ResearchPad - metalloproteases https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient <i>hsdRMS</i> restriction system broadening conjugation host range]]> https://www.researchpad.co/article/elastic_article_7739 Horizontal gene transfer is the main mechanism by which bacteria acquire and disseminate new traits, such as antibiotic resistance genes, that allow adaptation and evolution. Here we identified a gene, ardC, that enables a plasmid to increase its conjugative host range, and thus positively contributes to plasmid fitness. The crystal structure of the antirestriction protein ArdC revealed a fold different from other antirestriction proteins. Our results have wide implications for understanding how a gene enlarges the environments a plasmid can colonize and point to new targets to harness the bacterial DNA uptake control.

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<![CDATA[Matrix Metalloproteinases in COPD and atherosclerosis with emphasis on the effects of smoking]]> https://www.researchpad.co/article/5c785018d5eed0c484007c7f

Background

Matrix metalloproteinases (MMP´s) are known biomarkers of atherosclerosis. MMP´s are also involved in the pathophysiological processes underlying chronic obstructive pulmonary disease (COPD). Cigarette smoking plays an important role in both disease states and is also known to affect the concentration and activity of MMP´s systemically. Unfortunately, the epidemiological data concerning the value of MMP´s as biomarkers of COPD and atherosclerosis with special regards to smoking habits are limited.

Methods

450 middle-aged subjects with records of smoking habits and tobacco consumption were examined with comprehensive spirometry, carotid ultrasound examination and biomarker analysis of MMP-1, -3, -7, -10 and -12. Due to missing data 33 subjects were excluded.

Results

The remaining 417 participants were divided into 4 different groups. Group I (n = 157, no plaque and no COPD), group II (n = 136, plaque but no COPD), group III (n = 43, COPD but no plaque) and group IV (n = 81, plaque and COPD). Serum levels of MMP-1,-7,-10-12 were significantly influenced by smoking, and MMP-1, -3, -7 and-12 were elevated in subjects with COPD and carotid plaque. This remained statistically significant for MMP-1 and-12 after adjusting for traditional risk factors.

Conclusion

COPD and concomitant plaque in the carotid artery were associated with elevated levels of MMP-1 and -MMP-12 even when adjusting for risk factors. Further studies are needed to elucidate if these two MMP´s could be useful as biomarkers in a clinical setting. Smoking was associated with increased serum levels of MMP´s (except for MMP-3) and should be taken into account when interpreting serum MMP results.

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<![CDATA[Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease]]> https://www.researchpad.co/article/5c59febed5eed0c484135375

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities, specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage.

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<![CDATA[ADAMTS-1 in abdominal aortic aneurysm]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0203

Introduction

Extracellular matrix degradation is a hallmark of abdominal aortic aneurysm (AAA). Among proteases that are capable of degrading extracellular matrix are a disintegrin and metalloproteases with thrombospondin motifs (ADAMTS). Pathogenesis of these proteases in AAA has not been investigated until date.

Methods and results

Human aneurysmal and control aortas were collected and analyzed with RT-PCR measuring the ADAMTS-1, 4,5,6,8,9,10,13,17 and ADAMTSL-1. Expression of a majority of the investigated ADAMTS members on mRNA level was decreased in aneurysm compared to control aorta. ADAMTS-1 was one of the members that was reduced most. Protein analysis using immunohistochemistry and western blot for localization and expression of ADAMTS-1 revealed that ADAMTS-1 was present predominantly in areas of SMCs and macrophages in aneurysmal aorta and higher expressed in AAA compared to control aortas. The role of ADAMTS-1 in AAA disease was further examined using ADAMTS-1 transgenic/apoE-/- mice with the experimental angiotensin II induced aneurysmal model. Transgenic mice overexpressing ADAMTS-1 showed to be similar to ADAMTS-1 wild type mice pertaining collagen, elastin content and aortic diameter.

Conclusion

Several of the ADAMTS members, and especially ADAMTS-1, are down regulated at mRNA level in AAA, due to unknown mechanisms, at the same time ADAMTS-1 protein is induced. The cleavage of its substrates, don’t seem to be crucial for the pathogenesis of AAA but rather more important in the development of thoracic aortic aneurysm and atherosclerosis as shown in previous studies.

