Optimization of plasma-based BioID identifies plasminogen as a ligand of ADAMTS13.

ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.

[ADAMTS13-Mediated Proteolytic Cleavage of Unusually Large von Willebrand Factor Polymers on Endothelial Cells in the Absence of Fluid Shear Stress].

OBJECTIVE: To investigate the molecular mechanism of proteolytic cleavage of unusually large von Willebrand Factor(ULVWF) on endothelial cells by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) in the absence of fluid shear stress, so as to provide a theoretical basis for the pathogenesis of thrombotic thrombocytopenic purpura (TTP) and other thrombotic disorders. METHODS: The ADAMTS13-mediated proteolysis of ULVWF on the surface of endothelial cells in the absence of fluid shear stress was observed through immunofluorescence microscopy. The variation in VWF antigen levels in the conditioned media were determined by ELISA assay. The levels of VWF and the proteolytic fragments released into the conditioned media were determined by ELISA assay and Western blot in the absence and presence of fluid shear stress or FVIII. The effect of ADAMTS13-mediated ULVWF cleavage on the normal distribution of plasma VWF multimers was evaluated by multimer analysis. Histamine stimulated human umbilical vein endothelial cells (HUVECs) were incubated with ADAMTS13 and various N- and C-terminally truncated mutants. Then the ULVWF that maintained binding to the cells were observed through immunofluorescence microscopy and the soluble ULVWF released from endothelial cells was determined by ELISA, so as to demonstrate the domains of ADAMTS13 required for proteolysis of ULVWF on endothelial cells. RESULTS: The ULVWF strings on the endothelial cell surface were rapidly proteolyzed by recombinant and plasma ADAMTS13 in the absence of fluid shear stress. This proteolytic processing of ULVWF depended on incubation time and ADAMTS13 concentration, but not shear stress and FVIII. The distribution of VWF releaseded by ADAMTS13-mediated proteolysis was quite similar to that secreted by endothelial cells under histamine stimulation, suggesting the ULVWF cleavage occured at the cell surface. The proteolysis of the ULVWF on endothelial cells required the Cys-rich(CysR) and spacer domains, but not the TSP1 2-8 and CUB domains of ADAMTS13. CONCLUSION: The ULVWF polymers on endothelial cells are sensitive to ADAMTS13-mediated cleavage even in the absence of fluid shear stress. The findings provide novel insight into the molecular mechanism of ADAMTS13-mediated ULVWF cleavage at the cellular level and may contribute to understanding of the pathogenesis of TTP and other thrombotic disorders.

Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15.

In this study we used a spatial transcriptomics approach to identify genes specifically associated with either high or low outflow regions in the trabecular meshwork (TM) that could potentially affect aqueous humor outflow in vivo. High and low outflow regions were identified and isolated from organ cultured human anterior segments perfused with fluorescently-labeled 200 nm FluoSpheres. The NanoString GeoMx Digital Spatial Profiler (DSP) platform was then used to identified genes in the paraffin embedded tissue sections from within those regions. These transcriptome analyses revealed that 16 genes were statistically upregulated in high outflow regions and 57 genes were statistically downregulated in high outflow regions when compared to low outflow regions. Gene ontology enrichment analysis indicated that the top three biological categories of these differentially expressed genes were ECM/cell adhesion, signal transduction, and transcription. The ECM/cell adhesion genes that showed the largest differential expression (Log2FC ±1.5) were ADAM15, BGN, LDB3, and CRKL. ADAM15, which is a metalloproteinase that can bind integrins, was upregulated in high outflow regions, while the proteoglycan BGN and two genes associated with integrin signaling (LDB3, and CRKL) were downregulated. Immunolabeling studies supported the differential expression of ADAM15 and showed that it was specifically upregulated in high outflow regions along the inner wall of Schlemm's canal and in the juxtacanalicular (JCT) region of the TM. In addition to these genes, the studies showed that genes for decorin, a small leucine-rich proteoglycan, and the α8 integrin subunit were enriched in high outflow regions. These studies identify several novel genes that could be involved in segmental outflow, thus demonstrating that digital spatial profiling could be a useful approach for understanding segmental flow through the TM. Furthermore, this study suggests that changes in the expression of genes involved in regulating the activity and/or organization of the ECM and integrins in the TM are likely to be key players in segmental outflow.

