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Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application. [ABSTRACT FROM AUTHOR]

Fruit and vegetable are the raw materials for many popular fermented beverages with potential health benefits. In the present study, a novel pistachio beverage fermented with selected strains of lactic acid bacteria belonging to the species Leuconostoc pseudomesenteroides and Companilactobacillus paralimentarius was produced, and the extent of proteolysis was evaluated. Physico-chemical and microbiological analyses, together with peptidomics and proteomics, were carried out on the beverage after 24 h of fermentation, using both the non-inoculated and chemically acidified beverage as controls. Amino acid sequence of hundreds of peptides mainly released from 2S albumin, 11S and 7S globulin were characterized. The number and frequency of identified peptides were higher in beverage started with Leuconostoc pseudomesenteroides , followed by Companilactobacillus paralimentarius and acidic beverage. These results suggest that, in addition to endogenous proteases active at acidic pH, the proteolytic system of LAB directly participated to peptide degradation to some extent. According to the BIOPEP database, a group of 31 peptides were potentially bioactive, and primarily associated with antioxidative properties, ACE and DPP-IV inhibition. The beverage started with Leuconostoc pseudomesenteroides showed the highest amount and number of bioactive peptides. This study lays the foundations for the design of novel pistachio-fermented beverage with potential health properties. [Display omitted] • Proteolysis occurred in fermented pistachio beverage. • The majority of released peptides belong to 2S albumin, 7S and 11S globulin. • LAB proteases participate to peptide degradation, in addition to endogenous proteases. • Potentially bioactive peptides are released during fermentation process. • The peptide profiles of beverages vary among different fermenting LAB. [ABSTRACT FROM AUTHOR]

During the processing of insects for food and feed applications, thermal treatments are often used for decontamination purposes. However, these treatments denature proteins comprising their functional properties. Milder methods for microbiological decontamination can be used, such as microfiltration. Therefore, in this study dead-end microfiltration (0.2 µm poly-ethersulphone membrane) was tested for decontamination of soluble fractions of lesser mealworms and house crickets obtained at pH values of 3 and 8. The results showed that deadend microfiltration was successful in the removal of microorganisms (total viable count), removing the need for thermal treatments for the permeates. Protein recovery in the permeate was relatively low (14-43 %). Lesser mealworms at pH 3 gave the highest protein recovery in the permeate (43 %), as explained by the activity of endogenous proteases producing soluble peptides during microfiltration. Confocal imaging showed that the efficiency of microfiltration was reduced by membrane fouling caused by protein aggregation. The presence of lipids in the pre-filtrate does not hamper the permeate protein recovery. In conclusion, dead-end microfiltration with a 0.2 µm membrane is successful in achieving microbial stability. A pH of 3 is recommended during the extraction process to obtain a higher protein recovery. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) were believed to be a class of promising antimicrobials to combat the increasing resistant microbes. However, AMPs could be easily degraded by endogenous proteases, which limited their clinic use. Considering that lysine is the only one cationic amino acid residue in polybia-CP which were related to the cleavage of trypsin like serine protease's digestion, we introduced lysine's mimics which kept the key function group side chain –NH2 and only varied the methylene chains in the sequence of polybia-CP to investigate the effect of the introduction of non-proteinogenic amino acids on the activity and stability of antimicrobial peptides. In addition, two analogs in which lysine were substituted by the other two proteinogenic cationic amino acids arginine and histidine also were synthesized to evaluate the effect of fine tuning the function group in lysine on its antimicrobial activity and stability. We found that the introduction of amino acids with shortened side chain length of lysine could enhance the trypsin resistance of the derivatives of polybia-CP, while maintain the same or comparable antimicrobial activity with the parent peptide. In addition, while lysine was substituted by histidine, His-CP demonstrated comparable antimicrobial activity and dramatically improved trypsin resistance. Although the analog exhibited excellent antimicrobial activity while lysine was substituted by Arginine residue (Arg-CP), its enzymatic stability was almost the same as its parent peptide. These results suggest that the fine-tuning of side chain of lysine may offer a promising strategy to improve the tryptic stability and retain the antimicrobial activity of antimicrobial peptide. [ABSTRACT FROM AUTHOR]

Sleep disturbances and memory dysfunction are key characteristics across psychiatric disorders. Recent advances have revealed insight into the role of sleep in memory consolidation, pointing to key overlap between memory consolidation processes and structural and molecular abnormalities in psychiatric disorders. Ongoing research regarding the molecular mechanisms involved in memory consolidation has the potential to identify therapeutic targets for memory dysfunction in psychiatric disorders and aging. Recent evidence from our group and others points to extracellular matrix molecules, including chondroitin sulfate proteoglycans and their endogenous proteases, as molecules that may underlie synaptic dysfunction in psychiatric disorders and memory consolidation during sleep. These molecules may provide a therapeutic targets for decreasing strength of reward memories in addiction and traumatic memories in PTSD, as well as restoring deficits in memory consolidation in schizophrenia and aging. We review the evidence for sleep and memory consolidation dysfunction in psychiatric disorders and aging in the context of current evidence pointing to the involvement of extracellular matrix molecules in these processes. [ABSTRACT FROM AUTHOR]

Background: Bowman-Birk inhibitors (BBI) are a family of serine-type protease inhibitors that modulate endogenous plant proteolytic activities during different phases of development. They also inhibit exogenous proteases as a component of plant defense mechanisms, and their overexpression can confer resistance to phytophagous herbivores and multiple fungal and bacterial pathogens. Dicot BBIs are multifunctional, with a "double-headed" structure containing two separate inhibitory loops that can bind and inhibit trypsin and chymotrypsin proteases simultaneously. By contrast, monocot BBIs have a non-functional chymotrypsin inhibitory loop, although they have undergone internal duplication events giving rise to proteins with multiple BBI domains. Results: We used a Hidden Markov Model (HMM) profile-based search to identify 57 BBI genes in the common wheat (Triticum aestivum L.) genome. The BBI genes are unevenly distributed, with large gene clusters in the telomeric regions of homoeologous group 1 and 3 chromosomes that likely arose through a series of tandem gene duplication events. The genomes of wheat progenitors also contain contiguous clusters of BBI genes, suggesting this family underwent expansion before the domestication of common wheat. However, the BBI gene family varied in size among different cultivars, showing this family remains dynamic. Because of these expansions, the BBI gene family is larger in wheat than other monocots such as maize, rice and Brachypodium. We found BBI proteins in common wheat with intragenic homologous duplications of cysteine-rich functional domains, including one protein with four functional BBI domains. This diversification may expand the spectrum of target substrates. Expression profiling suggests that some wheat BBI proteins may be involved in regulating endogenous proteases during grain development, while others were induced in response to biotic and abiotic stresses, suggesting a role in plant defense. Conclusions: Genome-wide characterization reveals that the BBI gene family in wheat is subject to a high rate of homologous tandem duplication and deletion events, giving rise to a diverse set of encoded proteins. This information will facilitate the functional characterization of individual wheat BBI genes to determine their role in wheat development and stress responses, and their potential application in breeding. [ABSTRACT FROM AUTHOR]

Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered. [ABSTRACT FROM AUTHOR]

The addition of heavy protein to aquafeed causes low digestibility, high cost and significant nitrogen pollution in the water. The addition of protease is an effective way of improving protein absorption, but few proteases have been developed specifically for aquafeeds. The hydrolysis efficiency of protease additives for plant protein in the neutral gut environment and their synergy with endogenous proteases has not been sufficiently investigated. In this study, the performance of the recombinant neutral protease rNpI as an aquafeed additive to reduce the raw protein materials in the diet of Carassius carassius during 56 days of growth was evaluated. rNpI effectively hydrolysed soya bean protein isolates (SPI) with a high degree of hydrolysation and a high small‐peptide yield. The rNpI‐treated SPI exhibited a notable antioxidant activity, which benefited fish health. In vitro digestibility indicated that rNpI complemented trypsin in SPI digestion better than papain or Alcalase. The growth trial showed that the addition of rNpI could warrant a reduction in the crude protein and gross energy of feed while producing a lower feed‐conversion rate (FCR) and better growth performance. Crucian that were fed an RM‐wheat‐E diet (5.5% rapeseed meal and 5% wheat reduction) showed the best performance, with the lowest FCR and highest weight gain. These results indicate that rNpI could be an effective additive for the aquafeed industry and could provide economic benefits by reducing aquafeed protein requirements and nitrogen emissions. This study provides novel information and strategies for additive protease use and development in aquafeed. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus (SARSCoV), an enveloped virus with a positive-sense single-stranded RNA genome, facilitates the host cell entry through intricate interactions with proteins and lipids of the cell membrane. The detailed molecular mechanism involves binding to the host cell receptor and fusion at the plasma membrane or after being trafficked to late endosomes under favorable environmental conditions. A crucial event in the process is the proteolytic cleavage of the viral spike protein by the host’s endogenous proteases that releases the fusion peptide enabling fusion with the host cellular membrane system. The present review details the mechanism of viral fusion with the host and highlights the therapeutic options that prevent SARSCoV-2 entry in humans. [ABSTRACT FROM AUTHOR]

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo. Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD. [ABSTRACT FROM AUTHOR]

The process of thermal gelation involves protein denaturation, leading to the exposure of functional groups to form new interactions; these conformational changes favour protein-water- protein interactions and help to stabilise the gel. It is known that in muscle proteins, myofibrillar proteins such as myosin are responsible for the main functional properties; however, in invertebrate species, actin and paramyosin exert an influence on the rheological properties of the gels. Therefore, in the present work, the gelling property of the actomyosin-paramyosin complex was studied. There were significant differences (p < 0.05) in hardness and water-retention capacity, which was higher for actomyosin-paramyosin isolate (API) than for mantle proteins (MP). This may have been due to its structure being more porous than that of MP, which is agglomerated. The API system favoured protein-protein and water-protein interactions; these formed stronger cross-links, which in turn favoured gelling. Moreover, the presence of sarcoplasmic proteins may be more of a physical-chemical impediment rather than hydrolysis caused by endogenous proteases. [ABSTRACT FROM AUTHOR]

Protein C is a natural anticoagulant activated by thrombin in a reaction accelerated by the cofactor thrombomodulin. The zymogen to protease conversion of protein C involves removal of a short activation peptide that, relative to the analogous sequence present in other vitamin K-dependent proteins, contains a disproportionately high number of acidic residues. Through a combination of bioinformatic, mutagenesis and kinetic approaches we demonstrate that the peculiar clustering of acidic residues increases the intrinsic disorder propensity of the activation peptide and adversely affects the rate of activation. Charge neutralization of the acidic residues in the activation peptide through Ala mutagenesis results in a mutant activated by thrombin significantly faster than wild type. Importantly, the mutant is also activated effectively by other coagulation factors, suggesting that the acidic cluster serves a protective role against unwanted proteolysis by endogenous proteases. We have also identified an important H-bond between residues T176 and Y226 that is critical to transduce the inhibitory effect of Ca2+ and the stimulatory effect of thrombomodulin on the rate of zymogen activation. These findings offer new insights on the role of the activation peptide in the function of protein C. [ABSTRACT FROM AUTHOR]

At the time of reception of this article (April 2, 2020), efforts to develop a specific vaccine against SARS-Cov-2, the causative agent of the coronavirus disease 2019 (COVID-19), had just begun trial phase 1, but full validation of this and other current developments is likely to take many more months to reach completion. The ongoing pandemic constitutes a major health burden of world proportions that is also having a devastating impact on whole economies worldwide, the knock-on effects of which could be catastrophic especially in poorer countries. Alternative measures to ameliorate the impact and hamper or minimally slow down disease progression are urgently called for. This review discusses past and currently evolving data on the etiological agent of the current pandemic, SARS-CoV-2, and its host cell receptors with a view to disclosing alternative drugs for palliative or therapeutic approaches. Firstly, SARS-CoV-2 exhibits marked tropism for cells that harbor the membrane-bound metalloprotease angiotensin-converting enzyme 2 (ACE2) at their plasmalemma, predominantly in cells lining the oral cavity, upper respiratory tract, and bronchoalveolar cells, making these epithelial mucosae the most likely viral receptor cell targets and entry routes. Secondly, the crystal structures of several coronavirus spike proteins in complex with their cell host target receptors, and of SARS-Cov-2 in complex with an inhibitor, are now available at atomic resolution through X-ray diffraction and cryo-electron microscopy studies. Thirdly, viral entry of other viruses has been successfully blocked by inhibiting viral endogenous proteases or clathrin/dynamin-dependent endocytosis, the same internalization pathway followed by ACE2 and some viruses. Fourthly, the target cell-surface receptor molecules and SARS-CoV-2 possess other putative sites for drugs potentially modulating receptor activity or virus processing. A multi-pronged pharmacological approach attacking more than one flank of the viral-receptor interactions is worth considering as a front-line strategy. [ABSTRACT FROM AUTHOR]

In bacteria, the truncated forms of mRNAs, which usually lack a stop codon, are occasionally generated by premature termination of gene transcription and/or endo- or exonucleolytic cleavage events. Ribosomes proceeding on these molecules stall at the 3′ end of the chain and are rescued by a widely distributed mechanism known as trans-translation, which includes two essential elements, ssrA RNA (a special RNA) and SmpB (a small protein). Through this mechanism, the polypeptides translated from truncated mRNAs are marked by a short peptide, known as SsrA tag, at their C-termini and directed to the specific endogenous proteases for C-terminal proteolysis. Based on the deep understanding of the SsrA tagging and degradation mechanisms, recently a series of SsrA-based genetic tools have been developed for gene regulation on the level of post-translation. They are successfully applied for controllable regulation of biological circuits in bacteria. In the present article, we systematically summarize the history, structural characteristics, and functional mechanisms of the SsrA tagging and degrading machineries, as well as their technical uses and limitations. Key Points • SsrA system plays an important role in ribosome rescue in bacteria. • SsrA-based genetic tools are useful for controlling protein levels and activities. [ABSTRACT FROM AUTHOR]

