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Introduction Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline—Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia , and Daboia russelii were tested under inhibitory conditions using doxycycline. Materials and Methods Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii , and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. Results Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P  < .0001) (Fig. 1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P  < .0001) (Fig. 1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus  = 20.29 mg/kg (Fig. 2A), N. kaouthia  = 0.38 mg/kg (Fig. 2B), and D. russelii  = 7.92 mg/kg (Fig. 2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig. 3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P  < .0001) (Fig. 4). FIGURE 1. In vitro doxycycline inhibition of PLA, and SVMP activity in whole venom. The AUC was calculated for PLA and SVMP activities for 20-min and 4-h time courses, respectively using EnzChek Phospholipase A2, assay kits, and EnzChek Gelatinase (SVMP) assay kits. (A) Inhibition of PLA2, with doxycycline (Dox) was tested at concentrations of 0.32 mg/mL, 0.16 mg/mL, and 0.08 mg/mL against A. piscivorus (4 μg/mL), N. kaouthia (4 μg/mL), and D. russelii (8 μg/mL) venom. A venom-only control was included. (B) Inhibition of SVMP with doxycycline was tested at concentrations of 0.32 mg/mL, 0.16 mg/mL, and 0.08 mg/mL against A. piscivorus (120 μg/ml.), N. kaouthia (125 μg/ml.), and D. russelii (250 μg/ml.). A venom-only control was included. An ordinary 1-way ANOVA with Tukey's post-hoc test was performed for inhibition assays to compare treatments to their respective venom controls. All error bars show mean ± SD, n  = 3, (****, P  < .0001). Open in new tab Download slide FIGURE 2. Percent survival of mice receiving various dosages for each venom studied and LDs, calculation. Adult BALB/c mice received intramuscular injections of various venom dosages for each venom, (A) A. piscivorus , (B) N. kaouthia , and (C) D. russelii. The resulting percent survival was measured across 48 h and the lethal dose 50 (LD50) was calculated using the Spearman-Karber method. In each graph, each treatment group (venom dosage) has n  = 5. Open in new tab Download slide FIGURE 3. Percent survival of mice receiving various doxycycline dosages for each venom studied and ED calculation. Adult BALB/c mice received intramuscular injections of various doxycycline dosages combined with 3 times the LD50 of cach venom [ A. piscivorus (AP) (60.87 mg/kg), N. kaouthia (NK) (1.14 mg/kg). D. russelii (DR) (23.76 mg/kg)] and the resulting percent survival was measured across 48 hours and the effective dose 50 (ED50) was calculated using the Spearman–Karber method. Each treatment group has n  = 5. Open in new tab Download slide FIGURE 4. Venom-induced CK activity with doxycycline-mediated inhibition. The amount of CK produced by mice exposed to one-fourth of the LD50, strength of A. piscivorus (AP) (5.07 mg/kg), N. kaouthia (NK) (0.095 mg/kg), or D. russelli (DR) (1.98 mg/kg) venom either with or without doxycycline (64 mg/kg). All error bars show mean, ± SD, n  = 5 (NaCl—Dox, AP NaCl), n-4 (NK Dox), and n-3 (AP Dox. NK NaCl, DR Dox, DR + NaCl). Statistical differences were determined using an ordinary 1-way ANOVA with Tukey's post-hoc test comparing each venom group within itself and to the doxycycline only control (**** P  < .0001). (*** P  < .001), (** P  < .01). (* P  < .05). Open in new tab Download slide Conclusion Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species. [ABSTRACT FROM AUTHOR]

Nutritional deprivation triggers a switch from a saprotrophic to predatory lifestyle in soil-dwelling nematode-trapping fungi (NTF). In particular, the NTF Arthrobotrys oligospora secretes food and sex cues to lure nematodes to its mycelium and is triggered to develop specialized trapping devices. Captured nematodes are then invaded and digested by the fungus, thus serving as a food source. In this study, we examined the transcriptomic response of A. oligospora across the stages of sensing, trap development, and digestion upon exposure to the model nematode Caenorhabditis elegans. A. oligospora enacts a dynamic transcriptomic response, especially of protein secretion-related genes, in the presence of prey. Two-thirds of the predicted secretome of A. oligospora was up-regulated in the presence of C. elegans at all time points examined, and among these secreted proteins, 38.5% are predicted to be effector proteins. Furthermore, functional studies disrupting the t-SNARE protein Sso2 resulted in impaired ability to capture nematodes. Additionally, genes of the DUF3129 family, which are expanded in the genomes of several NTF, were highly up-regulated upon nematode exposure. We observed the accumulation of highly expressed DUF3129 proteins in trap cells, leading us to name members of this gene family as Trap Enriched Proteins (TEPs). Gene deletion of the most highly expressed TEP gene, TEP1, impairs the function of traps and prevents the fungus from capturing prey efficiently. In late stages of predation, we observed up-regulation of a variety of proteases, including metalloproteases. Following penetration of nematodes, these metalloproteases facilitate hyphal growth required for colonization of prey. These findings provide insights into the biology of the predatory lifestyle switch in a carnivorous fungus and provide frameworks for other fungal-nematode predator-prey systems.

BACKGROUND: The aim of this study was to address the diagnostic challenges associated with pancreatic disorders, such as chronic pancreatitis (CP) and pancreatic cancer (PC) and highlight the importance of protease activity as a crucial prognostic marker for these conditions. METHODS: Total proteolytic activity was determined using casein as a substrate. To measure the activity of metal-dependent and serine proteases, ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride were used. The content of low- and middle-molecular-weight (LMMW) substances was determined spectrophotometrically. The protein profile was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while zymography was performed to indicate the molecular weight of active enzymes. RESULTS: The pathogenesis of both pancreatic disorders was found to be accompanied by a proteolytic imbalance in pancreatic tissues, manifested in either the increase in the total proteolytic activity under pancreatitis or the decrease in the total proteolytic activity under pancreatic cancer. The increase in the levels of both metal-dependent and serine proteases was indicated in the blood plasma of patients in both studied groups. This proteolytic imbalance was accompanied by the accumulation of protein degradation fragments and LMMW substances. Results of zymography were also informative: pancreatic tissue and plasma samples from patients with either CP or PC were characterized by the presence of additional bands, indicating altered proteolytic activity in both pathological conditions. CONCLUSIONS: This study underscores the importance of proteolytic features in distinguishing CP and PC, offering potential diagnostic markers. Proteolytic shifts may aid in differentiating pathologies, detecting transitions, and developing specific protease inhibitors. Further research is crucial for advancing diagnostic and therapeutic strategies for CP and PC. [ABSTRACT FROM AUTHOR]

