Comparative Study on the Postmortem Proteolysis and Shear Force during Aging of Pork and Beef Semitendinosus Muscles.

The differences in the proteolytic patterns and shear force of pork and beef during aging were evaluated. Pork and beef semitendinosus muscles were obtained at 24 and 48 h postmortem, respectively, and aged at 4°C for 0 (Day 0), 7 (Day 7), and 14 days (Day 14). Changes in the electrical conductivity were observed in pork on Day 7 and beef on Day 14. The calpain activity increased in pork (p<0.05) after 14 days of aging, whereas that of beef decreased on Day 7 (p<0.05). The cathepsin B activity in pork and beef increased between Day 7 and 14 (p<0.05). The content of α-amino group in the 10% trichloroacetic acid-soluble fraction increased between Day 7 and 14 in pork (p<0.05), but increased steadily in beef throughout aging (p<0.05). The electrophoretogram of the myofibrillar proteins revealed a 30 kDa protein band only in the beef lane on Day 14. The cooked pork had no significant changes in the shear force during aging periods (p>0.05), while the gradual decrease in the shear force with the increasing aging periods was shown in the cooked beef (p<0.05). Circular dichroism analysis of myosin extracts from pork and beef revealed thermal denaturation temperatures of 55°C and 58°C, respectively. This study highlights the different post-mortem proteolytic patterns and thermal denaturation temperatures of myosin in pork and beef semitendinosus muscles, which contribute to distinct changes in the shear force during aging between pork and beef.

Luminescent Ln(III)-Metallopeptide Sensors for Monitoring Pseudomonas aeruginosa Elastase B Activity in Complex Biological Media.

The detection and monitoring of Pseudomonas aeruginosa and its virulence factors, such as the LasB protease, are crucial for managing bacterial infections. Traditional fluorescent sensors for this protease face limitations in bacterial cultures due to interference from pigments like pyoverdine secreted by this opportunistic pathogen. We report here a Ln(III)-metallopeptide that combines a DO3A-Ln(III) complex and a sensitizing unit via a short peptide sequence as a simple, tunable, and selective probe for detecting P. aeruginosa's LasB. The probe's luminescence switches off in the presence of P. aeruginosa's secretome due to LasB cleavage but remains stable in other bacterial environments, such as non-LasB-secreting P. aeruginosa strains or E. coli cultures. It also resists degradation by other proteases, like human leukocyte elastase and trypsin, and remains stable in the presence of bioanalytes related to P. aeruginosa infections, such as glutathione, H2O2, and pyocyanin, and in complex media like FBS. Importantly, time-gated experiments completely remove the background fluorescence of P. aeruginosa pigments, thus demonstrating the potential of the developed Ln(III)-metallopeptide for real-time monitoring of LasB activity in bacterial cultures.

A method for producing protease pS273R of the African swine fever virus.

The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in E. coli cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site in vivo. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates.

Qualitative and quantitative protease activity tests based on protein degradation in three-dimensional structures.

The pattern of the activity of proteases is related to distinct physiological states of living organisms. Often activity changes of a certain protease can be assigned to a specific disease. Hence, they are useful biomarkers and a simple and fast determination method of their activity could be a valuable tool for the efficient monitoring of numerous diseases. Here, two different methods for the qualitative and quantitative determination of protease activity are demonstrated using the model system of proteinase K. The first test system is based on a protein-modified and colored 3D silica structure that changes color when exposed to the enzyme. This method has also been used for the detection of matrix metallo-protease 2 (MMP2) with gelatine as protease substrate on the plates. The second detection system uses the decrease in the voltammetric signal of a cytochrome c/DNA multilayer electrode after incubation with a protease to quantitatively determine its proteolytic activity. While activities down to 0.15 U/ml can be detected with the first method, the second one provides detection limits of about 0.03U/ml (for proteinase K.) The functionality of both systems can be demonstrated and ways for further enhancement of sensitivity have been elucidated.

Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

Draft genome sequence of bacterium Bacillus proteolyticus strain IMGN4 from soil.