Background: Protease-activated receptor 4 (PAR4) is a seven transmembrane G-protein coupled receptor (GPCR) activated by endogenous proteases, such as thrombin. PAR4 is involved in various pathophysiologies including cancer, inflammation, pain, and thrombosis. Although regulators of G-protein signaling (RGS) are known to modulate GPCR/Gα-mediated pathways, their specific effects on PAR4 are not fully understood at present. We previously reported that RGS proteins attenuate PAR1- and PAR2-mediated signaling through interactions with these receptors in conjunction with distinct Gα subunits. Methods: We employed a bioluminescence resonance energy transfer technique and confocal microscopy to examine potential interactions among PAR4, RGS, and Gα subunits. The inhibitory effects of RGS proteins on PAR4-mediated downstream signaling and cancer progression were additionally investigated by using several assays including ERK phosphorylation, calcium mobilization, RhoA activity, cancer cell proliferation, and related gene expression. Results: In live cells, RGS2 interacts with PAR4 in the presence of Gαq while RGS4 binding to PAR4 occurs in the presence of Gαq and Gα12/13. Co-expression of PAR4 and Gαq induced a shift in the subcellular localization of RGS2 and RGS4 from the cytoplasm to plasma membrane. Combined PAR4 and Gα12/13 expression additionally promoted translocation of RGS4 from the cytoplasm to the membrane. Both RGS2 and RGS4 abolished PAR4-activated ERK phosphorylation, calcium mobilization and RhoA activity, as well as PAR4-mediated colon cancer cell proliferation and related gene expression. Conclusions: RGS2 and RGS4 forms ternary complex with PAR4 in Gα-dependent manner and inhibits its downstream signaling. Our findings support a novel physiological function of RGS2 and RGS4 as inhibitors of PAR4-mediated signaling through selective PAR4/RGS/Gα coupling. Video Abstract [ABSTRACT FROM AUTHOR]

Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be indicative of protease activities, and new methods for measuring protease activity in biological samples such as tumor biopsies are needed. Here we report a novel zymography-based technique, called the IHZTM assay, for the detection of specific protease activities in situ. The IHZ assay involves imaging the binding of a protease-activated monoclonal antibody prodrug, called a Probody® therapeutic, to tissue. Probody therapeutics are fully recombinant, masked antibodies that can only bind target antigen after removal of the mask by a selected protease. A fluorescently labeled Probody molecule is incubated with a biological tissue, thereby enabling its activation by tissue endogenous proteases. Protease activity is measured by imaging the activated Probody molecule binding to antigen present in the sample. The method was evaluated in xenograft tumor samples using protease specific substrates and inhibitors, and the measurements correlated with efficacy of the respective Probody therapeutics. Using this technique, a diverse profile of MMP and serine protease activities was characterized in breast cancer patient tumor samples. The IHZ assay represents a new type of in situ zymography technique that can be used for the screening of disease-associated proteases in patient samples from multiple pathological conditions. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of µ‐calpain oxidation on Coregonus peled myofibrillar protein degradation. In the present study, a hydroxyl radical oxidation system was selected to investigate oxidative modification on µ‐calpain activity and its degradation on C. peled myofibrillar protein. When subjected to oxidation, the carbonyl content of µ‐calpain significantly increased with the increasing of oxidation levels, and oxidation modification promoted the µ‐calpain activity. Incubation of C. peled myofibrillar protein with oxidized µ‐calpain resulted in the enhanced degradation of myosin heavy chains, actin, and troponin T, but the degradation of desmin at higher levels of oxidation was slightly inhibited, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. This study suggests that oxidation treatment of µ‐calpain could accelerate myofibrillar proteolysis through regulating the enzyme activity during postmortem aging. Practical Application: Endogenous proteases, especially µ‐calpain, are reported to be involved in fish softening during early postmortem storage, which is critical to muscle quality. The cysteine residues of proteins are particularly sensitive to oxidation. The investigation of the effect of oxidation on µ‐calpain (a cysteine protease) activity allows for the monitoring of its role in the postmortem proteolysis of fish myofibrils and the associated softening of fish meat, in an attempt to minimize this softening. [ABSTRACT FROM AUTHOR]

Proteases are involved in the processing and activation of the epithelial sodium channel (ENaC). The aim of the present study was to investigate whether the prototypical serine protease trypsin can activate ENaC in microdissected, split-open mouse renal distal tubules. Whole-cell patch-clamp recordings from principal cells of connecting tubules (CNT) or cortical collecting ducts (CCD) demonstrated that addition of trypsin (20 µg/ml) to the bath solution increased the ENaC-mediated amiloride-sensitive whole cell current (ΔIAmi) in the majority of cells. In contrast, trypsin applied in the presence of an excess of soybean trypsin inhibitor had no stimulatory effect. The ΔIAmi response to trypsin was variable, ranging from no apparent effect to a twofold increase in ΔIAmi with an average stimulatory effect of 31 or 37% in mice on low-Na+ or standard Na+ diet, respectively. In cultured M-1 mouse collecting duct cells, a robust stimulatory effect of trypsin on ΔIAmi was only observed in cells pretreated with protease inhibitors. This suggests that endogenous proteases contribute to ENaC activation in renal tubular cells and that the degree of ENaC prestimulation by endogenous proteases determines the magnitude of the stimulatory response to exogenous trypsin. In conclusion, we provide electrophysiological evidence that trypsin can stimulate ENaC activity in native renal mouse tubules. Thus, in the kidney, ENaC stimulation by extracellular proteases may be a relevant regulatory mechanism in vivo. [ABSTRACT FROM AUTHOR]

Ultrastructural changes and glutathione level were investigated in the pectoralis muscle of rats fed a low-protein diet. Electron microscopy demonstrated the ultrastructural changes of occasional myofibrils affected with protein deficiency that were characterized with the streaming and/ or disruption of the Z-line and disintegration of sarcomeric striation. In the affected sarcomeres, sarcomere length was often elongated and fragmented thick filaments were present together with dense amorphous materials flowing from the damaged Z-line. Glutathione level of muscle in the low-protein diet group (5%casein) was reduced to about one-third of that in the control diet group (20% casein). The study suggests that depletion of glutathione by protein malnutrition is responsible for inducing myofibrillar damage through the excess leaking of Ca[sup 2+] into the cytosol. [ABSTRACT FROM AUTHOR]

