Interdependency and differential expression of ERK1 and ERK2 in breast and melanoma cell lines.

BACKGROUND: Regulatory mechanism of ERK1 and ERK2, their mechanisms of action, and how they impact on development, growth, and homeostasis of different organisms have been given much emphasis for long. ERK1 and 2 though are isoforms of ERK mitogen-activated protein kinase but are coded by two different genes MAPK3 and MAPK1 respectively and show differential expressions and interdependency in different cancer cell lines. Our previous investigations substantially stated the effect of ERK1 and ERK2 on different extracellular molecules like MMPs and integrins, responsible for cell growth and differentiation. Here, we aim to study individual roles of ERK1 and ERK2 and their interdependency in progression and invasiveness in various cancer cell lines. METHODS: Different cancer cell lines namely B16F10 (melanoma), MCF7, and MDAMB231 (breast cancer) for studying this particular question were used. Methodologies like gelatin zymography, immunoprecipitation, Western blotting, cell invasion assay, wound healing assay, siRNA transfection, and double transfection procedures were followed for our study. RESULTS: Our findings suggest compensation for ERK2 deficiency by pERK1, clear ERK2 predominance in MCF7 cell line, ERK1-ERK2 interdependency in MDAMB231 cells with regard to compensating each other, and significant role of both ERK1 and ERK2 in modulation of MMP9. CONCLUSION: If summarized, our results prove the contribution of ERK2 in compensating ERK1 loss and vice versa and an evident role of ERK1 in cancer cell invasiveness.

Degradomics defines proteolysis information flow from human knee osteoarthritis cartilage to matched synovial fluid and the contributions of secreted proteases ADAMTS5, MMP13 and CMA1 to articular cartilage breakdown.

OBJECTIVES: Proteolytic cartilage extracellular matrix breakdown is a major mechanism of articular cartilage loss in osteoarthritis (OA) pathogenesis. We sought to determine the overlap of proteolytic peptides in matched knee OA cartilage and synovial fluid on a proteome-wide scale to increase the prospective biomarker repertoire and to attribute proteolytic cleavages to specific secreted proteases. DESIGN: Matched human knee OA cartilage and synovial fluid (n = 5) were analyzed by N-terminomics using Terminal Amine Isotopic Labeling of Substrates (TAILS), comprising labeling and enrichment of protein N-termini, high-resolution mass spectrometry and positional peptide mapping. Donor non-OA articular cartilage was digested with CMA1, MMP13 or ADAMTS5, and TAILS was used to identify cleavage sites, which were matched against cartilage and synovial fluid degradomes. RESULTS: Of over 20,000 cleaved peptides in the combined OA cartilage and synovial fluid degradomes, 677 peptides, originating from 153 proteins, were present in all cartilage and synovial fluid samples. CMA1, MMP13 and ADAMTS5 digestion of cartilage identified numerous cleavage sites for each protease and distinct cleavage site preferences. Peptides resulting from the activities of these proteases were detected in OA cartilage and synovial fluid. CONCLUSIONS: Proteolytic fragments from both cartilage and circulating proteins are detectable by synovial fluid degradomics. CMA1, MMP13 and ADAMTS5 activity profiles in cartilage are distinct from each other and the previously determined HtrA1 profile. This work expands the proteolytic biomarker space for OA investigation, suggests that multiple, diverse proteases contribute to cartilage destruction, and demonstrates that their specific contributions can each be defined by multiple biomarkers.

Rational design, synthesis and pharmacological characterization of novel aminopeptidase A inhibitors.

Aminopeptidase A (APA) is a membrane-bound zinc metallopeptidase involved in the production of angiotensin III, one effector peptide of the brain renin-angiotensin system, making brain APA a relevant pharmacological target for the development of novel therapeutic treatments against hypertension and heart failure. The structure-based design of new APA inhibitors is described, based on previously developed thiol-containing inhibitors and APA crystal structure. Chemical synthesis, in vitro assessment against APA activity, pharmacological and pharmacokinetic profiling were performed, ultimately leading to a potent and selective APA inhibitor.

Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania Donavani.