Inhibition of ADAM9 promotes the selective degradation of KRAS and sensitizes pancreatic cancers to chemotherapy.

Kirsten rat sarcoma virus (KRAS) signaling drives pancreatic ductal adenocarcinoma (PDAC) malignancy, which is an unmet clinical need. Here, we identify a disintegrin and metalloproteinase domain (ADAM)9 as a modulator of PDAC progression via stabilization of wild-type and mutant KRAS proteins. Mechanistically, ADAM9 loss increases the interaction of KRAS with plasminogen activator inhibitor 1 (PAI-1), which functions as a selective autophagy receptor in conjunction with light chain 3 (LC3), triggering lysosomal degradation of KRAS. Suppression of ADAM9 by a small-molecule inhibitor restricts disease progression in spontaneous models, and combination with gemcitabine elicits dramatic regression of patient-derived tumors. Our findings provide a promising strategy to target the KRAS signaling cascade and demonstrate a potential modality to enhance sensitivity to chemotherapy in PDAC.

Epigenetic Mechanisms Histone Deacetylase-Dependent Regulate the Glioblastoma Angiogenic Matrisome and Disrupt Endothelial Cell Behavior In Vitro.

Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.

Hsa_circ_0001583 fuels bladder cancer metastasis by promoting staphylococcal nuclease and tudor domain containing 1-mediated MicroRNA decay.

Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa_circ_0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa_circ_0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa_circ_0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa_circ_0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa_circ_0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa_circ_0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa_circ_0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets.

ADAM8 silencing suppresses the migration and invasion of fibroblast-like synoviocytes via FSCN1/MAPK cascade in osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disease that affects elderly populations worldwide, causing pain and disability. Alteration of the fibroblast-like synoviocytes (FLSs) phenotype leads to an imbalance in the synovial inflammatory microenvironment, which accelerates the progression of OA. Despite this knowledge, the specific molecular mechanisms of the synovium that affect OA are still unclear. METHODS: Both in vitro and in vivo experiments were undertaken to explore the role of ADAM8 playing in the synovial inflammatory of OA. A small interfering RNA (siRNA) was targeting ADAM8 to intervene. High-throughput sequencing was also used. RESULTS: Our sequencing analysis revealed significant upregulation of the MAPK signaling cascade and ADAM8 gene expression in IL-1ß-induced FLSs. The in vitro results demonstrated that ADAM8 blockade inhibited the invasion and migration of IL-1ß-induced FLSs, while also suppressing the expression of related matrix metallomatrix proteinases (MMPs). Furthermore, our study revealed that inhibiting ADAM8 weakened the inflammatory protein secretion and MAPK signaling networks in FLSs. Mechanically, it revealed that inhibiting ADAM8 had a significant effect on the expression of migration-related signaling proteins, specifically FSCN1. When siADAM8 was combined with BDP-13176, a FSCN1 inhibitor, the migration and invasion of FLSs was further inhibited. These results suggest that FSCN1 is a crucial downstream factor of ADAM8 in regulating the biological phenotypes of FLSs. The in vivo experiments demonstrated that ADAM8 inhibition effectively reduced synoviocytes inflammation and alleviated the progression of OA in rats. CONCLUSIONS: ADAM8 could be a promising therapeutic target for treating OA by targeting synovial inflammation.

ADAM9 deubiquitination induced by USP22 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition of trophoblast cells in preeclampsia.

INTRODUCTION: The dysregulation of deubiquitination has been shown to affect the development of pre-eclampsia (PE). A disintegrin and metalloprotease 9 (ADAM9) plays roles in diverse physiological contexts, including PE. Here, this study aimed to investigate whether ADAM9 regulated trophoblast cell dysfunction through ubiquitin-specific protease 22 (USP22) deubiquitinase-mediated deubiquitination during PE. METHODS: Levels of genes and proteins were tested via qRT-PCR and western blotting assays. Cell proliferation, migration, and invasion were detected using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell and wound healing assays, respectively. Epithelial-mesenchymal transition related markers were assayed using western blotting. Proteins between USP22 and ADAM9 were identified by co-immunoprecipitation assay. RESULTS: ADAM9 was highly expressed in PE patients, functionally, ADAM9 overexpression weakened the proliferation, migration, invasion, and EMT progression in trophoblast cells. Mechanistically, the deubiquitinase USP22 removed ubiquitination on ADAM9 and maintained its stability. Forced expression of USP22 also suppressed the proliferation and mobility in trophoblast cells. Moreover, the regulatory effects of USP22 on trophoblast cells were reversed by ADAM9 silencing. In addition, USP22 interacted with ADAM9 to regulate the activation of Wnt/ß-catenin pathway. DISCUSSION: ADAM9 was deubiquitinated and stabilized by USP22 and then suppressed the proliferation, migration, invasion, and EMT progression in trophoblast cells, indicating a new pathway of USP10/RUNX1 axis in PE process.