Prevalence of allergic diseases ranges from 20% to 30% worldwide and is increasing for the last few decades. Emerging studies have implicated proteases, both endogenous and exogenous in initiating, mediating, and exacerbating allergic responses. Mast Cells (MCs) are the critical effectors of the allergic diseases and upon activation release a wide variety of mediators and account for the majority of endogenous proteases including chymase, tryptase, and MC-carboxypeptidase A (MC-CPA). The substrates of MC proteases include extracellular matrix components, proenzymes, and cell surface receptors. These proteases are stored in a fully active state and upon release contribute to the tissue injury. Along with endogenous proteases, allergens having protease activity have also been implicated in the manifestation of allergic diseases. Protease allergens are known to modulate immune responses toward Th2 by (i) disrupting protease-antiprotease balance at the epithelial surfaces, (ii) disrupting airway epithelial barrier, (iii) activating airway epithelial cells, (iv) modulating the activity of immune cells, and (v) by cleaving cell surface receptors. As proteases play crucial roles in the manifestation of allergic reactions, they can be exploited as a target for the development of new generation therapies for allergic diseases. [ABSTRACT FROM AUTHOR]

An in vitro assay was developed to study protease activity during the maturation of beef postmortem. Myofibrils were purified from the semitendinosus and used as a sentinel for assessing the activity of endogenous proteases in longissimus thoracis et lumborum (LTL) and the extensor carpi radialis (ER) over time postmortem in beef carcasses. Samples were collected from each muscle at 0, 1, 2, 7, and 14 d of aging and snap frozen. Samples were powdered and added to an in vitro proteolysis assay containing buffer and purified myofibrils. Aliquots were collected at 0, 2, 120, 480, and 1440 min of incubation, and intact desmin and troponin-T were quantified. Digestions at 0 and 1 d using either muscle had little desmin degradation during the entire digestion period. In contrast, LTL muscle collected at 2, 7, and 14 d had the greatest proteolytic capacity as indicated by disappearance of intact desmin by 480 and 1440 min incubation. Though degradation ensued using powdered ER muscle, disappearance of intact proteins was limited. Degradation in vitro paralleled that observed in intact muscle. Addition of ethylene glycol tetra-acetic acid (EGTA), a cysteine protease inhibitor, and calpastatin inhibited proteolysis and suggest proteolytic activity observed in muscles and detected in our proteolysis assays is due to an active calpain protease. Collectively, our data show an active protease is minimal in bovine muscle until 48 h postmortem in the LTL muscle and suggest an in vitro assay containing purified myofibrils is a potential tool for studying temporal changes in proteolysis during the maturation and tenderization of beef across muscles. • Muscle-derived myofibril digestion reflects in vivo proteolysis. • Minimal proteolysis was observed with 0 and 24-h LTL muscle. • Proteolysis observed after 24 h postmortem is likely due to the proteolytic activity of an active calpain. [ABSTRACT FROM AUTHOR]

Aboveground plant material is subject to stress-induced senescence after harvest. For flowers, vegetables and forages, this process can severely impact quality. Proteases, in particular, are responsible for post-harvest protein degradation and contribute to cellular breakdown. Alfalfa is a high-quality forage and a perennial source of protein for dairy cows. Its nutritional value and suitability for sustainable agricultural production are also driving interest in the possible use of alfalfa for human consumption, however, the issue of rapid protein degradation after harvest needs to be addressed before the protein can be extracted for human use on a large scale. We have optimized a cost-effective, medium-throughput, fluorescence intensity-based protease activity assay specifically for aboveground plant tissue that balances precision and throughput, the latter of which has been a barrier to further study. This method will allow higher-throughput phenotyping of plant protease activity than is currently feasible: one person can process approximately 120 samples per day, compared to approximately 25 with previous methods. We have used this method in alfalfa to assess protease activity during wilting at pH 7.4 and pH 6.3. [ABSTRACT FROM AUTHOR]

As of 2023, there were 39.9 million people living with Human Immunodeficiency Virus type 1 (HIV-1). Although great strides have been made in treatment options for HIV-1, and our understanding of the HIV-1 life cycle has vastly improved since the start of this global health crisis, a functional cure remains elusive. One of the main barriers to a cure is latency, which allows the virus to persist despite combined antiretroviral therapy (cART). Recently, we have found that exosomes, which are small, membrane-enclosed particles released by virtually all cell types and known to mediate intercellular communication, caused an increase in RNA Polymerase II loading onto the HIV-1 promoter. This resulted in the production of both short- and long-length viral transcripts in infected cells under cART. This current study examines the effects of exosome-associated kinases on bystander cells. The phospho-kinase profiling of exosomes revealed differences in the kinase payload of exosomes derived from uninfected and HIV-1-infected cells, with CDK10, GSK3β, and MAPK8 having the largest concentration differences. These kinases were shown to be biologically active and capable of phosphorylating substrates, and they modulated changes in the cell cycle dynamics of exposed cells. Given the relevance of such effects for the immune response, our results implicate exosome-associated kinases as new possible key contributors to HIV-1 pathogenesis that affect bystander cells. These findings may guide new therapeutic avenues to improve the current antiretroviral treatment regimens. [ABSTRACT FROM AUTHOR]

High-pressure processing (HPP) is used as a non-thermal approach for controlling microbial viability. The purposes of this study were to (i) establish the decimal reduction times (D-values) for pathogenic bacteria during 350 MPa HPP treatment,; (ii) evaluate the impact of 350 MPa HPP on total plate count (TPC), yeasts and molds (YM), and lactic acid bacteria (LAB) in camel milk; (iii) investigate the behavior of several spoilage-causing bacteria during storage at 4 °C and 10 °C for up to 10 d post-HPP treatment; and (iv) assess the effect of HPP on the protein degradation of camel milk. The D-values for L. monocytogenes, E. coli O157:H7, and Salmonella spp. were 3.77 ± 0.36 min, 1.48 ± 0.08 min, and 2.10 ± 0.13 min, respectively. The HPP treatment decreased pathogenic microorganisms by up to 2 to 3 log cfu/mL (depending on treatment conditions). However, HPP reduced TPC, YM, and LAB by <1 log cfu/mL, regardless of the length of pressure exposure. HPP treatment, even at extended holding times, did not significantly alter either the proteolytic activity or casein micelle structure in camel milk. This study highlights HPP as a promising non-thermal technique for enhancing the microbiological safety of camel milk. [ABSTRACT FROM AUTHOR]

This study explores for the first time the impact of a 6-day germination process on the structure (FTIR), antioxidant activity, nutritional/safety attributes (ACE-I inhibitory activity, digestibility, and cytotoxicity), and functional properties of fractions of variable molecular weight (W > 5 kDa; 3 kDa < MW < 5 kDa; and MW < 3 kDa) isolated from proteins extracted from lentils. FTIR results indicated a substantial increase in β-sheet contents during germination. The digestibility of proteins increased from day 0 (16.32–17.04%) to day 6 of germination (24.92–26.05%) with variable levels of digestibility depending on their MW. ACE-I inhibitory activity improved during germination in all fractions, reaching IC50 values of 0.95, 0.83, and 0.69 mg/mL after 6 days of germination. All antioxidant activities analyzed notably increased, particularly in low-MW fractions (MW < 3 kDa). The functional properties of low-MW fractions were also the most promising, displaying the highest water and fat binding capacities and emulsifying and foaming capacities but lower foaming and emulsifying stability compared to high-MW fractions. Cytotoxicity tests on L929 cells revealed the slight adverse effects of low-MW fractions during germination. This study provides insights into the enhanced nutritional and functional attributes of lentil proteins following germination, emphasizing their potential application in functional foods. [ABSTRACT FROM AUTHOR]