AIM: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin [LTB] in the functional activity of L. donovani. BACKGROUND: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease [gp63] has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani has fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. RESULTS: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin [LTB] of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. CONCLUSION: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein [LTB] could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species­mediated MMP­9 activation and invasion in MCF­7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF­7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)­9 were assessed by reverse transcription­quantitative PCR and western blotting, and the activity of MMP­9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced MMP­9 expression and invasion of MCF­7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA­induced MMP­9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA­induced MMP­9 expression and invasion in MCF­7 cells. NOX1­ and NOX5­specific small interfering RNAs attenuated TPA­induced MMP­9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA­induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA­induced intracellular ROS levels, MMP­9 expression and NOX activity in MCF­7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA­induced MMP­9 expression and invasion of MCF­7 breast cancer cells. Furthermore, the present study indicated that TPA­induced MMP­9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti­invasive effects in breast cancer.

Replicative Endothelial Cell Senescence May Lead to Endothelial Dysfunction by Increasing the BH2/BH4 Ratio Induced by Oxidative Stress, Reducing BH4 Availability, and Decreasing the Expression of eNOS.

Vascular aging is associated with the development of cardiovascular complications, in which endothelial cell senescence (ES) may play a critical role. Nitric oxide (NO) prevents human ES through inhibition of oxidative stress, and inflammatory signaling by mechanisms yet to be elucidated. Endothelial cells undergo an irreversible growth arrest and alter their functional state after a finite number of divisions, a phenomenon called replicative senescence. We assessed the contribution of NO during replicative senescence of human aortic (HAEC) and coronary (CAEC) endothelial cells, in which accumulation of the senescence marker SA-ß-Gal was quantified by ß-galactosidase staining on cultured cells. We found a negative correlation in passaged cell cultures from P0 to P12, between a reduction in NO production with increased ES and the formation of reactive oxygen (ROS) and nitrogen (ONOO-) species, indicative of oxidative and nitrosative stress. The effect of ES was evidenced by reduced expression of endothelial Nitric Oxide Synthase (eNOS), Interleukin Linked Kinase (ILK), and Heat shock protein 90 (Hsp90), alongside a significant increase in the BH2/BH4 ratio, inducing the uncoupling of eNOS, favoring the production of superoxide and peroxynitrite species, and fostering an inflammatory environment, as confirmed by the levels of Cyclophilin A (CypA) and its receptor Extracellular Matrix Metalloprotease Inducer (EMMPRIN). NO prevents ES by preventing the uncoupling of eNOS, in which oxidation of BH4, which plays a key role in eNOS producing NO, may play a critical role in launching the release of free radical species, triggering an aging-related inflammatory response.

PCPE-2 (procollagen C-proteinase enhancer-2): the NON-IDENTICAL twin of PCPE-1.

PCPE-2 was discovered at the beginning of this century, and was soon identified as a close homolog of PCPE-1 (procollagen C-proteinase enhancer 1). After the demonstration that it could also stimulate the proteolytic maturation of fibrillar procollagens by BMP-1/tolloid-like proteinases (BTPs), PCPE-2 did not attract much attention as it was thought to fulfill the same functions as PCPE-1 which was already well-described. However, the tissue distribution of PCPE-2 shows both common points and significant differences with PCPE-1, suggesting that their activities are not fully overlapping. Also, the recently established connections between PCPE-2 (gene name PCOLCE2) and several important diseases such as atherosclerosis, inflammatory diseases and cancer have highlighted the need for a thorough reappraisal of the in vivo roles of this regulatory protein. In this context, the recent finding that, while retaining the ability to bind fibrillar procollagens and to activate their C-terminal maturation, PCPE-2 can also bind BTPs and inhibit their activity has substantially extended its potential functions. In this review, we describe the current knowledge about PCPE-2 with a focus on collagen fibrillogenesis, lipid metabolism and inflammation, and discuss how we could further advance our understanding of PCPE-2-dependent biological processes.

Associations of SEMA7A, SEMA4D, ADAMTS10, and ADAM8 with KRAS, NRAS, BRAF, PIK3CA, and AKT Gene Mutations, Microsatellite Instability Status, and Cytokine Expression in Colorectal Cancer Tissue.