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<![CDATA[Evaluating Soluble EMMPRIN as a Marker of Disease Activity in Multiple Sclerosis: Studies of Serum and Cerebrospinal Fluid]]> https://www.researchpad.co/article/5989da41ab0ee8fa60b8a00f

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is an inducer of matrix metalloproteinases and has roles in leukocyte activation and migration. We reported previously that in MS and its animal model, experimental autoimmune encephalomyelitis, cell surface-associated EMMPRIN was significantly elevated in leukocytes around inflammatory perivascular cuffs in the CNS. In this study we report that activated T-cells can secrete soluble form of EMMPRIN (sEMMPRIN) upon activation. As sEMMPRIN is also present in biological fluids, we determined whether sEMMPRIN is altered in the CSF and sera of MS subjects. Sera from individuals without neurological conditions served as controls, while CSFs collected from subjects undergoing discectomy, and without evidence of CNS pathology, were used as a comparator group. We found that serum levels of sEMMPRIN from clinically stable MS patients or other inflammatory conditions did not differ from control subjects. Paired serum and CSF samples demonstrated poor correlation of sEMMPRIN. Interestingly, sEMMPRIN levels were approximately 60% higher in CSFs compared to sera. sEMMPRIN CSF levels were significantly higher in secondary progressive compared to primary progressive subjects. Thus we conclude that measurement of sEMMPRIN in serum is not informative for disease activity in MS. The differential expression of sEMMPRIN in the CSF of primary and secondary progressive MS invites hypotheses of the still undefined roles of EMMPRIN in the CNS.

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<![CDATA[Association of Matrix Metalloproteinases -7, -8 and -9 and TIMP -1 with Disease Severity in Acute Pancreatitis. A Cohort Study]]> https://www.researchpad.co/article/5989da0eab0ee8fa60b7896d

Objectives

Several biomarkers for early detection of severe acute pancreatitis (SAP) have been presented. Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are released early in inflammation. We aimed to assess levels of MMP-7, -8, -9 and TIMP-1 in acute pancreatitis (AP) and explore their ability to detect disease severity. Our second aim was to find an association between MMPs, TIMP and creatinine.

Methods

We collected plasma samples for MMP-7, -8, -9 and TIMP-1 analyses from 176 patients presenting within 96 h from onset of acute pancreatitis (AP) symptoms. We used samples from 32 control subjects as comparison. The revised Atlanta Classification was utilised to assess severity of disease. Receiver operating characteristic curve analysis and Spearman´s Rho-test were utilised for statistical calculations.

Results

Compared with controls, patients showed higher levels of all studied markers. MMP-8 was higher in moderately severe AP than in mild AP (p = 0.005) and MMP-8, -9 and TIMP-1 were higher in severe than in mild AP (p<0.001, p = 0.005 and p = 0.019). MMP-8 detected SAP with an AUC of 0.939 [95% CI 0.894–0.984], LR+ 9.03 [5.30–15.39]. MMP-8, -9 and TIMP-1 failed to discern moderately severe AP from SAP. MMP-7 was not different between patient groups. MMP-7 and TIMP-1 correlated weakly with creatinine (Rho = 0.221 and 0.243). MMP-8 might be a useful biomarker in early detection of SAP.

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<![CDATA[A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbc7f

Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs), into adipocytes. Since matrix metalloproteinases (MMPs) play critical roles in the cell differentiation process, we conducted investigations to determine if a novel mercaptosulfonamide-based MMP inhibitor (MMPI), YHJ-7-52, could affect hMSC adipogenic differentiation and lipid accumulation. Enzyme inhibition assays, adipogenic differentiation experiments, and quantitative PCR methods were employed to characterize this inhibitor and determine its effect upon adipogenesis. YHJ-7-52 reduced lipid accumulation in differentiated cells by comparable amounts as a potent hydroxamate MMPI, GM6001. However, YHJ-7-82, a non-inhibitory structural analog of YHJ-7-52, in which the zinc-binding thiol group is replaced by a hydroxyl group, had no effect on adipogenesis. The two MMPIs (YHJ-7-52 and GM6001) were also as effective in reducing lipid accumulation in differentiated cells as T0070907, an antagonist of peroxisome-proliferator activated receptor gamma (PPAR-gamma), at a similar concentration. PPAR-gamma is a typical adipogenic marker and a key regulatory protein for the transition of preadiopocyte to adipocyte. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a PPAR-gamma agonist. Our results indicate that MMP inhibitors may be used as molecular tools for adipogenesis and obesity treatment research.