The protease ADAM17 at the crossroads of disease: revisiting its significance in inflammation, cancer, and beyond.

The protease A Disintegrin And Metalloproteinase 17 (ADAM17) plays a central role in the pathophysiology of several diseases. ADAM17 is involved in the cleavage and shedding of at least 80 known membrane-tethered proteins, which subsequently modulate several intracellular signaling pathways, and therefore alter cell behavior. Dysregulated expression and/or activation of ADAM17 has been linked to a wide range of autoimmune and inflammatory diseases, cancer, and cardiovascular disease. In this review, we provide an overview of the current state of knowledge from preclinical models and clinical data on the diverse pathophysiological roles of ADAM17, and discuss the mechanisms underlying ADAM17-mediated protein shedding and the potential therapeutic implications of targeting ADAM17 in these diseases.

Long non-coding RNA LINC00565 regulates ADAM19 expression through sponging microRNA-532-3p, thereby facilitating clear cell renal cell carcinoma progression.

Proven by publications, long non-coding RNAs (lncRNAs) play critical roles in the development of clear cell renal cell carcinoma (ccRCC). Although lncRNA LINC00565 has been implicated in the progression of various cancers, its biological effects on ccRCC remain unknown. This study aimed to investigate the biological functions of LINC00565, as well as its potential mechanism in ccRCC. Here, the expression data of mature microRNAs (miRNAs) (normal: 71, tumor: 545), messenger RNAs (mRNAs), and lncRNAs (normal: 72, tumor: 539) of ccRCC were acquired from The Cancer Genome Atlas (TCGA) database and subjected to differential expression analysis. Quantitative reverse transcriptase polymerase chain reaction analyzed the expression levels of LINC00565, miR-532-3p, and ADAM19 mRNA. TCGA database, dual-luciferase report detection, and Argonaute 2 RNA immunoprecipitation were utilized to confirm the relationships between LINC00565 and miR-532-3p and between miR-532-3p and ADAM19, respectively. The progression of ccRCC cells was determined via CCK-8, colony formation, scratch healing, and transwell assays. Western blot was applied to detect the protein levels of epithelial-mesenchymal transition markers and ADAM19. We herein suggested that LINC00565 was prominently upregulated in ccRCC tissues and cells. Knockdown of LINC00565 repressed cell progression. We further predicted and validated miR-532-3p as a target of LINC00565, and miR-532-3p could target ADAM19. Knockdown of LINC00565 resulted in ADAM19 level downregulation in ccRCC cells and suppressed miR-532-3p could restore ADAM19 level. Thus, the three RNAs constructed a ceRNA network. Overexpressed ADAM19 could eliminate the anticancer effects caused by knocking down LINC00565 on ccRCC cells. In conclusion, LINC00565 upregulated ADAM19 via absorbing miR-532-3p, thereby facilitating the progression of ccRCC cells.

Novel ADAMTS13 mutations in a patient with congenital thrombotic thrombocytopenic purpura.