Dry-fermented sausages are appreciated all over the world for their sensory characteristics. Carbohydrates (sugars) are added during the production process, the type and quantity of which directly affect the quality of this product. However, there are few data on the role of sugars on instrumental and sensory parameters of sausages derived from Iberian pork. The objective was to determine the effect of different carbohydrate formulas during the ripening time on the quality of dry-fermented Iberian pork sausages. Five batches were formulated with different combinations and concentrations of carbohydrates (dextrose, dextrin and glucose syrup), making 16 sausages per batch on which to perform repeated measurements throughout the ripening process. Physicochemical characteristics, instrumental colour, textural parameters and sensory attributes were analysed. The C− batch (without any carbohydrate addition) showed unstable pH evolution and high b* values throughout the ripening process and the lowest instrumental texture values (for hardness, springiness and chewiness). The D10d5 batch also presented an unstable pH evolution but low a* values, and it was the highest rated by consumers for sensory texture attributes. This batch, made with dextrose (in higher proportion) and dextrin, differed from the other Iberian pork sausages, suggesting that this carbohydrate formula may be more appreciated by Mediterranean consumers. [ABSTRACT FROM AUTHOR]

Chitin deacetylases (CDAs) are carbohydrate esterases associated with chitin metabolism and the conversion of chitin into chitosan. Studies have demonstrated that chitin deacetylation is essential for chitin organization and compactness and therefore influences the mechanical and permeability properties of chitinous structures, such as the peritrophic membrane (PM) and cuticle. In the present study, two genes (ApCDA5a and ApCDA5b) encoding CDA protein isoforms were identified and characterized in Chinese oak silkworm (Antheraea pernyi) larvae. Although five signature motifs were identified, CDA5 proteins only have the chitin-deacetylated catalytic domain. Spatiotemporal expression pattern analyses revealed that both transcripts presented the highest abundance in the anterior region of the midgut during the feeding period after molting, suggesting their role in chitin turnover and PM assembly. The down-regulation of ApCDA5a and ApCDA5b via RNA interference (RNAi) was correlated with the breakage of chitin microfibrils in the PM, suggesting that group V CDAs were essential for the growth and assembly of the chitinous layer. Additionally, ApCDA5a and ApCDA5b may have non-overlapping functions that regulate the morphological characteristics of PM chitin construction in different ways. Larvae injected with double-stranded RNA (dsRNA) against ApCDA5a and ApCDA5b transcripts were less resistant to infection by N. pernyi than those in the control groups. These results revealed that down-regulating ApCDA5a and ApCDA5b had independent effects on the PM structure and undermined the intactness of the PM, which disrupted the function of the PM against microsporidia infection per os. Our data provide new evidence for differentiating CDA functions among group V CDAs in lepidopteran insects. [ABSTRACT FROM AUTHOR]

Phosphate has been widely used in beef to improve processing characteristics such as tenderness and water-holding capacity. However, the effects of phosphates on the quality and especially the flavor of beef are not well understood. This study investigated the influence of eight different phosphate marinade solutions on the quality and flavor of prepared beef. The results revealed that the thawing loss in the control group was 11.47%, and NaCl with sodium hexametaphosphate (SYCP) had the lowest thawing loss, with a value of 2.13%, which was reduced by 81.43% as compared to the control group. The shear force of the control group was 3.85 kg, and the shear work was 10.03 kg. The best tenderness was recorded in the NaCl with sodium hexametaphosphate (SYST) group, which had a shear force of 1.14 kg and shear work of 3.34 kg. The incorporation of phosphates suppressed fat oxidation and increased the total free amino acid content. Additionally, the levels of certain key volatile flavor compounds, particularly those associated with fat oxidation, such as hexanal, heptanal, octanal, and nonanal, were reduced. In terms of sensory evaluation, juiciness, flavor, tenderness, and overall acceptability in the treatment group were significantly increased (p < 0.05). Overall, the results indicate that adding phosphates can enhance the quality of processed beef, inhibit lipid oxidation, and improve sensory evaluation. [ABSTRACT FROM AUTHOR]

Background: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory illness in humans and currently lacks an approved vaccine. The Newcastle disease virus (NDV) vector is a well-established, safe, and effective platform for vaccine development. With recent advancements in stabilizing coronavirus spike proteins to enhance their antigenicity, this study aimed to determine whether modifications to the MERS-CoV spike protein could improve its presentation on NDV particles, allowing the resulting virus to be used as an inactivated vaccine. Methods: We codon-optimized the gene encoding the ectodomain of the MERS-CoV spike protein and incorporated modifications at the S1/S2 and S2' cleavage sites, along with a proline substitution at residues V1060-L1061. This modified spike gene was inserted into the NDV genome to create the NDV-SMERS virus. After purification and inactivation, the vaccine's immunogenicity was assessed in mice. Results: Mice immunized with the inactivated NDV-SMERS vaccine developed robust anti-spike IgGs, neutralizing antibodies, and cellular immune responses. The study demonstrated that modifications to the MERS-CoV spike protein were essential for its effective presentation on NDV particles. Additionally, the spike gene insert remained stable through five egg passages, confirming the vector's stability. Conclusions: Engineering the MERS-CoV spike protein is crucial for its successful display on NDV particles. The strong immune responses elicited by the NDV-SMERS vaccine in mice highlight that NDV is a promising, safe, and effective platform for MERS-CoV vaccination. [ABSTRACT FROM AUTHOR]

Chinese local pig breeds have unique meat flavor. In this study, we investigated the meat quality traits and the characteristics of the nutritional flavor substances such as amino acids (AAs), fatty acids (FAs), thiamine (Vitamin B1, VB1), and inosine monophosphate (IMP) in four Shanghai local pig breeds (MMS, SW, PD, and SHW) and the commercial crossbred Duroc × Landrace × Yorkshire (DLY) breed. The results showed that the intramuscular fat (IMF) and protein content in the longissimus dorsi muscle (L) of Shanghai local breeds, especially Shanghai MMS and PD breeds, were significantly higher than those of the DLY breed (p-value < 0.01). The inter-breed differences in amino acid (AA) content were even more significant in the gluteal muscle (G). Total amino acids (TAAs), flavor amino acids (FAAs), and essential amino acids (EAAs) were significantly higher in the G muscle of the four Shanghai local breeds than that in the DLY breed. The results for fatty acids (FAs) revealed that the differences in polyunsaturated fatty acids (PUFAs) were all highly significant (p-value < 0.0001), especially for the MMS breed, where the content of PUFAs in the L and G muscle amounted to (14.86 ± 2.06) g/100 g and (14.64 ± 2.83) g/100 g, respectively, which were significantly higher than those of other breeds. The MMS breed was also found to have the highest IMP content and the lowest thiamine (VB1) content among several pig breeds. Therefore, these differences in meat nutritional flavor substances provide new insights into the characterization of meat flavor in Shanghai local pig breeds. [ABSTRACT FROM AUTHOR]