Here, we present the draft genome of Bacillus proteolyticus IMGN4, the gram-positive, soil-dwelling bacterium discovered in mountain Maemi, Republic of Korea in May 2019. The assembly resulted in 7 contigs, comprising a total of 6,063,502 base pairs and have 6,115 coding sequences.

Candida parapsilosis: Heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

Synthetic protein protease sensor platform.

Introduction: Protease activity can serve as a highly specific biomarker for application in health, biotech, and beyond. The aim of this study was to develop a protease cleavable synthetic protein platform to detect protease activity in a rapid cell-free setting. Methods: The protease sensor is modular, with orthogonal peptide tags at the N and C terminal ends, which can be uncoupled via a protease responsive module located in between. The sensor design allows for several different readouts of cleavage signal. A protein 'backbone' [Green fluorescent protein (GFP)] was designed in silico to have both a C-terminal Flag-tag and N-Terminal 6x histidine tag (HIS) for antibody detection. A protease cleavage site, which can be adapted for any known protease cleavage sequence, enables the uncoupling of the peptide tags. Three different proteases-Tobacco, Etch Virus (TEV), the main protease from coronavirus SARS-COV-2 (Mpro) and Matrix Metallopeptidase 9 (MMP9)-a cancer-selective human protease-were examined. A sandwich Enzyme-Linked Immunosorbent Assay (ELISA) was developed based on antibodies against the HIS and Flag tags. As an alternative readout, a C-terminal quencher peptide separable by protease cleavage from the GFP was also included. Purified proteins were deployed in cell-free cleavage assays with their respective protease. Western blots, fluorescence assays and immunoassay were performed on samples. Results: Following the design, build and validation of protein constructs, specific protease cleavage was initially demonstrated by Western blot. The novel ELISA proved to afford highly sensitive detection of protease activity in all cases. By way of alternative readout, activation of fluorescence signal upon protease cleavage was also demonstrated but did not match the sensitivity provided by the ELISA method. Discussion: This platform, comprising a protease-responsive synthetic protein device and accompanying readout, is suitable for future deployment in a rapid, low-cost, lateral flow setting. The modular protein device can readily accommodate any desired protease-response module (target protease cleavage site). This study validates the concept with three disparate proteases and applications-human infectious disease, cancer and agricultural crop infection.

Exploration of the shelf-life difference between chilled beef and pork with similar initial levels of bacterial contamination.

This study compared the shelf-life of beef and pork longissimus lumborum muscles (loins) that had the same initial bacterial loads and were held under the same chilled storage conditions. To identify the underlying pathways, comparisons were conducted from the perspective of the spoilage indicators; protease/lipase activity, and the volatile organic compounds (VOC) generated over 28 d of chilled storage. The initial total viable microbial count (TVC) on Day 0 for both type of meat was 4.3 log10 CFU/g. It was found that the TVC of beef and pork did not differ throughout the total chilled storage period and both ultimately exceeded 7 log10 CFU/g after 28 d. Based on total volatile basic nitrogen (TVB-N) guidelines, pork was spoilt after 21 d of chilled storage and therefore 7 d earlier than beef. Changes in the concentration of VOC spoilage biomarkers, including 1-octen-3-ol, 1-octanol, nonanal, and others, confirmed that pork had a shorter shelf-life than beef. An important reason for the difference in shelf-life between the two types of meat was that pork had a higher protease activity, although the beef had higher levels of total lipase activity. These findings help us understand the differences in the spoilage process of raw meat from different species and explore specific measures to control the spoilage of beef or pork.

Comparison of characteristics and antioxidant activities of sesame protein hydrolysates and their fractions.