Congenital thrombotic thrombocytopenic purpura (TTP) is a rare autosomal recessive genetic disorder caused by mutations in the ADAMTS13 gene. Approximately 200 mutations of the ADAMTS-13 gene have been identified, although only a few have been characterized through in vitro expression studies. We conducted an investigation on a male congenital TTP patient with reduced plasma levels of ADAMTS13 activity. DNA sequence analysis revealed two mutations on chromosome 9 (1.9q34.2) in the patient's ADAMTS13 gene. One mutation was a non-synonymous mutation (exon 5: c.A530G: p.Y177C), while the other was a nonsense mutation (exon 21: c.G2651A: p.W884X). Both mutations were found to be heterozygous. The patient's parents had no history of thrombocytopenia or neurological symptoms. DNA sequence analysis showed the patient's father was a heterozygote for the nonsense mutation of the ADAMTS13 gene (exon 21: c.G2651A: p.W884X), while the mother was a heterozygote for the non-synonymous mutation of the ADAMTS13 gene (exon 5: c.A530G: p.Y177C). To investigate the mechanism behind ADAMTS13 deficiency in this patient, wild type (WT), ADAMTS13 p.Y177C, and ADAMTS13 p.W884X were transiently expressed in 293-6E cells. Expression studies revealed a significant reduction in enzyme activity and secretion, although the protease was detected within the cells. The 3D structures of the natural and mutated ADAMTS-13 proteins were partially reconstructed using the Phyre2 web server. The mutation that replaces the tyrosine residue at amino acid position 177 with cysteine may result in decreased steric hindrance and a looser structure. This mutation affects the binding of calcium ions and the secretion of the enzyme from intracellular to extracellular compartments.

miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion, and migration in miscarriage†.

A large proportion of miscarriages are classified as unexplained miscarriages since no cause is identified. No reliable biomarkers or treatments are available for these pregnancy losses. While our transcriptomic sequencing has revealed substantial upregulation of miR-146b-5p in unexplained miscarriage villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy, miR-146b-5p was significantly elevated in villous tissues from unexplained miscarriage patients and displayed promising diagnostic potential. Moreover, miR-146b-5p agomir contributed to higher rates of embryonic resorption in ICR mice. When overexpressed in HTR-8/SVneo cells, miR-146b-5p attenuated the proliferative, invasive, and migratory activity of these cells while suppressing the expression of MMP9 and immune inflammation-associated cytokines, including IL1B, IL11, CXCL1, CXCL8, and CXCL12. Conversely, inhibition of its expression enhanced proliferation, migration, and invasion abilities. Mechanistically, IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19 were identified as miR-146b-5p targets regulating trophoblast function, and silencing IL-1 receptor-associated kinase-1 had similar effects as miR-146b-5p overexpression, while IL-1 receptor-associated kinase-1 overexpression could partially reverse the inhibitory impact of this microRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for unexplained miscarriage.

Cattle-FRETS71, a novel fluorogenic substrate with broad applicability for characterizing ADAMTS13 properties and function.

BACKGROUND: Current ADAMTS13 activity assays are important for diagnosing thrombotic thrombocytopenic purpura (TTP) but are unreliable to assay ADAMTS13 activity in animal models. The Cattle-FRETS71 assay is capable of detecting ADAMTS13 activity in plasma from multiple animal species, making it a potentially useful reagent at all stages of clinical research. The performance of Cattle-FRETS71 in TTP diagnosis is not yet known. OBJECTIVES: We evaluated the performance of the Cattle-FRETS71 substrate against the human FRETS-rVWF71 and the FRETS-VWF73 commercial substrates in human plasma and serum samples to validate its utility in diagnosing TTP in patients. METHODS: Internal validation was performed using heparinized plasma samples (n = 81). External validation was a blinded study using serum samples from the Oklahoma TTP Registry (n = 118, collected 2004-2014) that had been initially assayed by FRETS-VWF73 within 1 year of collection. Additional validation was performed with citrated plasma samples with variable ADAMTS13 activities (n = 32) that were analyzed by FRETS-VWF73. RESULTS: There was an excellent correlation (r = 0.94) between Cattle-FRETS71 and FRETS-rVWF71 for assayed heparinized plasma samples (n = 81). Assay results between Cattle-FRETS71 and FRETS-VWF73 of Oklahoma TTP Registry serum samples (n = 118) and citrated plasma samples (n = 32) were comparably good (r = 0.81 and r = 0.85, respectively). CONCLUSION: The Cattle-FRETS71 assay is comparable with other assays in quantifying ADAMTS13 activity in human plasma collected from patients with documented or suspected TTP. The versatility of Cattle-FRETS71, combined with its specificity and sensitivity, makes it a useful tool for the standardization of ADAMTS13 activity across basic and clinical research paradigms.

Chemotherapy-induced release of ADAM17 bearing EV as a potential resistance mechanism in ovarian cancer.

Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell-derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)-itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient-derived EV induce AREG shedding and restore chemoresistance in ADAM17-deficient cells. Confirming that ADAM17-containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.