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide‐specific phospholipase C (PI‐PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI‐PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI‐PLC γ1 was produced. The decrease of PI‐PLC γ1 was associated with the decrease of metalloprotease‐3 (MMP‐3), and MMP‐13, and ADAMTS‐5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI‐PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI‐PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI‐PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine. Significant Statement: This manuscript focuses on the modulation of phosphoinositide‐specific phospholipase C (PI‐PLC) γ1 in human primary fibroblast‐like synoviocytes, which leads to the modulation of metalloprotease‐3 (MMP‐3), MMP‐13, and ADAMTS‐5, involved in the degradation of extracellular matrix in degenerative joint diseases. We found that in some synoviocyte samples, the PI‐PLC γ1 mRNA incurs in the intron retention (IR) phenomenon, which results in an unsuccessful translation of the proteins associated with the decrease of MMP enzymes. Moreover, IR is increased after cell treatment with Harpagophytum procumbens extract (HPE), traditionally used to treat osteoarthritis. Considering that HPE results are effective in modulating MMPs only in synoviocyte samples where the IR phenomenon is present, it is necessary to evaluate individual variability in the personalized medicine field. [ABSTRACT FROM AUTHOR]

Envenomation by Loxosceles spiders can result in local and systemic pathologies. Systemic loxoscelism, which can lead to death, is characterized by intravascular hemolysis, platelet aggregation, and acute kidney injury. Sphingomyelinase D (SMase D) in Loxosceles spider venom is responsible for both local and systemic pathologies, and has been shown to induce metalloprotease activity. As the complement system is involved in many renal pathologies and is involved in hemolysis in systemic loxoscelism, the aim of this study was to investigate its role and the role of complement regulators and metalloproteases in an in vitro model of Loxosceles venom induced renal pathology. We investigated the effects of the venom/SMase D and the complement system on the HK-2 kidney cell line. Using cell viability assays, western blotting, and flow cytometry, we show that human serum, as a source of complement, enhanced the venom/SMase D induced cell death and the deposition of complement components and properdin. Inhibitors for ADAM-10 and ADAM-17 prevented the venom induced release of the of the complement regulator MCP/CD46 and reduced the venom/SMase D induced cell death. Our results show that the complement system can contribute to Loxosceles venom induced renal pathology. We therefore suggest that patients experiencing systemic loxoscelism may benefit from treatment with metalloproteinase inhibitors and complement inhibitors, but this proposition should be further analyzed in future pre-clinical and clinical assays. [ABSTRACT FROM AUTHOR]

Purpose: Low doses of systemic doxycycline (LD-SD) inhibit angiogenesis and the expression of matrix metalloproteases, which are determinants of pterygium progression. This study aimed to compare the recurrence rate and visual outcome of pterygium excision in patients undergoing chronic treatment with LD-SD for chronic refractory blepharitis and LD-SD-naive patients. Methods: A retrospective analysis of patients that underwent surgical excision and conjunctival graft apposition was conducted. Patients were divided in a TETRA group (under LD-SD treatment at the moment of surgery) and a control group. The main outcome was the rate of recurrence at 1 year postoperatively. Secondary outcomes were the comparisons of surface regularity, visual quality, and dry-eye symptoms at 6-week, 6-month, and 1-year follow-up in the two groups. Results: The TETRA group showed a significantly lower rate of 1-year recurrence both in primary (p = 0.034) and recurrent (p < 0.001) pterygia. The best corrected visual acuity (BCVA), astigmatic error, corneal total root mean square (RMS), and ocular surface disease index (OSDI) significantly reduced during the follow-up in both groups. The surface asymmetry index and high-order aberrations (HOAs) significantly reduced only in the TETRA group. The final BCVA was significantly higher, while the OSDI score and total RMS and HOAs were significantly lower in the TETRA group compared to the control. Conclusions: Patients under treatment with LD-SD showed a lower rate of recurrence at 1-year follow-up compared to controls. These patients also experienced higher BCVA and surface regularity and less dry-eye symptoms. [ABSTRACT FROM AUTHOR]

Regenerative endodontic procedures (REP) aim at reestablishing tooth vitality by replacing the irreversibly damaged dental pulp removed by the dental practitioner with a new functional one. The current treatment of advanced caries relies on the replacement of the inflamed or necrosed dental pulp with an inert filling material. This leads to a functional but non-vital tooth, which lacks the ability to sense dental tissue damage, and to protect from further bacterial attack. Therapeutic strategies inspired by tissue engineering called REP propose to regenerate a fully functional dental pulp directly in the canal space. Promising results were obtained using dental pulp mesenchymal stem cells (DP-MSCs) in combination with bio-inspired artificial and temporary 3D hydrogels made of extracellular matrix molecules such as collagen and fibrin biomacromolecules. However, the uncontrolled mechanisms of DP regeneration from DP-MSCs in 3D biomacromolecules fail to regenerate a fully functional DP and can induce fibrotic scarring or mineralized tissue formation to a non-negligible extent. The lack of knowledge regarding the early molecular mechanisms initiated by DP-MSCs seeded in ECM-made hydrogels is a scientific lock for REP. In this study, we investigated the early DP-MSC–response in a 3D fibrin hydrogel. DP-MSCs isolated from human third molars were cultured for 24 h in the fibrin hydrogel. The differential transcript levels of extracellular and cell surface genes were screened with 84-gene PCR array. Out of the 84 genes screened, 9 were found to be overexpressed, including those coding for the integrin alpha 2 subunit, the collagenase MMP1 and stromelysins MMP3, MMP10 and MMP12. Over-expression of ITGA2 was confirmed by RT-qPCR. The expression of alpha 2 integrin subunit protein was assessed over time by immunoblot and immunofluorescence staining. The increase in the transcript level of MMP1, MMP3, MM10 and MMP12 was confirmed by RT-qPCR. The overexpression of MMP1 and 3 at the protein level was assessed by immunoblot. MMP3 expression by DP-MSCs was observed by immunofluorescence staining. This work demonstrates overexpression of ITGA2 and of MMP1, 3, 10 and 12 by DP-MSCs cultured in a fibrin hydrogel. The main preliminary extracellular and cell surface response of the DP-MSCs to fibrin hydrogel seems to rely on a ITGA2/MMP3 axis. Further investigations are needed to precisely decipher the role of this axis in dental pulp tissue building. Nevertheless, this work identifies extracellular and cell surface molecules that could be potential checkpoints to be targeted to guide proper dental pulp tissue regeneration.