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<![CDATA[The effect of biological sealants and adhesive treatments on matrix metalloproteinase expression during renal injury healing]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf71e

Background

Renal injuries are relatively common in cases of abdominal trauma. Adhesives and sealants can be used to repair and preserve damaged organs. Using a rat model, this study explores the activity of different matrix metalloproteinases (MMP) during the healing of renal injuries treated by two biological adhesives (TachoSil and GelitaSpon) and a new synthetic elastic cyanoacrylate (Adhflex).

Methods

Renal traumatic injuries were experimentally induced in 90 male Wistar rats by a Stiefel Biopsy Punch in the anterior aspect of the left kidney. Animals were divided into five groups: 1, sham non-injured (n = 3); 2, non-treated standard punch injury (n = 6); 3, punch injury treated with TachoSil (n = 27); 4, punch injury treated with GelitaSpon (n = 27); and, 5, punch injury treated with Adhflex (n = 27). Wound healing was evaluated 2, 6, and 18 days after injury by determining the expression of MMPs, and the histopathological evolution of lesions.

Findings

Histologically, the wound size at 6 days post-injury was larger in Adhflex-treated samples than in the other treatments, but the scarring tissue was similar at 18 days post-injury. Only the MMPs subtypes 1, 2, 8, 9, and 13 were sufficiently expressed to be quantifiable. Both time since injury and treatment type had a significant influence on MMPs expression. Two days after injury, the expression of MMP8 and MMP9 was predominant. MMP2 expression was greater 6 days after injury. The Adhflex-treated group had a significantly higher MMPs expression than the other treatment groups at all healing stages.

Conclusions

All three sealant treatments induced almost similar expression of MMPs than untreated animals indicating a physiological healing process. Given that all renal trauma injuries must be considered emergencies, both biological and synthetic adhesives, such as Adhflex, should be considered as a treatment options.

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<![CDATA[Monoclonal antibodies specific for Bacteroides fragilis enterotoxins BFT1 and BFT2 and their use in immunoassays]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbe20

We have developed 22 mouse IgG1 monoclonal antibodies (mAbs) against Bacteroides fragilis zinc metalloprotease toxins 1 and 2 (BFT1 and BFT2). Mice were immunized with recombinant BFT1 or BFT2 proteins with metalloprotease activity. Eight of the mAbs bind specifically to BFT1. One mAb, 2H6, binds specifically to BFT2. The remaining 13 mAbs bind to both BFT1 and BFT2. The eight BFT1-specific mAbs recognize at least five different epitopes on the toxin. Four of the BFT1-specific mAbs neutralized rBFT1 metalloprotease activity. Only one of these four mAbs, 1D9, neutralizes the cytotoxic effect of BFT1. Here, we describe the development of enzyme-linked immunosorbent assays (ELISAs) to detect BFT1 or BFT2 toxin in an isotype-specific manner. The sandwich ELISAs have a detection limit of 20 to 40 ng/ml when purified recombinant BFT protein is diluted into PBS. The sandwich ELISA can be used to distinguish and quantify levels of rBFT1 and rBFT2 in stool. This ELISA can be an important tool to investigate the association between BFT expression by enterotoxigenic B. fragilis and diseases such as diarrhea, inflammatory bowel disease and colorectal cancer.