The objective of the present work was to examine the effect of incorporating spirulina powder (SP) in merguez-type sausages made exclusively with camel meat, as well as to evaluate its physicochemical, microbiological, and sensory quality attributes and its prebiotic potential. The final purpose was to offer an innovative meat product to increase camel meat consumption. Several innovative fresh sausage formulations were developed using SP (00, 100, 250, and 500 mg/kg) and stored under vacuum conditions with refrigeration at 1 ± 1 °C for 35 days. A control group of camel sausage without SP was also stored overwrapped (OW) under aerobic conditions, to serve as the negative control. The addition of SP to the vacuum-packed camel sausages extended their shelf life by 20 to 35 days compared to the control group, which was completely spoiled by the fifth day of storage. These results were more pronounced the higher the percentage of SP incorporated into the camel sausage formulation, as indicated by the following parameters: 2-thiobarbituric acid-reactive substances TBARS (1.46 vs. 2.89 mg MDA/kg), CIE a* (14.65 vs. 10.12), total volatile basic nitrogen TVB-N (13.02 vs. 15.09 mg/kg), total psychrotrophic bacteria TPB (5.71 vs. 6.34 log CFU/g), and overall acceptability score (3.17 vs. 2.5). The study of prebiotic potential suggested that the addition of SP to camel sausages promoted the growth of probiotic strains, which in turn were able to inhibit the growth of pathogenic microorganisms such as S. aureus and E. coli O157:H7. In conclusion, this study highlighted how SP, as a clean label ingredient, based on its rich composition and its antioxidant, antibacterial, and prebiotic effects, may represent a source of beneficial substances for human health and offer an alternative approach to producing a new traditional merguez-type sausage with improved acceptance. [ABSTRACT FROM AUTHOR]

Genetic differences typically cause differences in the structure and function of proteins in meat. The objective of this research was to examine the biochemical characteristics and functional behavior of proteins in fresh composite meat from Thai Ligor hybrid chicken (LC) and commercial broiler chicken (BC). The composite meat samples, which comprise minced breast and thigh without skin from 20 chicken carcasses in a 1:1 (w/w) ratio, were randomly selected for analysis using the completely randomized design (CRD). Results showed that BC meat exhibited higher ultimate pH after 24 h, Ca2+-ATPase activity, and trichloroacetic acid (TCA)-soluble peptide content compared to LC meat (p < 0.05). While both meat types showed non-significant differences in reactive sulfhydryl (SH) levels (p > 0.05), LC meat exhibited higher hydrophobicity compared to BC meat (p < 0.05). Differential scanning calorimetry (DSC) analysis revealed a single transition peak in all samples. LC meat exhibited higher thermal stability than BC meat, with transition peaks at 91 °C and 81 °C, respectively, in non-sodium chloride (NaCl) treated samples. Samples treated with 2.5% NaCl exhibited transition peaks around 70 °C for BC and 79 °C for LC. LC meat showed higher storage modulus (G′) and loss modulus (G″) values than BC meat, suggesting a stronger gel-forming tendency. LC meat gels exhibited higher hardness, cohesiveness, gumminess, and chewiness, and a slightly lower pH (6.14 vs. 5.97) compared to BC meat gels (p < 0.05). LC meat gels displayed larger expressible moisture content (p < 0.05), although the value was approximately 6%. Compared to LC meat gels, BC meat gels appeared slightly whiter (p < 0.05). To compare the lipid oxidation of BC and LC meat gels day by day, the thiobarbituric acid reactive substances (TBARS) of gels stored at 4 °C in polyethylene bags were measured on Days 0, 4, and 8. Both BC and LC meat gels showed acceptable lipid oxidation-based rancid off-flavor after short-term storage at 4 °C, with TBARS values below 2 mg malondialdehyde (MDA) equivalent/kg on Day 8. Understanding these variations in biochemical properties and functional behavior can help optimize processing methods and produce meat products of superior quality that meet consumer preferences. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in the early stage of infection. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro). The test system consists of a peptide that fluoresces when cleaved by SARS PLpro that is active in crude, unprocessed lysates from human tongue scrapes and saliva. Test results are obtained in 30 minutes or less using widely available fluorescence plate readers, or a battery-operated portable instrument for on-site testing. Proof-of-concept was obtained in a study on clinical specimens collected from patients with COVID-19 like symptoms who tested positive (n = 10) or negative (n = 10) with LIAT RT-PCR using nasal mid turbinate swabs. When saliva from these patients was tested with in-house endpoint RT-PCR, 17 were positive and only 5 specimens were negative, of which 2 became positive when tested 5 days later. PLpro activity correlated in 17 of these cases (3 out of 3 negatives and 14 out of 16 positives, with one invalid specimen). Despite the small number of samples, the agreement was significant (p value = 0.01). Two false negatives were detected, one from a sample with a late Ct value of 35 in diagnostic RT-PCR, indicating that an active infection was no longer present. The PLpro assay is easily scalable and expected to detect all viable SARS-CoV-2 variants, making it attractive as a screening and surveillance tool. Additionally, we show feasibility of the platform as a new homogeneous phenotypic assay for rapid screening of SARS-CoV-2 antiviral drugs and neutralizing antibodies. [ABSTRACT FROM AUTHOR]

Fishmeal (FM) is a key component of commercial fish feeds, but due its unsustainable supply, the search for quality alternatives of FM has become a significant area of investigation worldwide. The insect-based proteins such as black soldier fly larvae (BSFL) are being recognized as an alternative ingredient. However, anti-nutritional factors in these alternatives may negatively affect nutrient utilization in fish. Incorporating exogenous protease enzymes as feed additives could be a promising way to improve the digestibility of these alternative ingredients. Therefore, this study aimed to evaluate the impact of dietary inclusion of BSFL meal, combined with protease supplementation, on various parameters of striped catfish (Pangasius hypophthalmus). Five diets were formulated: a control diet (FM-based), two diets with 30% and 60% BSFL inclusion (BSF30 and BSF60), and two diets with the same inclusion levels plus the incorporation of exogenous protease (BSF30P and BSF60P). One hundred fifty fish (8.26±0.02 g) were arbitrarily allocated to five groups, each with three replicates. The fish were provided with their respective diets twice per day for 60 days. In comparison to the control, fish-fed diets supplemented with protease demonstrated statistically significant lower levels of feed conversion ratio and higher growth performance regarding the final body weight and weight gain. Lipase activity in the BSF60P group was notably greater than in the control group. Protease supplementation significantly enhanced the apparent digestibility coefficient of protein, intestinal protease activity, and crude protein content in the whole body. Most hematological and biochemical parameters remained unaffected except for substantially lower triglyceride and cholesterol levels in the highest BSFL inclusion groups. These groups also showed a reduction in crude fat contents. While glutathione peroxidase and malondialdehyde levels did not change significantly from the control, the liver tissues of fish fed BSF60P diets showed considerably higher levels of antioxidant enzymes such as catalase and superoxide dismutase. The findings suggest that including 60% of a BSFL based diet, along with exogenous protease supplementation, is feasible without compromising the growth performance and health of striped catfish. [ABSTRACT FROM AUTHOR]