Background: An acquired cholesteatoma is a benign but locally aggressive lesion in the middle ear. It is characterized by chronic inflammation and the destruction of surrounding bone. Therefore, the aim of this study was to compare defensins HβD-2 and HβD-4; pro- and anti-inflammatory cytokines IL-1α and IL-10; proliferation marker Ki-67; transcription factor NF-κβ; angiogenetic factor VEGF; Sonic hedgehog gene protein SHH; and remodeling factors MMP-2, MMP-9, TIMP-2, and TIMP-4 in adult and pediatric cholesteatoma tissue, and to compare these groups with control skin tissue. Methods: The study included 25 cholesteatoma tissue material samples from children, 25 from adults, and 7 deep external ear canal skin samples from cadavers. The tissues were stained immunohistochemically and evaluated using semi-quantitative methods. Nonparametric tests, such as the Kruskal–Wallis test and Spearman rank correlation, were used. Results: There were no statistically discernible differences between the adult and children groups when comparing the relative numbers of factor-positive cells. Conclusions: There are no histopathological differences between adult and children cholesteatoma tissues. [ABSTRACT FROM AUTHOR]

Respiratory diseases in ruminants are a main cause of economic losses to farmers worldwide. Approximately 25% of ruminants experience at least one episode of respiratory disease during the first year of life. Mannheimia haemolytica is the main etiological bacterial agent in the ruminant respiratory disease complex. M. haemolytica can secrete several virulence factors, such as leukotoxin, lipopolysaccharide, and proteases, that can be targeted to treat infections. At present, little information has been reported on the secretion of M. haemolytica A2 proteases and their host protein targets. Here, we obtained evidence that M. haemolytica A2 proteases promote the degradation of hemoglobin, holo-lactoferrin, albumin, and fibrinogen. Additionally, we performed biochemical characterization for a specific 110 kDa Zn-dependent metalloprotease (110-Mh metalloprotease). This metalloprotease was purified through ion exchange chromatography and characterized using denaturing and chaotropic agents and through zymography assays. Furthermore, mass spectrometry identification and 3D modeling were performed. Then, antibodies against the 110 kDa-Mh metalloprotease were produced, which achieved great inhibition of proteolytic activity. Finally, the antibodies were used to perform immunohistochemical tests on postmortem lung samples from sheep with suggestive histology data of pneumonic mannheimiosis. Taken together, our results strongly suggest that the 110-Mh metalloprotease participates as a virulence mechanism that promotes damage to host tissues. [ABSTRACT FROM AUTHOR]

Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

The tumor microenvironment in glioblastoma (GB) is considered to be "cold", i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent "imprinting" mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage–tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8. [ABSTRACT FROM AUTHOR]

The chemokine fractalkine (FKN, CX3CL1), a member of the CX3C subfamily, contributes to neuron--glia interaction and the regulation of microglial cell activation. Fractalkine is expressed by neurons as a membrane-bound protein (mCX3CL1) that can be cleaved by extracellular proteases generating several sCX3CL1 forms. sCX3CL1, containing the chemokine domain, and mCX3CL1 have high affinity by their unique receptor (CX3CR1) which, physiologically, is only found in microglia, a resident immune cell of the CNS. The activation of CX3CR1contributes to survival and maturation of the neural network during development, glutamatergic synaptic transmission, synaptic plasticity, cognition, neuropathic pain, and inflammatory regulation in the adult brain. Indeed, the various CX3CL1 forms appear in some cases to serve an anti-inflammatory role of microglia, whereas in others, they have a pro-inflammatory role, aggravating neurological disorders. In the last decade, evidence points to the fact that sCX3CL1 and mCX3CL1 exhibit selective and differential effects on their targets. Thus, the balance in their level and activity will impact on neuron--microglia interaction. This review is focused on the description of factors determining the emergence of distinct fractalkine forms, their age-dependent changes, and how they contribute to neuroinflammation and neurodegenerative diseases. Changes in the balance among various fractalkine forms may be one of the mechanisms on which converge aging, chronic CNS inflammation, and neurodegeneration. [ABSTRACT FROM AUTHOR]

Prostate cancer represents the most common type of cancer among males and the second leading cause of cancer death in men in Western society. In most cases (~70%), PC has a slow and symptom-free growth, whereas it is more aggressive in the remaining patients. Current PC therapies prevalently target the proliferative function of the androgen receptor and may only be effective within short periods, beyond which the disease will progress to metastatic and castration-resistant phenotype. Preclinical and clinical studies are aimed at investigating the molecular basis for prostate cancer spreading. Although considerable efforts have been made to dissect the programs that foster prostate cancer spreading, few biomarkers predictive of metastatic phenotype have yet been identified and few therapeutic options are available for treatment of the metastatic disease. In the present paper, we will discuss innovative aspects of prostate cancer biology, which impinge on the role of cancer-associated fibroblasts and the released matrix metalloproteinases in the disease progression. Investigating these aspects might allow the discovery of clinically actionable biomarkers to target in the advanced stages of prostate cancer.

The aim of the present work was to evaluate if the use of grape pomace (GP) in the feeding of dairy ewes can improve the content of phenolic compounds (PCs) in the milk and affect the anti-inflammatory and antioxidative status of the milk. For this purpose, 46 ewes were randomly assigned to two groups of 23 animals each: a control group (Ctrl) that received a standard diet and an experimental group (GP+), whose diet was been formulated with 10% GP on a dry matter (DM) basis. At the end of the 60 days of the trial, from 10 ewes selected randomly from each group, individual milk samples were collected and analyzed for the identification and the quantification of phenolic compounds through an ultra-high-performance liquid chromatography system, and milk anti-inflammatory and antioxidative status were evaluated by enzyme-linked immunosorbent assay, determining the activity of GPx and CAT and the levels of IL-1 and TNFα. In addition, gelatinolytic activity of Type IV collagenases (MMP-2/MMP-9) was evaluated by the fluorometric method and zymographic approach. The results obtained showed that the diet with GP affects the phenolic profile of milk, inducing milk enrichment of phenolic compounds without, however, having a significant impact on milk antioxidant and inflammatory status. However, a lower activity of MMP-9 was found in GP+ milk. The use of the molecular docking approach showed the ability of luteolin to approach the catalytic pocket of the enzyme, interfering with the recruitment of the substrate, and therefore, slowing down their hydrolytic activity. [ABSTRACT FROM AUTHOR]

BackgroundPulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates the management of these deep-seated infections. Pathological inflammation during infection is driven by interleukin-1β (IL-1β), but the molecular processes involved are not fully understood.MethodsWe examined IL-1β activation in a pulmonary model infection of Pseudomonas aeruginosa and in vitro using genetics, specific inhibitors, recombinant proteins, and targeted reporters of protease activity and IL-1β bioactivity.FindingsCaspase-family inflammasome proteases canonically regulate maturation of this proinflammatory cytokine, but we report that plasticity in IL-1β proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1β activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection.InterpretationPreservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Discovery of this IL-1β regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such as matrix metalloprotease inhibitors that inhibit LasB and limit inflammation and pathology during P. aeruginosa pulmonary infections.FundingFull details are provided in the Acknowledgements section.

Cryptococcus neoformans (Cn) is the leading cause of fungal meningitis, a deadly disease with limited therapeutic options. Dissemination to the central nervous system hinges on the ability of Cn to breach the blood-brain barrier (BBB) and is considered an attribute of Cn virulence. Targeting virulence instead of growth for antifungal drug development has not been fully exploited despite the benefits of this approach. Mpr1 is a secreted fungal metalloprotease not required for fungal growth, but rather, it functions as a virulence factor by facilitating Cn migration across the BBB. This central role for Mpr1, its extracellular location, and lack of expression in mammalian cells make Mpr1 a high-value target for an antivirulence approach aimed at developing therapeutics for cryptococcal meningitis. To test this notion, we devised a large-scale screen to identify compounds that prohibited Cn from crossing the BBB by selectively blocking Mpr1 proteolytic activity, without inhibiting the growth of Cn A phytochemical natural product-derived library was screened to identify new molecular scaffolds of prototypes unique to a Cn microecosystem. Of the 240 pure natural products examined, 3 lead compounds, abietic acid, diosgenin, and lupinine inhibited Mpr1 proteolytic activity with 50% inhibitory concentration (IC50) values of

Tissue inhibitor of metalloproteinase (TIMP) 3 is a naturally occurring inhibitor of a broad range of proteases, with key roles in extracellular matrix turnover and in the pathogenesis of various diseases. In this study, we investigated the response of mice lacking TIMP3 (TIMP3-/-) to hepatic ischemia/reperfusion injury (IRI). We report here that TIMP3-/- mice showed an enhanced inflammatory response, exacerbated organ damage, and further impaired liver function after IRI when compared with their wild-type littermates. Loss of TIMP3 led to the cleavage and shedding of E-cadherin during hepatic IRI; the full-length 120-kDa E-cadherin and the ratio of 38-kDa C-terminal fragment/120-kDa E-cadherin were decreased and increased, respectively, in TIMP3-/- livers after IRI. Moreover, GI254023X, a potent inhibitor of a disintegrin and metalloprotease (ADAM) 10, was capable of partially rescuing the expression of E-cadherin in the TIMP3-null hepatocytes. The proteolysis of E-cadherin in the TIMP3-/- livers was also linked to the loss of β-catenin from the hepatocyte membranes and to an increased susceptibility to apoptosis after liver IRI. In a similar fashion, depression of the E-cadherin/β-catenin complex mediated by TIMP3 deletion and knockdown of β-catenin by small interfering RNA were both capable of inducing caspase activation in isolated hepatocytes subjected to H2 O2 oxidative stress. Hence, these results support a protective role for TIMP3 expression in sheltering the hepatocyte E-cadherin/β-catenin complex from proteolytic processing and inhibiting apoptosis after hepatic IRI.

Abstract Background Metastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis. Methods We used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA. Results We found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines. Conclusions We conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs. Video Abstract

Objective: Platelet-rich plasma (PRP) is used in multiple coagulation disorders. Its therapeutic effectiveness relies on technical procedures related to PRP procurement and preservation because free radicals induce platelet activation and aging. This work aims to elucidate the oxidative mechanisms involved in activation of platelets obtained from PRP during storage. Materials and Methods: One hundred ten PRP batches were obtained from healthy donors and kept under stirring at a temperature of 20-24 °C. Protein extraction was performed from platelet homogenates and plasma at different times of storage from day 1 to 20. The activities of antioxidant markers such as catalase (CAT), superoxide dismutase, and ceruloplasmin, as well as fibrinolytic protein activity metalloproteases 2 and 3, plasmin, and urokinase plasminogen activator, were analyzed by zymography assays. Oxidized proteins were also determined. Results: Significant activity of antioxidant enzymes and fibrinolytic molecules was observed on day 5 of storage in PRP homogenates, which increased over time and was concomitantly correlated with oxidized protein levels. Reverse enzymatic activity patterns were observed in plasma, except for CAT, which remained unchanged. Conclusion: Storage conditions of platelets from PRP for up to 5 days induced in vitro platelet activation by oxidative damage and proteolysis. This finding confirms the need for proper management of these blood products to preserve their viability and functionality.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane‐anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP‐3 and MMP‐7. Moreover, we found that MMP‐2 and MMP‐12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described. [ABSTRACT FROM AUTHOR]

Background: Autism spectrum disorder (ASD) encompasses a group of disorders characterized by difficulties with social interaction and repetitive behavior. The condition is supposed to originate from early shifts in brain development, while the underlying processes are unknown. Moreover, a considerable number of patients with ASD experience digestive difficulties. Metalloproteases (ADAMs) are a class of enzymes capable of cleaving membrane-bound proteins. Members of this family, ADAM17 and ADAM22, have the ability to cleave proteins like the pro-inflammatory cytokine TNF-ά and glutamate synaptic molecules, which are both engaged in neuro-inflammation and glutamate excitotoxicity as crucial etiological mechanisms in ASD. ADAM17 and ADAM22 may also have a role in ASD microbiota–gut–brain axis connections by regulating immunological and inflammatory responses in the intestinal tract. Subjects and Methods: Using ELISA kits, the plasma levels of ADAM17 and ADAM22 were compared in 40 children with ASD and 40 typically developing children. All of the autistic participants' childhood autism rating scores (CARS), social responsiveness scales (SRS), and short sensory profiles (SSP) were evaluated as indicators of ASD severity. Results: Our results showed that plasma levels of ADAM17 were significantly lower in ASD children than in control children, while ADAM22 demonstrated non-significantly lower levels. Our data also indicate that while ADAM17 correlates significantly with age, ADAM22 correlates significantly with CARS as a marker of ASD severity. Conclusions: Our interpreted data showed that alteration in ADAM17 and ADAM22 might be associated with glutamate excitotoxicity, neuroinflammation, and altered gut microbiota as etiological mechanisms of ASD and could be an indicator of the severity of the disorder. [ABSTRACT FROM AUTHOR]