Genome-wide identification of bovine ADAMTS gene family and analysis of its expression profile in the inflammatory process of mammary epithelial cells.

ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS) are secreted, multi-domain matrix-related zinc endopeptidases that play a role in organogenesis, assembly and degradation of extracellular matrix (ECM), cancer and inflammation. Genome-wide identification and analysis of the bovine ADAMTS gene family has not yet been carried out. In this study, 19 ADAMTS family genes were identified in Bos taurus by genome-wide bioinformatics analysis, and they were unevenly distributed on 12 chromosomes. Phylogenetic analysis shows that the Bos taurus ADAMTS are divided into eight subfamilies, with highly consistent gene structures and motifs within the same subfamily. Collinearity analysis showed that the Bos taurus ADAMTS gene family is homologous to other bovine subfamily species, and many ADAMTS genes may be derived from tandem replication and segmental replication. In addition, based on the analysis of RNA-seq data, we found the expression pattern of ADAMTS gene in different tissues. Meanwhile, we also analyzed the expression profile of ADAMTS gene in the inflammatory response of bovine mammary epithelial cells (BMECs) stimulated by LPS by qRT-PCR. The results can provide ideas for understanding the evolutionary relationship and expression pattern of ADAMTS gene in Bovidae, and clarify the theoretical basis of the function of ADAMTS in inflammation.

ADAMTS Proteases: Importance in Animal Reproduction.

Many reproductive physiological processes, such as folliculogenesis, ovulation, implantation, and fertilization, require the synthesis, remodeling, and degradation of the extracellular matrix (ECM). The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) family genes code for key metalloproteinases in the remodeling process of different ECM. Several genes of this family encode for proteins with important functions in reproductive processes; in particular, ADAMTS1, 4, 5 and 9 are genes that are differentially expressed in cell types and the physiological stages of reproductive tissues. ADAMTS enzymes degrade proteoglycans in the ECM of the follicles so that the oocytes can be released and regulate follicle development during folliculogenesis, favoring the action of essential growth factors, such as FGF-2, FGF-7 and GDF-9. The transcriptional regulation of ADAMTS1 and 9 in preovulatory follicles occurs because of the gonadotropin surge in preovulatory follicles, via the progesterone/progesterone receptor complex. In addition, in the case of ADAMTS1, pathways involving protein kinase A (PKA), extracellular signal regulated protein kinase (ERK1/2) and the epidermal growth factor receptor (EGFR) might contribute to ECM regulation. Different Omic studies indicate the importance of genes of the ADAMTS family from a reproductive aspect. ADAMTS genes could serve as biomarkers for genetic improvement and contribute to enhance fertility and animal reproduction; however, more research related to these genes, the synthesis of proteins encoded by these genes, and regulation in farm animals is needed.

Evaluation of two fully automated ADAMTS13 activity assays in comparison to manual FRET assay.

INTRODUCTION: The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity assays for clinical diagnostic decision-making and to compare their performance. METHODS: Two automated ADAMTS13 activity assays (Werfen HemosIL® AcuStar ADAMTS13 Activity, Technoclone Technofluor ADAMTS13 Activity) were compared with a manual FRET assay (BioMedica ACTIFLUOR ADAMTS13 Activity). The following samples were used: 13 acute phase TTP (thrombotic thrombocytopenic purpura) samples from 11 different patients, one sample from a patient with congenital ADAMTS13 deficiency, 16 samples from control patients, three follow-up samples from TTP patients in long-term remission and one sample from a patient with stem cell transplantation related thrombotic microangiopathy (TMA). The WHO 1st International Standard for ADAMTS13 and several dilutions of normal plasma with ADAMTS13-depleted normal plasma were also tested. Statistical analysis included descriptive statistics, sensitivity and specificity, Passing & Bablok regression and Bland-Altman plot. RESULTS: The quantitative comparison between the HemosIL® (x) and Technofluor (y) methods showed a strong correlation (Pearson r = 0.98, n = 49). When considering an ADAMTS13 activity of <10% as a hallmark for the diagnosis of TTP, two fully automated assays were both able to identify all TTP- and non-TTP-samples correctly, resulting in sensitivities and specificities of 100%. CONCLUSION: Both fully automated ADAMTS13 activity assays showed a good diagnostic performance and quantitative correlation among themselves, discriminating reliably between TTP- and non-TTP-patients.