Sesame meal is a by-product obtained from oil extraction. We investigated the characteristics and antioxidant activities of a sesame protein hydrolysate (SPH-B), as well as its peptide fractions. Four peptide fractions (F1; >100 kDa, F2; 10-100 kDa, F3; 1-10 kDa, and F4; <1 kDa) of SPH-B were prepared. The characteristics and antioxidant properties of SPH-B and its peptide fractions were evaluated. Sesame protein (SP) contained protein fractions with molecular weights ranging from 10 to 44 kDa, whereas SPH-B had peptide fractions ranging from 8 to 44 kDa. The peptide fractions had molecular weight ranging from 7 to 10 kDa. The four peptide fractions had a higher α-helix content and lower surface hydrophobicity than SPH-B and SP. They exhibited better antioxidant properties, with higher ABTS and DPPH radical scavenging activities, higher metal chelating activity, and greater inhibition of linoleic acid peroxidation, suggesting that sesame peptide fractions can use as plant-based functional ingredients and potentially health-promoting properties.

Lactoferrin and Lysozyme Inhibit the Proteolytic Activity and Cytopathic Effect of Naegleria fowleri Enzymes.

Naegleria fowleri is a ubiquitous free-living amoeba that causes primary amoebic meningoencephalitis. As a part of the innate immune response at the mucosal level, the proteins lactoferrin (Lf) and lysozyme (Lz) are secreted and eliminate various microorganisms. We demonstrate that N. fowleri survives the individual and combined effects of bovine milk Lf (bLf) and chicken egg Lz (cLz). Moreover, amoebic proliferation was not altered, even at 24 h of co-incubation with each protein. Trophozoites' ultrastructure was evaluated using transmission electron microscopy, and these proteins did not significantly alter their organelles and cytoplasmic membranes. Protease analysis using gelatin-zymograms showed that secreted proteases of N. fowleri were differentially modulated by bLf and cLz at 3, 6, 12, and 24 h. The bLf and cLz combination resulted in the inhibition of N. fowleri-secreted proteases. Additionally, the use of protease inhibitors on bLf-zymograms demonstrated that secreted cysteine proteases participate in the degradation of bLf. Nevertheless, the co-incubation of trophozoites with bLf and/or cLz reduced the cytopathic effect on the MDCK cell line. Our study suggests that bLf and cLz, alone or together, inhibited secreted proteases and reduced the cytopathic effect produced by N. fowleri; however, they do not affect the viability and proliferation of the trophozoites.

NS2B-D55E and NS2B-E65D Variations are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model.

Japanese Encephalitis Virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and a NS2B cofactor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residue 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. These observations gain an insight into the role of NS2B in regulation of NS3 protease activities, which is useful for understanding the replication of JEV GI and GIII viruses.

Characterising the Gene Expression, Enzymatic Activity and Subcellular Localisation of Arabidopsis thaliana Metacaspase 5 (AtMCA-IIb).

Metacaspases are a class of proteases found in plants that have gained attention in recent years due to their involvement in programmed cell death (PCD) and other essential cellular processes. Although structurally homologous to caspases found in animals, metacaspases have distinct properties and functions. There are nine metacaspase genes in the Arabidopsis thaliana genome; these can be type I or type II, and working out the function of each member of the gene family is challenging. In this study, we report the characterisation of one Arabidopsis type II metacaspase, metacaspase-5 (AtMC5; AtMCA-IIb). We detected the expression of AtMC5 only under specific conditions with a strong upregulation by ER stress and oxidative stress at a narrow 6 h time point. Recombinant AtMC5 was successfully purified from E. coli, with the recombinant AtMC5 working optimally at pH 7, using an optimised reaction buffer containing 10 mM calcium chloride together with 15% sucrose. Like other metacaspases, AtMC5 cleaved after arginine residue and demonstrated a substrate preference towards VRPR. Additionally, AtMC5-RFP was shown to be localised in the cytosol and nucleus of transfected cells. We found no evidence of a strong link between AtMC5 and PCD, and the data provide additional insights into the function of metacaspases in plants and will aid in future research toward further understanding their mode of action.

Lead Toxicity-Mediated Growth and Metabolic Alterations at Early Seedling Stages of Maize (Zea mays L.).