Prostate cancer represents the most common type of cancer among males and the second leading cause of cancer death in men in Western society. In most cases (~70%), PC has a slow and symptom-free growth, whereas it is more aggressive in the remaining patients. Current PC therapies prevalently target the proliferative function of the androgen receptor and may only be effective within short periods, beyond which the disease will progress to metastatic and castration-resistant phenotype. Preclinical and clinical studies are aimed at investigating the molecular basis for prostate cancer spreading. Although considerable efforts have been made to dissect the programs that foster prostate cancer spreading, few biomarkers predictive of metastatic phenotype have yet been identified and few therapeutic options are available for treatment of the metastatic disease. In the present paper, we will discuss innovative aspects of prostate cancer biology, which impinge on the role of cancer-associated fibroblasts and the released matrix metalloproteinases in the disease progression. Investigating these aspects might allow the discovery of clinically actionable biomarkers to target in the advanced stages of prostate cancer. [ABSTRACT FROM AUTHOR]

Background and Objective: Biological regulators of periodontal inflammation, collagen degradation, and insulin resistance have not been determined in association with severity of periodontitis and response to periodontal treatment in diabetics. Our objective was to determine whether type 2 diabetes (T2DM) patients with periodontal disease present a distinct salivary biomarker profile compared with T2DM patients without periodontal disease and healthy subjects (without diabetes and periodontitis) pre‐ and post‐nonsurgical therapy. Methods: Clinical parameters of periodontal health and whole unstimulated saliva were collected from 92 participants (31 Not Periodontitis, NP; 32 T2DM without periodontitis, DWoP; and 29 with T2DM with periodontitis, DWP) at baseline. The T2DM groups received scaling and root planning (SRP) and provided saliva at 6‐week follow‐up. Salivary concentrations of interleukin (IL)‐1β, IL‐6, matrix metalloproteinase‐8 (MMP‐8), and resistin were measured by immunoassay. Results: The DWP group had significantly more disease and higher salivary concentrations at baseline for IL‐1β, MMP‐8, and resistin (p's <.01) compared with DWoP and NP. SRP resulted in significant improvement in periodontal parameters for the T2DM groups; however, more disease persisted (p <.001), and IL‐1β, MMP‐8, and resistin concentrations remained significantly higher in the DWP than the DWoP group (p <.01) at 6 weeks post‐treatment. Principal component analysis demonstrated the DWoP group appeared more biologically similar to the NP group than the DWP group. Concentrations of these salivary biomarkers increased with increasing periodontal disease severity (p <.05) in this study population. Conclusion: Salivary concentrations of IL‐1β, MMP‐8, and resistin appear to serve as biomarkers of periodontal status pre‐ and post‐treatment, irrespective of diabetes status. [ABSTRACT FROM AUTHOR]

Subgingival microbiome dysbiosis promotes the development of periodontitis, an irreversible chronic inflammatory disease associated with metabolic diseases. However, studies regarding the effects of a hyperglycemic microenvironment on host–microbiome interactions and host inflammatory response during periodontitis are still scarce. Here, we investigated the impacts of a hyperglycemic microenvironment on the inflammatory response and transcriptome of a gingival coculture model stimulated with dysbiotic subgingival microbiomes. HGF-1 cells overlaid with U937 macrophage-like cells were stimulated with subgingival microbiomes collected from four healthy donors and four patients with periodontitis. Pro-inflammatory cytokines and matrix metalloproteinases were measured while the coculture RNA was submitted to a microarray analysis. Subgingival microbiomes were submitted to 16s rRNA gene sequencing. Data were analyzed using an advanced multi-omics bioinformatic data integration model. Our results show that the genes krt76, krt27, pnma5, mansc4, rab41, thoc6, tm6sf2, and znf506 as well as the pro-inflammatory cytokines IL-1β, GM-CSF, FGF2, IL-10, the metalloproteinases MMP3 and MMP8, and bacteria from the ASV 105, ASV 211, ASV 299, Prevotella, Campylobacter and Fretibacterium genera are key intercorrelated variables contributing to periodontitis-induced inflammatory response in a hyperglycemic microenvironment. In conclusion, our multi-omics integration analysis unveiled the complex interrelationships involved in the regulation of periodontal inflammation in response to a hyperglycemic microenvironment. [ABSTRACT FROM AUTHOR]

• Limited proteolysis of thrombospondins is an important post-translational mechanism to modulate the function in the extracellular space. • Current data from the literature and databases have been collected to summarize the current knowledge of TSP processing by proteases, mainly metalloproteases and serine proteases. • We present an overview of the cleavage of TSPs by proteases, a map of TSP cleavage sites, and considerations on the biological role of the generated fragments in pathological settings, with a particular focus on cancer. Limited proteolysis of thrombospondins is a powerful mechanism to ensure dynamic tuning of their activities in the extracellular space. Thrombospondins are multifunctional matricellular proteins composed of multiple domains, each with a specific pattern of interactions with cell receptors, matrix components and soluble factors (growth factors, cytokines and proteases), thus with different effects on cell behavior and responses to changes in the microenvironment. Therefore, the proteolytic degradation of thrombospondins has multiple functional consequences, reflecting the local release of active fragments and isolated domains, exposure or disruption of active sequences, altered protein location, and changes in the composition and function of TSP-based pericellular interaction networks. In this review current data from the literature and databases is employed to provide an overview of cleavage of mammalian thrombospondins by different proteases. The roles of the fragments generated in specific pathological settings, with particular focus on cancer and the tumor microenvironment, are discussed. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a multifactorial and polygenic disease, which affects joints as well as the periarticular tissue, and it represents a huge economic cost to public health systems worldwide. Numerous genes that are involved in the pathogenesis of the disease are already identified. More recently, the epigenetic mechanisms are being researched as the potential risk factors for osteoarthritis. MicroRNAs (miRNAs) are small non-coding molecules which play a role in the post-transcriptional regulation of gene expression. Their genes make up to 1-2% of the total genome and it is thought that they could potentially regulate up to 30% of the coding genes. MiRNAs have important role in the pathogenesis of some cancers, cardiovascular, autoimmune, skin and neurological diseases. Furthermore, the above mentioned molecules have an important role in the pathogenesis of osteoarthritis. Various miRNA molecules are expressed differently in the cartilage affected by osteoarthritis as compared to the healthy cartilage. Certain miRNA molecules have also been shown to regulate enzymes responsible for cartilage degradation, some were associated with the regulation of chondrocyte apoptosis and some involved in cartilage regeneration processes. Due to these findings, some of the following studies tried to prove the connection between certain single nucleotide polymorphisms of some miRNA genes and the proneness to osteoarthritis. Given that all current therapies for osteoarthritis aim at treating the consequences of the disease rather than curing the cause, further research into etiopathogenesis of osteoarthritis is required. The MiRNA molecules could play an important role in the pathogenesis of the osteoarthritis and might easily be among the potential targets for the new pharmaceutical therapies. [ABSTRACT FROM AUTHOR]