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<![CDATA[Diagnostic Accuracy of the Enhanced Liver Fibrosis (ELF®) Score Using HCV-Infected Serum Samples Cryopreserved for up to 25 Years]]> https://www.researchpad.co/article/5989da6cab0ee8fa60b931b8

Introduction & Aims

Cryopreservation of serum samples is a standard procedure for biomedical research in tertiary centers. However, studies evaluating the long-term biological stability of direct liver fibrosis markers using cryopreserved samples are scarce.

Methods

We compared the stability of hyaluronic acid (HA), tissue inhibitor of metalloproteinases (TIMP-1) and amino-terminal propeptide of type III procollagen (PIIINP) in 225 frozen serum samples of HCV-infected patients with a paired liver biopsy for up to 25 years (1990–2014). Moreover, we assessed the diagnostic accuracy (AUROC) of the Enhanced Liver Fibrosis (ELF®) score to identify significant fibrosis (F2-4) and its predictive capacity to identify clinical events during follow-up.

Results

Seventy-six patients (39,8%) had mild fibrosis (F0-1) and 115 (60,2%) significant fibrosis (F2-4). HA, PIIINP and TIMP-1 values remained stable during the period from 1995 to 2014 while those of 1990–94 were slightly higher. We did not find significant differences in the median ELF® values during the 20-year period from 1995–2014 in patients with mild (from 8,4 to 8,7) and significant fibrosis (from 9,9 to 10,9) (p = ns between periods and fibrosis stages). The AUROCs of ELF® to identify significant fibrosis were high in all the periods (from 0,85 to 0,91). The ELF® score showed a good predictive capability to identify clinical events during follow-up.

Conclusions

The biological stability of direct serum markers (HA, PIIINP and TIMP-1) using HCV-infected samples cryopreserved for 20 years is good. Therefore, the diagnostic accuracy of the ELF® score to identify significant fibrosis and clinical events during follow-up is very high.

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<![CDATA[Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells]]> https://www.researchpad.co/article/5989da61ab0ee8fa60b9114b

Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.

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<![CDATA[Structural Basis of Substrate Selectivity of E. coli Prolidase]]> https://www.researchpad.co/article/5989da36ab0ee8fa60b863b9

Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.

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<![CDATA[ADAMTS-1 Is Found in the Nuclei of Normal and Tumoral Breast Cells]]> https://www.researchpad.co/article/5989dac8ab0ee8fa60bb3387

Proteins secreted in the extracellular matrix microenvironment (ECM) by tumor cells are involved in cell adhesion, motility, intercellular communication and invasion. The tumor microenvironment is expansively modified and remodeled by proteases, resulting in important changes in both cell-cell and cell-ECM interactions and in the generation of new signals from the cell surface. Metalloproteinases belonging to the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family have been implicated in tissue remodeling events observed in cancer development, growth and progression. Here we investigated the subcellular localization of ADAMTS-1 in normal-like (MCF10-A) and tumoral (MCF7 and MDA-MB-231) human breast cells. ADAMTS-1 is a secreted protease found in the extracellular matrix. However, in this study we show for the first time that ADAMTS-1 is also present in the nuclei and nucleoli of the three mammary cell lines studied here. Our findings indicate that ADAMTS-1 has proteolytic functions in the nucleus through its interaction with aggrecan substrate.

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<![CDATA[Distinct Interactions with Cellular E-Cadherin of the Two Virulent Metalloproteinases Encoded by a Bacteroides fragilis Pathogenicity Island]]> https://www.researchpad.co/article/5989dab1ab0ee8fa60bab750