To investigate the toxic effects of lead (Pb) on key metabolic activities essential for proper germination and seedling growth of maize seeds, experiments were carried out with different levels of Pb (0 to 120 mg of Pb L-1 as PbCl2) applied through growth medium to two maize hybrids H-3310S and H-6724. The research findings indicated that growth and metabolic activities were adversely affected by increased Pb contamination in growth medium; however, a slow increase in these parameters was recorded with increasing time from 0 to 120 h. Protease activity decreased with an increase in the level of Pb contamination but increased with time; consequently, a reduction in seed proteins and an increase in total free amino acids were observed with time. Similarly, α-amylase activity decreased with an increase in Pb concentration in growth medium while it increased with increasing time from 0 to 120 h; consequently, reducing and non-reducing sugars increased with time but decreased with exposure to lead. The roots of both maize hybrids had higher Pb contents than those of the shoot, which decreased the uptake of nitrogen, phosphorus, and potassium. All these nutrients are essential for optimal plant growth; therefore, the reduction in growth and biomass of maize seedlings could be due to Pb toxicity that altered metabolic processes, as sugar and amino acids are necessary for the synthesis of metabolic compounds, rapid cell division, and proper functioning of enzymes in the growing embryo, but all were dramatically reduced due to suppression of protease and α-amylase by toxicity of Pb. In general, hybrid H-3310S performed better in Pb-contaminated growth medium than H-6724.

The Effects of Tea Polyphenol on Chicken Protein Digestion and the Mechanism under Thermal Processing.

Meat product is the main food and major source of daily protein intake. Polyphenols are always introduced into many meat products during processing. Some complex interactions may occur between polyphenol and meat protein during the processing, especially thermal processing, which may affect the digestion of protein. In this experiment, chicken protein and tea polyphenol were interacted in simulated systems to explore the effects of the interaction between meat protein and polyphenols on the digestion of meat protein. The mechanism of tea polyphenol inhibiting chicken protein digestion was studied by analyzing the changes of chicken protein in intrinsic fluorescence, surface plasmon resonance (SPR), reactive sulfhydryl group, and solubility in different solvents. The results showed that the chicken protein digestion had a negative correlation with tea polyphenol concentration and interaction temperature, and the meat protein has a higher affinity to EGCG than protease. The mechanism of tea polyphenol inhibiting chicken protein digestion was related to the changing spatial structure of chicken protein and the decreasing activity of proteases. In the simulation system, at low-concentration tea polyphenol, the inhibition of the tea polyphenol on the digestibility of chicken protein might be mainly caused by the changes in chicken protein structure, while at high concentration, the changes in protein structure and the inhibition of proteases activity played a role together. This experiment revealed the effect and the mechanism of polyphenols on the digestion performance of meat protein and provide more references for the further application of polyphenols in meat processing.

BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis.

BACKGROUND: Airway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583. METHODS: Binding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model. RESULTS: BI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma. CONCLUSION: BI 1291583-mediated inhibition of CatC is expected to restore the protease-antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.

The influence of subclinical mastitis on the protein composition and protease activities of raw milk from lactating Thai-crossbred dairy cows.

Background and Aim: Mastitis in dairy cattle is associated with a high rate of morbidity and death, which has major implications for milk production and quality. This study aimed to investigate the protein component and the activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in raw milk samples with different testing scores determined using the California mastitis test (CMT). Materials and Methods: Thirty cows were employed in the study, and milk from each quarter was tested for subclinical mastitis (SCM). According to the results of CMT, raw milk samples were classified into five categories: Healthy (score 0), trace (score T), weakly positive (score 1), distinctly positive (score 2), and strongly positive (score 3) for somatic cell count (SCC). The total milk protein was analyzed using the Bio-Rad protein assay, and the milk protein composition was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In addition, gelatin zymography was used to evaluate changes in proteolytic abilities. Results: Milk samples with CMT scores of 1 and 3 had the highest total milk protein levels (32.25 ± 12.60 g/L and 32.50 ± 7.67 g/L, respectively), while the samples from healthy cows (CMT score 0) were only 6.75 ± 1.64 g/L. Globulin and lactoferrin were significantly increased in samples with a CMT score of 3 compared with those with other CMT scores. The bovine serum albumin level in samples with a CMT score of 2 was significantly (p < 0.05) higher than those with other CMT scores. No significant differences in casein abundance were found among samples with different CMT scores. Results from analysis of proteolytic activities demonstrated that the level of MMP-9 in samples with a CMT score of 3 was significantly (p < 0.05) higher than those with other CMT scores. Conclusion: The protein content and gelatinolytic activity of milk were drastically altered by the number of SCC, mainly due to SCM.