Semaphorins (SEMAs), ADAM, and ADAMTS family members are implicated in various cancer progression events within the tumor microenvironment across different cancers. In this study, we aimed to evaluate the expression of SEMA7A, SEMA4D, ADAM8, and ADAMTS10 in colorectal cancer (CRC) in relation to the mutational landscape of KRAS, NRAS, BRAF, PIK3CA, and AKT genes, microsatellite instability (MSI) status, and clinicopathological features. We also examined the associations between the expression of these proteins and selected cytokines, chemokines, and growth factors, assessed using a multiplex assay. Protein concentrations were quantified using ELISA in CRC tumors and tumor-free surgical margin tissue homogenates. Gene mutations were evaluated via RT-PCR, and MSI status was determined using immunohistochemistry (IHC). GSEA and statistical analyses were performed using R Studio. We observed a significantly elevated expression of SEMA7A in BRAF-mutant CRC tumors and an overexpression of ADAM8 in KRAS 12/13-mutant tumors. The expression of ADAMTS10 was decreased in PIK3CA-mutant CRC tumors. No significant differences in the expression of the examined proteins were observed based on MSI status. The SEMA7A and SEMA4D expressions were correlated with the expression of numerous cytokines associated with various immune processes. The potential immunomodulatory functions of these molecules and their suitability as therapeutic targets require further investigation.

Insights into the Evolutionary Dynamics: Characterization of Disintegrin and Metalloproteinase Proteins in the Venom Gland Transcriptome of the Hemiscorpius lepturus Scorpion.

BACKGROUND: The Disintegrin and Metalloproteinase (ADAM) family, also known as the metalloproteinase/disintegrin/cysteine-rich (MDC) proteins, includes both secreted and transmembrane molecules involved in critical biological processes, such as cell migration, adhesion, and signaling. This study aimed to investigate the evolutionary relationships and structural characteristics of disintegrin and metalloproteinase proteins identified in the venom gland transcriptome of the scorpion Hemiscorpius lepturus. METHODS: Using bioinformatics tools, we analyzed the open reading frame, conserved motifs, and primary, secondary, and tertiary structures of these proteins. Five proteins, named HLDis- Met1, HLDisMet2, HLDisMet3, HLDisMet4, and HLDisMet5, were identified. Their predicted 3- D structures were within normal ranges (Z-score between -4 to -9). RESULTS: Phylogenetic analysis revealed that HLDisMet1 shares similarities with proteins from various spider species (Nephila pilipes, Argiope bruennichi, Araneus ventricosus, and Trichonephila inaurata madagascariensis), HLDisMet2 with the scorpion Centruroides sculpturatus, HLDis- Met4 with the scorpion Tityus serrulatus, and HLDisMet5 with several snake species (Python bivittatus, Vipera anatolica senliki, Protobothrops mucrosquamatus, and Naja naja). CONCLUSION: These findings highlight the significant similarities between HLDisMet proteins and those found in other venomous species, suggesting a complex and diverse evolutionary pathway for venom components. The cross-species conservation observed may indicate a convergent evolutionary strategy, where different species independently develop similar venom components to adapt to similar ecological niches or prey types. This study highlights the evolutionary significance of venom diversification and its potential applications in understanding venom biology across different species.

Structural basis of divergent substrate recognition and inhibition of human neurolysin.

A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1-13) potently inhibits the Nln activity while dynorphin A (1-8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.

Apo-Lactoferrin Inhibits the Proteolytic Activity of the 110 kDa Zn Metalloprotease Produced by Mannheimia haemolytica A2.

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Furin, ADAM, and γ-secretase: Core regulatory targets in the Notch pathway and the therapeutic potential for breast cancer.

The activation of the Notch pathway promotes the occurrence and progression of breast cancer. The Notch signal plays different roles in different molecular subtypes of breast cancer. In estrogen receptor-positive (ER+) breast cancer, the Notch pathway regulates the activity of estrogen receptors. In human epidermal growth factor receptor 2-positive (HER2+) breast cancer, crosstalk between Notch and HER2 enhances HER2 signal expression. In triple-negative breast cancer (TNBC), Notch pathway activation is closely linked to tumor invasion and drug resistance. This article offers a comprehensive review of the structural domains, biological functions, and key targets of Notch with a specific focus on the roles of Furin protease, ADAM metalloprotease, and γ-secretase in breast cancer and their potential as therapeutic targets. We discuss the functions and mutual regulatory mechanisms of these proteinases in the Notch pathway as well as other potential targets in the Notch pathway, such as the glycosylation process and key transcription factors. This article also introduces new approaches in the treatment of breast cancer, with a special focus on the molecular characteristics and treatment response differences of different subtypes. We propose that the core regulatory molecules of the Notch pathway may become key targets for development of personalized treatment, which may significantly improve treatment outcomes and prognosis for patients with breast cancer.