In this study, silver carp surimi products enriched with Antarctic krill oil high internal phase emulsions (AKO-HIPEs) were cooked using steaming (STE), microwave heating (MIC), and air-frying (AIR), respectively. The gel and flavor properties, lipid quality and stability were investigated. Compared to the MIC and AIR groups, the STE surimi gel added with HIPEs had better texture properties, exhibiting higher water-holding capacity and a more homogeneous structure, while the air-frying treatment resulted in visually brighter surimi products. The degree of lipid oxidation during cooking was in an order of STE < MIC < AIR as determined by electron paramagnetic resonance spectrometer and thiobarbituric acid reactive substances. HIPE-added surimi gels retained more nutrients and flavor when cooked by AIR compared to STE and MIC. Results imply that the texture properties and lipid stability of surimi products fortified with AKO-HIPEs were better than those of the oil group under any cooking method. In conclusion, surimi products added with AKO-HIPEs had better gel properties and retained more fatty acids and flavor than AKO-SO. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of multi-enzyme (alkaline protease, xylanase, glucanase, β-mannanase, cellulase, acid protease, glucoamylase, and α-galactosidase) on antioxidant capacity, egg quality, amino acid profiles in yolk, cecal microflora and metabolites in laying hens. A total of 384 Jingfen No.6 laying hens aged 65 weeks were randomly divided into 4 treatments groups (6 replicates per group) and fed diets containing 0, 150, 300, or 600 mg kg−1 multi-enzyme over an 8-week feeding duration. Our findings revealed that supplementation with 600 mg kg−1 of multi-enzyme significantly increased the albumen height (P < 0.05) and haugh unit (P < 0.05). Moreover, as the levels of multi-enzyme supplementation in the diet increased, there were significant increases in activities of total antioxidant capacity (T-AOC) in serum (P < 0.05) and glutathione peroxidase (GSH-Px) in the liver (P < 0.05). Different levels of multi-enzyme supplementation significantly affected the composition of amino acid profiles in the yolk. Furthermore, the results from 16S rRNA sequencing and untargeted metabolomics analysis of cecal content revealed that multi-enzyme supplementation altered the cecal microflora and metabolite profiles. We found the relative abundance of the Bacteroidota phyla in T600 group was significantly increased (P < 0.05) compared to CON and T150 groups, but the relative abundance of the Firmicutes phylum in T600 group were significantly lower than T150 group (P < 0.05). At the genus level, the relative abundance of the Parabacteroides genera in T300 group, the Faecalibacterium genera in T300 and T600 groups, the norank_f_Prevotellaceae genera in treatment groups (T150, T300 and T600), the norank_f_Peptococcaceae genera in T600 group, and the Monoglobus genera in T1 group were significantly increased. The KEGG pathway analysis showed that the common enrichment metabolic pathways of each treatment group compared to the CON group were glycine, serine and threonine metabolism, foxo signaling pathway and mTOR signaling pathway, and the enrichment metabolic pathways shared by T300 vs CON and T600 vs CON was galactose metabolism and glycolysis/gluconeogenesis pathways. Correlation analysis identified notable relationships between specific microbes and metabolites with T-AOC in serum, GSH-Px activity in the liver, amino acids in yolk, albumen height, and haugh units. Overall, this study suggests that multi-enzyme supplementation regulated the cecal microbial community and metabolism, potentially influencing amino acid profiles in yolk, antioxidant capacity, and egg quality. [ABSTRACT FROM AUTHOR]

Background: The serine protease like (Spl) proteases of Staphylococcus aureus are a family of six proteases whose function and impact on virulence are poorly understood. Here we propose alpha-1-antitrypsin (AAT), an important immunomodulatory serine protease inhibitor as target of SplD, E and F. AAT is an acute phase protein, interacting with many proteases and crucial for prevention of excess tissue damage by neutrophil elastase during the innate immune response to infections. Methods: We used MALDI-TOF-MS to identify the cleavage site of Spl proteases within AAT's reactive center loop (RCL) and LC-MS/MS to quantify the resulting peptide cleavage product in in vitro digestions of AAT and heterologous expressed proteases or culture supernatants from different S. aureus strains. We further confirmed proteolytic cleavage and formation of a covalent complex with Western Blots, investigated AAT's inhibitory potential against Spls and examined the NETosis inhibitory activity of AAT-Spl-digestions. Results: SplD, E and F, but not A or B, cleave AAT in its RCL, resulting in the release of a peptide consisting of AAT's C-terminal 36 amino acids (C36). Synthetic C36, as well as AAT-SplD/E/F-digestions exhibit NETosis inhibition. Only SplE, but not D or F, was partly inhibited by AAT, forming a covalent complex. Conclusion: We unraveled a new virulence trait of S. aureus , where SplD/E/F cleave and inactivate AAT while the cleavage product C36 inhibits NETosis. [ABSTRACT FROM AUTHOR]

Elevated neutrophil counts in broncho-alveolar lavage (BAL) fluids of lung transplant (LTx) patients with chronic lung allograft dysfunction (CLAD) are associated with disease pathology. However, phenotypical characteristics of these cells remained largely unknown. Moreover, despite enhanced levels of the most potent human neutrophil-attracting chemokine CXCL8 in BAL fluid, no discrimination had been made between natural NH2-terminally truncated CXCL8 proteoforms, which exhibit up to 30-fold differences in biological activity. Therefore, we aimed to characterize the neutrophil maturation and activation state, as well as proteolytic activation of CXCL8, in BAL fluids and peripheral blood of LTx patients with CLAD or infection and stable LTx recipients. Flow cytometry and microscopy revealed a high diversity in neutrophil maturity in blood and BAL fluid, ranging from immature band to hypersegmented aged cells. In contrast, the activation phenotype of neutrophils in BAL fluid was remarkably homogeneous. The highly potentiated NH2-terminally truncated proteoforms CXCL8(6–77), CXCL8(8–77) and CXCL8(9–77), but also the partially inactivated CXCL8(10–77), were detected in BAL fluids of CLAD and infected LTx patients, as well as in COVID-19 and influenza patient cohorts by tandem mass spectrometry. Moreover, the most potent proteoform CXCL8(9–77) specifically correlated with the neutrophil counts in the LTx BAL fluids. Finally, rapid proteolysis of CXCL8 in BAL fluids could be inhibited by a combination of serine and metalloprotease inhibitors. In conclusion, proteolytic activation of CXCL8 promotes neutrophilic inflammation in LTx patients. Therefore, application of protease inhibitors may hold pharmacological promise for reducing excessive neutrophil-mediated inflammation and collateral tissue damage in the lungs. [ABSTRACT FROM AUTHOR]

