fungal protease FP-II. The protein and lipid contents of Gemfish and Hoki roes were 23.8% and 7.6%; and 17.9% and 10.1%, respectively. The lipid fraction consisted mainly of monounsaturated fatty acid (MUFA) in both Gemfish roe (41.5%) and Hoki roe (40.2%), and docosahexaenoic (DHA) was the dominant polyunsaturated fatty acid (PUFA) in Gemfish roe (21.4%) and Hoki roe (18.6%). Phosphatidylcholine was the main phospholipid in Gemfish roe (34.6%) and Hoki roe (28.7%). Alcalase achieved the most extensive hydrolysis, and its hydrolysate displayed the highest 2,2-dipheny1-1-picrylhydrazyl (DPPH)˙ and 2,2\'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidant power (FRAP). The combination of defatting and freeze-drying treatments reduced DPPH˙ scavenging activity (by 38%), ABTS˙ scavenging activity (by 40%) and ferric (Fe3+) reducing power by18% (p < 0.05). These findings indicate that pre-processing treatments of delipidation and freeze-drying could negatively impact the effectiveness of enzymatic hydrolysis in extracting valuable compounds from low grade roe.

Nattokinase (NK) is an enzyme that has been recognized as a new potential thrombolytic drug due to its strong thrombolytic activity. However, it is difficult to maintain the enzyme activity of NK during high temperature environment of industrial production. In this study, we constructed six NK mutants with potential for higher thermostability using a rational protein engineering strategy integrating free energy-based methods and molecular dynamics (MD) simulation. Then, wild-type NK and NK mutants were expressed in Escherichia coli (E. coli), and their thermostability and thrombolytic activity were tested. The results showed that, compared with wild-type NK, the mutants Y256P, Q206L and E156F all had improved thermostability. The optimal mutant Y256P showed a higher melting temperature (Tm) of 77.4 °C, an increase of 4 °C in maximum heat-resistant temperature and an increase of 51.8 % in activity at 37 °C compared with wild-type NK. Moreover, we also explored the mechanism of the increased thermostability of these mutants by analysing the MD trajectories under different simulation temperatures.

OBJECTIVE: Cancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors.
METHODS: In this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors.
RESULTS: We observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer.
CONCLUSIONS: This study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.

The Jatropha curcas cake, a protein-rich by-product of biofuel production, was the subject of our study. We identified and quantified the ACE inhibitory, antioxidant, and antidiabetic activities of bioactive peptides from a Jatropha curcas L. var Sevangel protein isolate. The protein isolate (20.44% recovered dry matter, 38.75% protein content, and 34.98% protein yield) was subjected to two enzyme systems for hydrolysis: alcalase (PEJA) and flavourzyme (PEJF), recording every 2 h until 8 h had passed. The highest proteolytic capacity in PEJA was reached at 2 h (4041.38 ± 50.89), while in PEJF, it was reached at 6 h (3435.16 ± 59.31). Gel electrophoresis of the PEJA and PEJF samples showed bands corresponding to peptides smaller than 10 kDa in both systems studied. The highest values for the antioxidant capacity (DPPH) were obtained at 4 h for PEJA (56.17 ± 1.14), while they were obtained at 6 h for PEJF (26.64 ± 0.52). The highest values for the antihypertensive capacity were recorded at 6 h (86.46 ± 1.85) in PEJF. The highest antidiabetic capacity obtained for PEJA and PEJF was observed at 6 h, 68.86 ± 8.27 and 52.75 ± 2.23, respectively. This is the first report of their antidiabetic activity. Notably, alcalase hydrolysate outperformed flavourzyme hydrolysate and the cereals reported in other studies, confirming its better multi-bioactivity.

BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31.
METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX.
RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity.
CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity.
CONCLUSIONS: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.