The C-type lectin domain of CD62P (P-selectin) functions as an integrin ligand.

Recognition of integrins by CD62P has not been reported and this motivated a docking simulation using integrin αvß3 as a target. We predicted that the C-type lectin domain of CD62P functions as a potential integrin ligand and observed that it specifically bound to soluble ß3 and ß1 integrins. Known inhibitors of the interaction between CD62P-PSGL-1 did not suppress the binding, whereas the disintegrin domain of ADAM-15, a known integrin ligand, suppressed recognition by the lectin domain. Furthermore, an R16E/K17E mutation in the predicted integrin-binding interface located outside of the glycan-binding site within the lectin domain, strongly inhibited CD62P binding to integrins. In contrast, the E88D mutation that strongly disrupts glycan binding only slightly affected CD62P-integrin recognition, indicating that the glycan and integrin-binding sites are distinct. Notably, the lectin domain allosterically activated integrins by binding to the allosteric site 2. We conclude that CD62P-integrin binding may function to promote a diverse set of cell-cell adhesive interactions given that ß3 and ß1 integrins are more widely expressed than PSGL-1 that is limited to leukocytes.

Tnfa , Il6 , Cxcl1 , and Adam8 Genes Are the Early Markers After Mouse Tail Intervertebral Disc Injury.

OBJECTIVES: The early molecular events after intervertebral disc injury remain unclear. In this study, we aimed to compare inflammatory markers from 1 day to 4 wks after injury to have a comprehensive understanding of the intervertebral disc response to injury. DESIGN: Mouse tail intervertebral disc injury was induced by a needle puncture. Inflammatory marker gene expression and morphological changes were recorded at 1 day, 1 wk, and 4 wks after injury. RESULTS: Tnfa , Il6 , and Cxcl1 gene expression peaked at day 1 post-needle puncture of the mouse intervertebral disc, Adam8 gene expression peaked at 1-wk time point, while Tipe2 gene expression was upregulated at week 4 postinjury. F4/80 positive cells, likely to be macrophages, are present as early as day 1 in the injured intervertebral discs and consistently present at week 4 postinjury. Loss of Safranin O staining and increased histological scores of the injured intervertebral discs are consistent with progressive degeneration after injury. CONCLUSIONS: Inflammatory cytokines including Tnfa precede Tipe2 , suggesting that Tipe2 is likely induced by Tnfa . Upregulation of Adam8 and Cxcl1 gene expression persisted at week 4, suggesting that they play a role in the transition to chronic phase of intervertebral disc degeneration.

Brain metastasis in breast cancer: focus on genes and signaling pathways involved, blood–brain barrier and treatment strategies

Breast cancer (BC) is one of the most prevalent types of cancer in women. Despite advancement in early detection and efficient treatment, recurrence and metastasis continue to pose a significant risk to the life of BC patients. Brain metastasis (BM) reported in 17–20 percent of BC patients is considered as a major cause of mortality and morbidity in these patients. BM includes various steps from primary breast tumor to secondary tumor formation. Various steps involved are primary tumor formation, angiogenesis, invasion, extravasation, and brain colonization. Genes involved in different pathways have been reported to be associated with BC cells metastasizing to the brain. ADAM8 gene, EN1 transcription factor, WNT, and VEGF signaling pathway have been associated with primary breast tumor; MMP1, COX2, XCR4, PI3k/Akt, ERK and MAPK pathways in angiogenesis; Noth, CD44, Zo-1, CEMIP, S0X2 and OLIG2 are involved in invasion, extravasation and colonization, respectively. In addition, the blood–brain barrier is also a key factor in BM. Dysregulation of cell junctions, tumor microenvironment and loss of function of microglia leads to BBB disruption ultimately resulting in BM. Various therapeutic strategies are currently used to control the BM in BC. Oncolytic virus therapy, immune checkpoint inhibitors, mTOR-PI3k inhibitors and immunotherapy have been developed to target various genes involved in BM in BC. In addition, RNA interference (RNAi) and CRISPR/Cas9 are novel interventions in the field of BCBM where research to validate these and clinical trials are being carried out. Gaining a better knowledge of metastasis biology is critical for establishing better treatment methods and attaining long-term therapeutic efficacies against BC. The current review has been compiled with an aim to evaluate the role of various genes and signaling pathways involved in multiple steps of BM in BC(AU)