Bacteroides fragilis causes the majority of Gram-negative anaerobic infections in the humans. The presence of a short, 6-kb, pathogenicity island in the genome is linked to enterotoxigenic B. fragilis (ETBF). The role of the enterotoxin in B. fragilis virulence, however, remains to be determined, as the majority of clinical isolates lack ETBF genes and healthy individuals carry enterotoxin-positive B. fragilis. The island encodes secretory metalloproteinase II (MPII) and one of three homologous enterotoxigenic fragilysin isoenzymes (FRA; also termed B. fragilis toxin or BFT). The secretory metalloproteinases expressed from the genes on the B. fragilis pathogenicity island may have pathological importance within the gut, not linked to diarrhea. MPII and FRA are counter-transcribed in the bacterial genome, implying that regardless of their structural similarity and overlapping cleavage preferences these proteases perform distinct and highly specialized functions in the course of B. fragilis infection. The earlier data by us and others have demonstrated that FRA cleaves cellular E-cadherin, an important adherens junction protein, and weakens cell-to-cell contacts. Using E-cadherin-positive and E-cadherin–deficient cancer cells, and the immunostaining, direct cell binding and pull-down approaches, we, however, demonstrated that MPII via its catalytic domain efficiently binds, rather than cleaves, E-cadherin. According to our results, E-cadherin is an adherens junction cellular receptor, rather than a proteolytic target, of the B. fragilis secretory MPII enzyme. As a result of the combined FRA and MPII proteolysis, cell-to-cell contacts and adherens junctions are likely to weaken further.

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<![CDATA[Temporal Gene Expression Analysis and RNA Silencing of Single and Multiple Members of Gene Family in the Lone Star Tick Amblyomma americanum]]> https://www.researchpad.co/article/5989dab1ab0ee8fa60bab9b8

Saliva is an integral factor in the feeding success of veterinary and medically important ticks. Therefore, the characterization of the proteins present in tick saliva is an important area of tick research. Here, we confirmed previously generated sialotranscriptome data using quantitative real-time PCR. The information obtained in this in-depth study of gene expression was used to measure the effects of metalloprotease gene silencing on tick feeding. We analyzed the temporal expression of seven housekeeping genes and 44 differentially expressed salivary molecules selected from a previously published Amblyomma americanum sialotranscriptome. Separate reference genes were selected for the salivary glands and midgut from among the seven housekeeping genes, to normalize the transcriptional expression of differentially expressed genes. The salivary gland reference gene, ubiquitin, was used to normalize the expression of 44 salivary genes. Unsurprisingly, each gene family was expressed throughout the blood meal, but the expression of specific genes differed at each time point. To further clarify the complex nature of the many proteins found in the saliva, we disrupted the translation of several members of the metalloprotease family. Intriguingly, the nucleotide sequence similarity of the reprolysin metalloprotease gene family is so homologous that a single synthesized dsRNA sequence knocked down multiple members of the family. The use of multigene knockdown yielded a more significant picture of the role of metalloproteases in tick feeding success, and changes were observed in the female engorgement weight and larval hatching success. Interestingly, the depletion of metalloprotease transcripts also reduced the total number of bacteria present in the salivary glands. These data provide insight into the expression and functions of tick salivary proteins expressed while feeding on its host.

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<![CDATA[TIMP1 and MMP9 are predictors of mortality in septic patients in the emergency department and intensive care unit unlike MMP9/TIMP1 ratio: Multivariate model]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdba05

Introduction

Matrix metalloproteinases and tissue inhibitors of metalloproteinases could be promising biomarkers for establishing prognosis during the development of sepsis. It is necessary to clarify the relationship between matrix metalloproteinases and their tissue inhibitors. We conducted a cohort study with 563 septic patients, in order to elucidate the biological role and significance of these inflammatory biomarkers and their relationship to the severity and mortality of patients with sepsis.

Materials and methods

A multicentric prospective cohort was performed. The sample was composed of patients who had sepsis as defined by the International Conference 2001. Serum procalcitonin, creatinine, urea nitrogen, C-Reactive protein, TIMP1, TIMP2, MMP2 and MMP9 were quantified; each patient was followed until death or up to 30 days. A descriptive analysis was performed by calculating the mean and the 95% confidence interval for continuous variables and proportions for categorical variables. A multivariate logistic regression model was constructed by the method of intentional selection of covariates with mortality at 30 days as dependent variable and all the other variables as predictors.

Results

Of the 563 patients, 68 patients (12.1%) died within the first 30 days of hospitalization in the ICU. The mean values for TIMP1, TIMP2 and MMP2 were lower in survivors, MMP9 was higher in survivors. Multivariate logistic regression showed that age, SOFA and Charlson scores, along with TIMP1 concentration, were statistically associated with mortality at 30 days of septic patients; serum MMP9 was not statistically associated with mortality of patients, but was a confounder of the TIMP1 variable.