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies. [ABSTRACT FROM AUTHOR]

The chemokine fractalkine (FKN, CX3CL1), a member of the CX3C subfamily, contributes to neuron–glia interaction and the regulation of microglial cell activation. Fractalkine is expressed by neurons as a membrane-bound protein (mCX3CL1) that can be cleaved by extracellular proteases generating several sCX3CL1 forms. sCX3CL1, containing the chemokine domain, and mCX3CL1 have high affinity by their unique receptor (CX3CR1) which, physiologically, is only found in microglia, a resident immune cell of the CNS. The activation of CX3CR1contributes to survival and maturation of the neural network during development, glutamatergic synaptic transmission, synaptic plasticity, cognition, neuropathic pain, and inflammatory regulation in the adult brain. Indeed, the various CX3CL1 forms appear in some cases to serve an anti-inflammatory role of microglia, whereas in others, they have a pro-inflammatory role, aggravating neurological disorders. In the last decade, evidence points to the fact that sCX3CL1 and mCX3CL1 exhibit selective and differential effects on their targets. Thus, the balance in their level and activity will impact on neuron–microglia interaction. This review is focused on the description of factors determining the emergence of distinct fractalkine forms, their age-dependent changes, and how they contribute to neuroinflammation and neurodegenerative diseases. Changes in the balance among various fractalkine forms may be one of the mechanisms on which converge aging, chronic CNS inflammation, and neurodegeneration.

Purpose: Low doses of systemic doxycycline (LD-SD) inhibit angiogenesis and the expression of matrix metalloproteases, which are determinants of pterygium progression. This study aimed to compare the recurrence rate and visual outcome of pterygium excision in patients undergoing chronic treatment with LD-SD for chronic refractory blepharitis and LD-SD-naive patients. Methods: A retrospective analysis of patients that underwent surgical excision and conjunctival graft apposition was conducted. Patients were divided in a TETRA group (under LD-SD treatment at the moment of surgery) and a control group. The main outcome was the rate of recurrence at 1 year postoperatively. Secondary outcomes were the comparisons of surface regularity, visual quality, and dry-eye symptoms at 6-week, 6-month, and 1-year follow-up in the two groups. Results: The TETRA group showed a significantly lower rate of 1-year recurrence both in primary (p = 0.034) and recurrent (p < 0.001) pterygia. The best corrected visual acuity (BCVA), astigmatic error, corneal total root mean square (RMS), and ocular surface disease index (OSDI) significantly reduced during the follow-up in both groups. The surface asymmetry index and high-order aberrations (HOAs) significantly reduced only in the TETRA group. The final BCVA was significantly higher, while the OSDI score and total RMS and HOAs were significantly lower in the TETRA group compared to the control. Conclusions: Patients under treatment with LD-SD showed a lower rate of recurrence at 1-year follow-up compared to controls. These patients also experienced higher BCVA and surface regularity and less dry-eye symptoms.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (

ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used “surface-spanning enrichment with click-sugars (SUSPECS)” proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding.

Introduction: Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized. Methods: Genetically identified L3 of A. pegreffii were maintained for 24 h at 37° C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis. Results and discussion: EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts. [ABSTRACT FROM AUTHOR]

Background: Metastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis. Methods: We used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA. Results: We found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines. Conclusions: We conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs. A1m_Mu21TUHDLKeRdJvkXh Video Abstract [ABSTRACT FROM AUTHOR]

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2, which requires cleavage of its spike protein (S) at two sites: S1/S2 and S2′. This study investigates the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. The assays used were cell-to-cell fusion in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increased cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduced cell-to-cell fusion by promoting the cellular degradation of ACE2. SKI-1 activity led to enhanced S2′ formation, which was attributed to increased metalloprotease activity as a response to enhanced cholesterol levels via activated SREBP-2. However, high metalloprotease activity resulted in the shedding of S2′ into a new C-terminal fragment (S2″), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2″ formation abolished S2′ and cell-to-cell fusion, as well as pseudoparticle entry, indicating that the formation of S2″ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested the binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms. [ABSTRACT FROM AUTHOR]

Background and Objectives. The aim of this study was to compare the distribution of proliferation markers (Ki-67, NF-κβ), tissue-remodeling factors (MMP-2, MMP-9, TIMP-2, TIMP-4), vascular endothelial growth factor (VEGF), interleukins (IL-1 and IL-10), human beta defensins (HβD-2 and HβD-4) and Sonic hedgehog gene protein in cholesteatoma and control skin. Methods. Nineteen patient cholesteatoma tissues and seven control skin materials from cadavers were included in the study and stained immunohistochemically. Results. Statistically discernible differences were found between the following: the Ki-67 in the matrix and the Ki-67 in the skin epithelium (p = 0.000); the Ki-67 in the perimatrix and the Ki-67 in the connective tissue (p = 0.010); the NF-κβ in the cholesteatoma matrix and the NF-κβ in the epithelium (p = 0.001); the MMP-9 in the matrix and the MMP-9 in the epithelium (p = 0.008); the HβD-2 in the perimatrix and the HβD-2 in the connective tissue (p = 0.004); and the Shh in the cholesteatoma's perimatrix and the Shh in the skin's connective tissue (p = 0.000). Conclusion. The elevation of Ki-67 and NF-κβ suggests the induction of cellular proliferation in the cholesteatoma. Intercorrelations between VEGF, NF-κβ and TIMP-2 induce neo-angiogenesis in adult cholesteatoma. The similarity in the expression of IL-1 and IL-10 suggests the dysregulation of the local immune status in cholesteatoma. The overexpression of the Sonic hedgehog gene protein in the cholesteatoma proves the selective local stimulation of perimatrix development. [ABSTRACT FROM AUTHOR]