Adamts9 is required for the development of primary ovarian follicles and maintenance of female sex in zebrafish.

Previous studies have suggested that adamts9 (a disintegrin and metalloprotease with thrombospondin type-1 motifs, member 9), an extracellular matrix (ECM) metalloprotease, participates in primordial germ cell (PGC) migration and is necessary for female fertility. In this study, we found that adamts9 knockout (KO) led to reduced body size, and female-to-male sex conversion in late juvenile or adult zebrafish; however, primary sex determination was not affected in early juveniles of adamts9 KO. Overfeeding and lowering the rearing density rescued growth defects in female adamts9 KO fish but did not rescue defects in ovarian development in adamts9 KO. Delayed PGC proliferation, significantly reduced number and size of Stage IB follicles (equivalent to primary follicles) in early juveniles of adamts9 KO, and arrested development at Stage IB follicles in mid- or late-juveniles of adamts9 KO are likely causes of female infertility and sex conversion. Via RNAseq, we found significant enrichment of differentially expressed genes involved in ECM organization during sexual maturation in ovaries of wildtype fish; and significant dysregulation of these genes in adamts9 KO ovaries. RNAseq analysis also showed enrichment of inflammatory transcriptomic signatures in adult ovaries of these adamts9 KO. Taken together, our results indicate that adamts9 is critical for development of primary ovarian follicles and maintenance of female sex, and loss of adamts9 leads to defects in ovarian follicle development, female infertility, and sex conversion in late juveniles and mature adults. These results show that the ECM and extracellular metalloproteases play major roles in maintaining ovarian follicle development in zebrafish.

Insight into the role of Streptococcus suis zinc metalloprotease C from the new serotype causing meningitis in piglets.

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 - implications for post COVID-19 conditions.

BACKGROUND: Severe COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC). METHODS: The IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10. RESULTS: Forty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals. CONCLUSIONS: This work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Fibroblast-like synoviocytes orchestrate daily rhythmic inflammation in arthritis.

Rheumatoid arthritis is a chronic inflammatory disease that shows characteristic diurnal variation in symptom severity, where joint resident fibroblast-like synoviocytes (FLS) act as important mediators of arthritis pathology. We investigate the role of FLS circadian clock function in directing rhythmic joint inflammation in a murine model of inflammatory arthritis. We demonstrate FLS time-of-day-dependent gene expression is attenuated in arthritic joints, except for a subset of disease-modifying genes. The deletion of essential clock gene Bmal1 in FLS reduced susceptibility to collagen-induced arthritis but did not impact symptomatic severity in affected mice. Notably, FLS Bmal1 deletion resulted in loss of diurnal expression of disease-modulating genes across the joint, and elevated production of MMP3, a prognostic marker of joint damage in inflammatory arthritis. This work identifies the FLS circadian clock as an influential driver of daily oscillations in joint inflammation, and a potential regulator of destructive pathology in chronic inflammatory arthritis.

Comparison of MMP-2, MMP-9, COX-2, and PGP Expression in Feline Injection-Site and Feline Noninjection-Site Sarcomas-Pilot Study.

Feline injection-site sarcomas (FISSs) are aggressive neoplasms that have been associated mostly with vaccination. Feline noninjection-site sarcomas (non-FISSs) are less frequently observed in cats and may arise in any anatomic site. This study aimed to determine the differences in the expression of the selected proteins (matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), and P-glycoprotein (PGP)) and their correlation with the mitotic count in FISS and non-FISS, in order to characterize their immunohistochemical features. A preliminary study of eleven samples of FISS and eight samples of non-FISS was performed using immunohistochemistry. Among all the tested sarcomas, 80.4% of the tumors were positive for COX-2, 90.2% were positive for MMP-9, and 100% were positive for PGP. The results showed that the expressions of COX-2, MMP-9, and PGP were significantly higher in FISS than in non-FISS (COX-2-p ≤ 0.001; MMP-9-p ≤ 0.05; and PGP-p ≤ 0.05). A Spearman rank correlation analysis showed a moderate negative correlation between the expression of COX-2 and MMP-9 in FISS (r = -0.52). A strong negative correlation between COX-2 and PGP (r = -0.81), a moderate positive correlation between MMP-2 and MMP-9 (r = +0.69), and a moderate negative correlation between MMP-2 and PGP (r = -0.44) were observed in non-FISS. In summary, our study presents the immunohistochemical profile of the proteins involved with inflammation and carcinogenesis in FISS and non-FISS, which can contribute to expanding the knowledge of tumor biology.