Conclusion

It could be argued that plasma levels of TIMP1 should be considered as a promising prognostic biomarker in the setting of sepsis. Additionally, this study, like other studies with large numbers of septic patients does not support the predictive value of TIMP1 / MMP9.

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<![CDATA[Hyaluronic Acid Suppresses the Expression of Metalloproteinases in Osteoarthritic Cartilage Stimulated Simultaneously by Interleukin 1β and Mechanical Load]]> https://www.researchpad.co/article/5989db1bab0ee8fa60bce313

Purpose

In patients with osteoarthritis (OA), intraarticular injection of hyaluronic acid (HA) frequently results in reduced pain and improved function for prolonged periods of time, i.e. more than 6 months. However, the mechanisms underlying these effects are not fully understood. Our underlying hypothesis is that HA modifies the enzymatic breakdown of joint tissues.

Methods

To test this hypothesis, we examined osteochondral cylinders from 12 OA patients. In a bioreactor, these samples were stimulated by interleukin 1β (Il1ß) (2 ng/ml) plus mechanical load (2.0 Mpa at 0.5 Hz horizontal and 0.1 Hz vertical rotation), thus the experimental setup recapitulated both catabolic and anabolic clues of the OA joint.

Results

Upon addition of HA at either 1 or 3 mg/ml, we observed a significant suppression of expression of metalloproteinase (MMP)-13. A more detailed analysis based on the Kellgren and Lawrence (K&L) OA grade, showed a much greater degree of suppression of MMP-13 expression in grade IV as compared to grade II OA. In contrast to the observed MMP-13 suppression, treatment with HA resulted in a suppression of MMP-1 expression only at 1 mg/ml HA, while MMP-2 expression was not significantly affected by either HA concentration.

Conclusion

Together, these data suggest that under concurrent catabolic and anabolic stimulation, HA exhibits a pronounced suppressive effect on MMP-13. In the long-run these findings may benefit the development of treatment strategies aimed at blocking tissue degradation in OA patients.

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<![CDATA[Localization, Shedding, Regulation and Function of Aminopeptidase N/CD13 on Fibroblast like Synoviocytes]]> https://www.researchpad.co/article/5989da77ab0ee8fa60b9725c

Aminopeptidase N/CD13 is highly expressed by fibroblast like synoviocytes (FLS) and may play a role in rheumatoid arthritis (RA). CD13 was previously detected in human synovial fluid where it was significantly increased in RA compared to osteoarthritis. In this study we found that CD13 in biological fluids (plasma, synovial fluid, FLS culture supernatant) is present as both a soluble molecule and on extracellular vesicles, including exosomes, as assessed by differential ultracentrifugation and density gradient separation. Having determined CD13 could be released as a soluble molecule from FLS, we examined potential mechanisms by which CD13 might be shed from the FLS membrane. The use of protease inhibitors revealed that CD13 is cleaved from the FLS surface by metalloproteinases. siRNA treatment of FLS revealed one of those proteases to be MMP14. We determined that pro-inflammatory cytokines (TNFα, IFNγ, IL-17) upregulated CD13 mRNA in FLS, which may contribute to the increased CD13 in RA synovium and synovial fluid. Inhibition of CD13 function by either inhibitors of enzymatic activity or anti-CD13 antibodies resulted in decreased growth and diminished migration of FLS. This suggests that CD13 may be involved in the pathogenic hyperplasia of RA FLS. This data expands potential roles for CD13 in the pathogenesis of RA.

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<![CDATA[A Deep Insight into the Sialome of Rhodnius neglectus, a Vector of Chagas Disease]]> https://www.researchpad.co/article/5989db0dab0ee8fa60bcad78

Background

Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immune responses.

Methods/Principal Findings

Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva. We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant.

Conclusions/Significance

The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts.

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<![CDATA[Structural and Biochemical Characterization of the Cop9 Signalosome CSN5/CSN6 Heterodimer]]> https://www.researchpad.co/article/5989db28ab0ee8fa60bd0c78

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.

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