Objective: Platelet-rich plasma (PRP) is used in multiple coagulation disorders. Its therapeutic effectiveness relies on technical procedures related to PRP procurement and preservation because free radicals induce platelet activation and aging. This work aims to elucidate the oxidative mechanisms involved in activation of platelets obtained from PRP during storage. Materials and Methods: One hundred ten PRP batches were obtained from healthy donors and kept under stirring at a temperature of 20-24 °C. Protein extraction was performed from platelet homogenates and plasma at different times of storage from day 1 to 20. The activities of antioxidant markers such as catalase (CAT), superoxide dismutase, and ceruloplasmin, as well as fibrinolytic protein activity metalloproteases 2 and 3, plasmin, and urokinase plasminogen activator, were analyzed by zymography assays. Oxidized proteins were also determined. Results: Significant activity of antioxidant enzymes and fibrinolytic molecules was observed on day 5 of storage in PRP homogenates, which increased over time and was concomitantly correlated with oxidized protein levels. Reverse enzymatic activity patterns were observed in plasma, except for CAT, which remained unchanged. Conclusion: Storage conditions of platelets from PRP for up to 5 days induced in vitro platelet activation by oxidative damage and proteolysis. This finding confirms the need for proper management of these blood products to preserve their viability and functionality. [ABSTRACT FROM AUTHOR]

Mexico has the highest diversity of snake species in the world, following Australia when considering just venomous snakes. Specifically, in Sonora, the second largest state in the country, more than 15 highly venomous species occur, including the northern black-tailed rattlesnake (Crotalus molossus). This specie's venom has not been as thoroughly researched in contrast with other Mexican vipers, nevertheless some studies report its biological activity and even pharmacological potential with antibacterial and cytotoxic activity. In this study we identified the main protein components from a pool of C. molossus venom through a gel-free proteomics approach, reporting ∼140 proteins belonging to the SVMP (38.76%), PLA 2 (28.75%), CTL (11.93%), SVSP (6.03%) and LAAO (5.67%) toxin families. To study its biological activities, we evaluated its hemolytic, antibacterial, and cytotoxic activity in red blood cells, Gram positive and negative bacteria and a luminal A breast carcinoma cell line (T47D), respectively, in vitro. We report that concentrations <100 μg/mL are potentially not hemolytic and reduced the bacteria viability of E. coli and S. aureus with an IC 50 of 10.27 and 11.51 μg/mL, respectively. Finally, we determined the C. molossus venom as cytotoxic against the T47D breast carcinoma cell line, with an IC 50 of 1.55 μg/mL. We suggest that the evaluated cytotoxicity was due to a high abundance of SVMPs and PLA 2 s, since it's been reported that they affect the extracellular matrix and membrane permeation. This may provide a useful tool for pharmaceutical screening in the future. [Display omitted] • C. molossus venom is highly diverse, with over 140 proteins found in it. • The most abundant protein toxin families found were PLA 2 s, SVMPs, CTLs and LAAOs. • C. molossus whole venom had antibacterial activity in both Gram-positive and Gram-negative bacteria. • C. molossus whole venom was cytotoxic (IC 50 : 1.55 μg/mL) in T47D breast carcinoma cells. [ABSTRACT FROM AUTHOR]

Introduction:The main goal of our study was to describe the transcription factor (NF-κβ), angiogenetic factor (VEGF), and remodeling markers (MMP-9 and TIMP-4) of the cholesteatoma tissue compared to control skin tissue. There are still uncertainties how transcription, angiogenetic and remodeling factors affect the cholesteatoma course. Materials and Methods:Eight cholesteatoma tissue specimens were retrieved from children, seven – from adults, seven skin controls – from cadavers. Obtained material immunohistochemically were stained for NF-κβ, MMP-9, TIMP-4, VEGF. Non-para­metric statistic methods were used. Results:A statistically significant higher numbers of NF-κβ and TIMP-4 immunoreactive cells in the cholesteatoma compared to control group. A very strong positive correlation between MMP-9 and TIMP-4 was seen in the patient group. A strong positive correlation - between MMP-9 in matrix and MMP-9, VEGF in perimatrix, between TIMP-4 in matrix and TIMP-4 in perimatrix, NF-κβ in the matrix and VEGF; between TIMP-4 in perimatrix and NF-κβ in the matrix. Conclusions:Correlation between MMP-9 and TIMP-4 suggests that TIMP-4 in cholesteatoma tissue intercorrelates to MMP-9. TIMP-4 likely regulates the development of cholesteatoma. Disbalance between MMPs and TIMPs affects NF-κβ and causes uncontrolled cell proliferation and immune response in this tumor. There is a lack of VEGF strong expression in cholesteatoma perimatrix.

Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.

Estrogen receptor α (ERα) action plays an important role in pancreatic β-cell function and survival; thus, it is considered a potential therapeutic target for the treatment of type 2 diabetes in women. However, the mechanisms underlying the protective effects of ERα remain unclear. Because ERα regulates mitochondrial metabolism in other cell types, we hypothesized that ERα may act to preserve insulin secretion and promote β-cell survival by regulating mitochondrial-endoplasmic reticulum (EndoRetic) function. We tested this hypothesis using pancreatic islet-specific ERα knockout (PERαKO) mice and Min6 β-cells in culture with Esr1 knockdown (KD). We found that Esr1-KD promoted reactive oxygen species production that associated with reduced fission/fusion dynamics and impaired mitophagy. Electron microscopy showed mitochondrial enlargement and a pro-fusion phenotype. Mitochondrial cristae and endoplasmic reticulum were dilated in Esr1-KD compared with ERα replete Min6 β-cells. Increased expression of Oma1 and Chop was paralleled by increased oxygen consumption and apoptosis susceptibility in ERα-KD cells. In contrast, ERα overexpression and ligand activation reduced both Chop and Oma1 expression, likely by ERα binding to consensus estrogen-response element sites in the Oma1 and Chop promoters. Together, our findings suggest that ERα promotes β-cell survival and insulin secretion through maintenance of mitochondrial fission/fusion-mitophagy dynamics and EndoRetic function, in part by Oma1 and Chop repression.