Potential of Marine Bacterial Metalloprotease A69 in the Preparation of Peanut Peptides with Angiotensin-Converting Enzyme (ACE)-Inhibitory and Antioxidant Properties.

Marine bacterial proteases have rarely been used to produce bioactive peptides, although many have been reported. This study aims to evaluate the potential of the marine bacterial metalloprotease A69 from recombinant Bacillus subtilis in the preparation of peanut peptides (PPs) with antioxidant activity and angiotensin-converting enzyme (ACE)-inhibitory activity. Based on the optimization of the hydrolysis parameters of protease A69, a process for PPs preparation was set up in which the peanut protein was hydrolyzed by A69 at 3000 U g-1 and 60 °C, pH 7.0 for 4 h. The prepared PPs exhibited a high content of peptides with molecular weights lower than 1000 Da (>80%) and 3000 Da (>95%) and contained 17 kinds of amino acids. Moreover, the PPs displayed elevated scavenging of hydroxyl radical and 1,1-diphenyl-2-picryl-hydrazyl radical, with IC50 values of 1.50 mg mL-1 and 1.66 mg mL-1, respectively, indicating the good antioxidant activity of the PPs. The PPs also showed remarkable ACE-inhibitory activity, with an IC50 value of 0.71 mg mL-1. By liquid chromatography mass spectrometry analysis, the sequences of 19 ACE inhibitory peptides and 15 antioxidant peptides were identified from the PPs. These results indicate that the prepared PPs have a good nutritional value, as well as good antioxidant and antihypertensive effects, and that the marine bacterial metalloprotease A69 has promising potential in relation to the preparation of bioactive peptides from peanut protein.

Role of iRhom2 in Olfaction: Implications for Odorant Receptor Regulation and Activity-Dependent Adaptation.

The cell surface metalloprotease ADAM17 (a disintegrin and metalloprotease 17) and its binding partners iRhom2 and iRhom1 (inactive Rhomboid-like proteins 1 and 2) modulate cell-cell interactions by mediating the release of membrane proteins such as TNFα (Tumor necrosis factor α) and EGFR (Epidermal growth factor receptor) ligands from the cell surface. Most cell types express both iRhoms, though myeloid cells exclusively express iRhom2, and iRhom1 is the main iRhom in the mouse brain. Here, we report that iRhom2 is uniquely expressed in olfactory sensory neurons (OSNs), highly specialized cells expressing one olfactory receptor (OR) from a repertoire of more than a thousand OR genes in mice. iRhom2-/- mice had no evident morphological defects in the olfactory epithelium (OE), yet RNAseq analysis revealed differential expression of a small subset of ORs. Notably, while the majority of ORs remain unaffected in iRhom2-/- OE, OSNs expressing ORs that are enriched in iRhom2-/- OE showed fewer gene expression changes upon odor environmental changes than the majority of OSNs. Moreover, we discovered an inverse correlation between the expression of iRhom2 compared to OSN activity genes and that odor exposure negatively regulates iRhom2 expression. Given that ORs are specialized G-protein coupled receptors (GPCRs) and many GPCRs activate iRhom2/ADAM17, we investigated if ORs could activate iRhom2/ADAM17. Activation of an olfactory receptor that is ectopically expressed in keratinocytes (OR2AT4) by its agonist Sandalore leads to ERK1/2 phosphorylation, likely via an iRhom2/ADAM17-dependent pathway. Taken together, these findings point to a mechanism by which odor stimulation of OSNs activates iRhom2/ADAM17 catalytic activity, resulting in downstream transcriptional changes to the OR repertoire and activity genes, and driving a negative feedback loop to downregulate iRhom2 expression.