Immune and Reproductive Biomarkers in Female Sea Urchins Paracentrotus lividus under Heat Stress.

The functioning of the immune and reproductive systems is crucial for the fitness and survival of species and is strongly influenced by the environment. To evaluate the effects of short-term heat stress (HS) on these systems, confirming and deepening previous studies, female sea urchin Paracentrotus lividus were exposed for 7 days to 17 °C, 23 and 28 °C. Several biomarkers were detected such as the ferric reducing power (FRAP), ABTS-based total antioxidant capacity (TAC-ABTS), nitric oxide metabolites (NOx), total thiol levels (TTL), myeloperoxidase (MPO) and protease (PA) activities in the coelomic fluid (CF) and mitochondrial membrane potential (MMP), H2O2 content and intracellular pH (pHi) in eggs and coelomocytes, in which TAC-ABTS and reactive nitrogen species (RNS) were also analyzed. In the sea urchins exposed to HS, CF analysis showed a decrease in FRAP levels and an increase in TAC-ABTS, TTL, MPO and PA levels; in coelomocytes, RNS, MMP and H2O2 content increased, whereas pHi decreased; in eggs, increases in MMP, H2O2 content and pHi were found. In conclusion, short-term HS leads to changes in five out of the six CF biomarkers analyzed and functional alterations in the cells involved in either reproductive or immune activities.

Serine proteases autotransporter of Enterobacteriaceae: Structures, subdomains, motifs, functions, and targets.

Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors, resembling the trypsin-like superfamily of serine proteases. SPATEs accomplish multiple functions associated to disease development of their hosts, which could be the consequence of SPATE cleavage of host cell components. SPATEs have been divided into class-1 and class-2 based on structural differences and biological effects, including similar substrate specificity, cytotoxic effects on cultured cells, and enterotoxin activity on intestinal tissues for class-1 SPATEs, whereas most class-2 SPATEs exhibit a lectin-like activity with a predilection to degrade a variety of mucins, including leukocyte surface O-glycoproteins and soluble host proteins, resulting in mucosal colonization and immune modulation. In this review, the structure of class-1 and class-2 are analyzed, making emphasis on their putative functional subdomains as well as a description of their function is provided, including prototypical mechanism of action.

Zingiber officinale-Derived Extracellular Vesicles Attenuate Bleomycin-Induced Pulmonary Fibrosis Trough Antioxidant, Anti-Inflammatory and Protease Activity in a Mouse Model.

Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe idiopathic interstitial pneumonia. It is a chronic and progressive disease with a poor prognosis and is a major cause of morbidity and mortality. This disease has no cure; therefore, there is a clinical need to search for alternative treatments with greater efficacy. In this study, we aimed to evaluate the effect of extracellular vesicles (EVs) from Zingiber officinale (EVZO) in a murine model of bleomycin (BLM)-induced IPF administered through an osmotic minipump. EVZO had an average size of 373 nm and a spherical morphology, as identified by scanning electron microscopy. Label-free proteomic analysis of EVZOs was performed by liquid chromatography coupled to mass spectrometry, and 20 proteins were identified. In addition, we demonstrated the protease activity of EVZO by gelatin-degrading zymography assay and the superoxide dismutase (SOD) activity of EVZO by an enzymatic assay. In the BLM-induced IPF mouse model, nasal administration of 50 µg of EVZO induced recovery of alveolar space size and decreased cellular infiltrate, collagen deposition, and expression of α-SMA-positive cells. Additionally, EVZO inhibited inflammatory markers such as iNOS and COX-2, lipid peroxidation, and apoptotic cells. These results show that EVZO may represent a novel natural delivery mechanism to treat IPF.