This study explored the nutritional qualities of black mash bean flour impacted by fermentation, gelatinization and their combination. Protease activity, in vitro protein digestibility (IVPD), and protein solubility were evaluated for both fermented and unfermented black mash bean flour samples. Interestingly, when compared to fermented samples and gelatinized mash bean flour (GMBF), raw mash bean flour (RMBF) showed the highest protein solubility. Due to enzymatic activity and protein hydrolysis, fermentation with Lactobacillus E14 and Saccharomyces cerevisiae MK-157 enhanced protein solubility. Prolonged fermentation timeframes were positively correlated with protease activity and IVPD, suggesting the degradation of complex proteins. The highest levels of IVPD (90.12%) and protease activity (5546.78 U/g) were detected in GMBF fermented by S. cerevisiae MK-157, indicating the impact of fermentation on protein breakdown. The Lactobacillus E14-fermented RMBF exhibited 5521.45 U/g of protease activity and 87.54% IVPD. The mineral content of the fermented samples was substantially greater than that of the unfermented GMBF. The increased calcium, magnesium, potassium, sodium, iron, zinc, and copper levels for the GMBF fermented by S. cerevisiae MK-157 indicated that fermentation facilitated the release of minerals from chelated compounds. Microscopic examination revealed higher protein quantities in the fermented flour as well as hydrolyzed and broken starch granules embedded in the protein matrix. Large mash bean proteins were shown to be proteolyzed during fermentation into smaller peptides by SDS PAGE. Storage proteins were extensively broken down during fermentation with S. cerevisiae MK-157, whereas Lactobacillus E14 displayed a more focused pattern of hydrolysis. The nutritional profile of black mash bean flour was improved by fermentation with Lactobacillus E14 and S. cerevisiae MK-157. [ABSTRACT FROM AUTHOR]

Volumetric muscle loss (VML) is a serious problem in healthcare that requires innovative solutions. Collagen and its derivatives are promising biomaterials for muscle tissue replacement due to their high biocompatibility, biodegradability, and lack of toxicity. This review comprehensively discusses collagen from various sources, its structural characteristics, cross-linking methods to obtain hydrogels, and approaches to incorporating various therapeutic molecules to create a biocomposite system with controlled release. Collagen-based scaffolds are promising constructs in tissue engineering and regenerative medicine. They can both perform their function independently and act as a depot for various biologically active substances (drugs, growth factors, genetic material, etc.). Collagen-based scaffolds for muscle volume restoration are three-dimensional constructs that support cell adhesion and proliferation and provide controlled release of therapeutic molecules. Various mechanical and biological properties of scaffolds can be achieved by cross-linking agents and bioactive molecules incorporated into the structure. This review highlights recent studies on collagen-based hydrogels for restoration of volumetric muscle loss. [ABSTRACT FROM AUTHOR]

To address the limited supply of serum albumin (SA) and potential pathogen contamination, focus has been concentrated on the heterologous expression of human serum albumin (HSA), particularly in Pichia pastoris. However, there are rare studies on feline serum albumin (FSA), which requires a large amount in pet foods and clinical treatment. In this work, the codon-optimized recombinant feline serum albumin (rFSA) sequence was designed and transferred into P. pastoris GS115 for recombinant expression. The high-level expression strain was selected by a high concentration of G418, followed by plate and shaker screening. At the 5 L fermenter level, the total protein concentration reached 3.89 mg/mL after 113 h of induction. At lower concentrations (1–4 μM), rFSA exhibited a potent free radical scavenging capacity, reaching 99% and 60% for ABTS+• and •O2−, respectively, which surpassed that of natural plasma-derived FSA. The secondary structure and stability of rFSA were found to be consistent with those of FSA. Additionally, an in vivo safety assay in mice showed no significant difference between the rFSA group and the normal saline group in terms of body weight changes, complete blood count, serum biochemistry, inflammatory factors, and tissue sections. These results above indicate that P. pastoris is the optimal host for the high preparation of rFSA. Furthermore, rFSA has been demonstrated to be relatively safe, which paves the way for subsequent industrialized production and its application in pet foods and veterinary clinics. [ABSTRACT FROM AUTHOR]

Tetanus neurotoxins (TeNT) and botulinum neurotoxins (BoNTs) are closely related ~150 kDa protein toxins that together comprise the group of clostridial neurotoxins (CNTs) expressed by various species of Clostridia. While TeNT is expressed as a single polypeptide, BoNTs are always produced alongside multiple non-toxic proteins that form a stabilizing complex with BoNT and are encoded in a conserved toxin gene cluster. It is unknown how tent evolved without a similar gene cluster and why complex-free TeNT is secreted as a stable and soluble protein by C. tetani, whereas complexing proteins appear to be essential for BoNT stability in culture supernatants of C. botulinum. To assess whether the stability of TeNT is due to an innate property of the toxin or is a result of C. tetani's intra- and extra-cellular environment, both TeNT and complex-free BoNT/A1ERY were expressed recombinantly in atoxic C. tetani and analyzed for expression and stability. The strong clostridial ferredoxin (fdx) promotor resulted in the expression of recombinant TeNT at greater levels and earlier time points than endogenously produced TeNT. Recombinant BoNT/A1ERY was similarly expressed by atoxic C. tetani, although partial degradation was observed. The rBoNT/A1ERY produced in C. tetani was also partially proteolytically processed to the dichain form. Investigations of bacterial growth media and pH conditions found that the stability of rTeNT and rBoNT/A1ERY in spent media of C. tetani or C. botulinum was affected by growth media but not by pH. These data indicate that the distinct metabolism of C. tetani or C. botulinum under various growth conditions is a primary factor in creating a more or less favorable environment for complex-free CNT stability. [ABSTRACT FROM AUTHOR]

Baker's yeast is a key starting material for producing extracts with diverse compositions and applications. This study investigates the effect of pulsed electric field (PEF) pretreatment, which induces irreversible electropermeabilization, on the enzymatic hydrolysis of yeast. Cell suspensions were exposed to monopolar rectangular pulses in a continuous flow system followed by 4 h of incubation with Alcalase at concentrations of 0.2% and 0.5%. PEF pretreatment significantly improved enzymatic hydrolysis, with maximum intracellular content recovery under electrical conditions resulting in outlet temperatures of 56–58 °C. The released protein reached 163.7 ± 13 mg per gram of dry cell weight (DCW). SDS-PAGE analysis showed that the extracts predominantly contained peptides with molecular masses below 4.7 kDa. The phenolic content was comparable to that of cell lysates obtained after mechanical disruption. The free α-amino nitrogen content and total antioxidant activity reached 218.2 ± 26 mg/gDCW and 53.4 ± 4.6 mg TE/gDCW, respectively, representing 3.2-fold and 2.65-fold increases compared to cell lysates. The hydrolysates from PEF-pretreated cells demonstrated a positive effect on the proliferation of the human keratinocyte cell line HaCat. The obtained data lead to the conclusion that PEF pretreatment is a promising approach to enhance the production of yeast hydrolysates with various applications. [ABSTRACT FROM AUTHOR]