Cryptococcal meningoencephalitis, the most common form of cryptococcosis, is caused by the opportunistic fungal pathogen, Cryptococcus neoformans. Molecular strategies used by C. neoformans to invade the central nervous system (CNS) have been the focus of several studies. Recently, the role of a novel secreted metalloprotease (Mpr1) in the pathogenicity of C. neoformans was confirmed by studies demonstrating that Mpr1 mediated the migration of fungal cells into the CNS. Given this central function, the aim here was to identify the molecular determinants of Mpr1 activity and resolve their role in the migration of cryptococci across the blood-brain barrier (BBB). The Mpr1 protein belongs to an understudied group of metalloproteases of the M36 class of fungalysins unique to fungi. They are generally synthesized as propeptides with fairly long prodomains and highly conserved regions within their catalytic core. Through structure-function analysis of Mpr1, our study identified the prodomain cleavage sites of Mpr1 and demonstrated that when mutated, the prodomain appears to remain attached to the catalytic C-terminus of Mpr1 rendering a nonfunctional Mpr1 protein and an inability for cryptococci to cross the BBB. We found that proteolytic activity of Mpr1 was dependent on the coordination of zinc with two histidine residues in the active site of Mpr1, since amino acid substitutions in the HExxH motif abolished Mpr1 proteolytic activity and prevented the migration of cryptococci across the BBB. A phylogenetic analysis of Mpr1 revealed a distinct pattern likely reflecting the neurotropic nature of C. neoformans and the specific function of Mpr1 in breaching the BBB. This study contributes to a deeper understanding of the molecular regulation of Mpr1 activity and may lead to the development of specific inhibitors that could be used to restrict fungal penetration of the CNS and thus prevent cryptococcal meningoencephalitis-related deaths.

Metalloproteinases (MPs) is a large family of proteinases with metal ions in their active centers. According to the different domains metalloproteinases can be divided into a variety of subtypes mainly including Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteases (ADAMs) and ADAMs with Thrombospondin Motifs (ADAMTS). They have various functions such as protein hydrolysis, cell adhesion and remodeling of extracellular matrix. Metalloproteinases expressed in multiple types of cancers and participate in many pathological processes involving tumor genesis and development, invasion and metastasis by regulating signal transduction and tumor microenvironment. In this review, based on the current research progress, we summarized the structure of MPs, their expression and especially immunomodulatory role and mechanisms in cancers. Additionally, a relevant and timely update of recent advances and future directions were provided for the diagnosis and immunotherapy targeting MPs in cancers. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 cell–cell fusion and syncytiation is an emerging pathomechanism in COVID-19, but the precise factors contributing to the process remain ill-defined. In this study, we show that metalloproteases promote SARS-CoV-2 spike protein-induced syncytiation in the absence of established serine proteases using in vitro cell–cell fusion assays. We also show that metalloproteases promote S2′-activation of the SARS-CoV-2 spike protein, and that metalloprotease inhibition significantly reduces the syncytiation of SARS-CoV-2 variants of concern. In the presence of serine proteases, however, metalloprotease inhibition does not reduce spike protein-induced syncytiation and a combination of metalloprotease and serine protease inhibition is necessitated. Moreover, we show that the spike protein induces metalloprotease-dependent ectodomain shedding of the ACE2 receptor and that ACE2 shedding contributes to spike protein-induced syncytiation. These observations suggest a benefit to the incorporation of pharmacological inhibitors of metalloproteases into treatment strategies for patients with COVID-19. [ABSTRACT FROM AUTHOR]

Early-onset pre-eclampsia is believed to arise from defective placentation in the first trimester, leading to placental ischaemia/reperfusion (I/R) and oxidative stress. However, our current understanding of the effects of I/R and oxidative stress on trophoblast function is ambiguous in part due to studies exposing trophoblasts to hypoxia instead of I/R, and which report conflicting results. Here, we present a model of simulated ischaemia/reperfusion (SI/R) to recapitulate the pathophysiological events of early-onset pre-eclampsia (PE), by exposing first trimester cytotrophoblast HTR-8/SVneo cells to a simulated ischaemia buffer followed by reperfusion. We examined different ischaemia and reperfusion times and observed that 1 h ischaemia and 24 h reperfusion induced an increase in reactive oxygen species (ROS) production (P<0.0001) and oxygen consumption rate (P<0.01). SI/R-exposed trophoblast cells exhibited deficits in migration, proliferation, and invasion (P<0.01). While the deficits in migration and proliferation were rescued by antioxidants, suggesting an ROS-dependent mechanism, the loss of invasion was not affected by antioxidants, which suggests a divergent ROS-independent pathway. In line with this, we observed a decrease in MMP-9, the key regulatory enzyme necessary for trophoblast invasion (P<0.01), which was similarly unaffected by antioxidants, and pharmacological inhibition of MMP-9 replicated the phenotype of deficient invasion (P<0.01). Collectively, these data demonstrate that I/R impairs trophoblast migration and proliferation via a ROS-dependent mechanism, and invasion via an ROS-independent loss of MMP-9, disambiguating the role of oxidative stress and providing insights into the response of trophoblasts to I/R in the context of early-onset PE., (© 2024 The Author(s).)

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Original covalent probes with an N-acyl-N-alkyl sulfonamide cleavable linker were developed to target a broad set of human Matrix Metalloproteases (MMPs). The electrophilicity of this cleavable linker was modulated to improve the selectivity of the probes as well as reduce their unspecific reactivity in complex biological matrices. We first demonstrated that targeting the S 3 subsite of MMPs enables access to broad-spectrum affinity-based probes that exclusively react with the active version of these proteases. The probes were further assessed in proteomes of varying complexity, where human MMP-13 was artificially introduced at known concentration and the resulting labeled MMP was imaged by in-gel fluorescence imaging. We showed that the less reactive probe was still able to covalently modify MMP-13 while exhibiting reduced off-target unspecific reactivity. This study clearly demonstrated the importance of finely controlling the reactivity of the NASA warhead to improve the selectivity of covalent probes in complex biological systems., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)