For decades, disintegrin and metalloproteinase 17 (ADAM17) has been the object of deep investigation. Since its discovery as the tumor necrosis factor convertase, it has been considered a major drug ...target, especially in the context of inflammatory diseases and cancer. Nevertheless, the development of drugs targeting ADAM17 has been harder than expected. This has generally been due to its multifunctionality, with over 80 different transmembrane proteins other than tumor necrosis factor α (TNF) being released by ADAM17, and its structural similarity to other metalloproteinases. This review provides an overview of the different roles of ADAM17 in disease and the effects of its ablation in a number of in vivo models of pathological conditions. Furthermore, here, we comprehensively encompass the approaches that have been developed to accomplish ADAM17 selective inhibition, from the newest non-zinc-binding ADAM17 synthetic inhibitors to the exploitation of iRhom2 to specifically target ADAM17 in immune cells.
Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix ...metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.
In recent years, probiotics have been emerging as an attractive therapeutic strategy for several diseases. In orthopedics, probiotics seem to be a promising supplementation for treatment of ...osteoporosis, osteoarthritis, muscle loss-related disease, wound and ulcer issues, and prevention of surgical antibiotic prophylaxis side effects. Although probiotics are still not included in guidelines for these conditions, several studies have reported theoretical benefits of their administration. Further high-level clinical trials are necessary to convert research into solid clinical practice. However, probiotics represent a cost-effective future perspective and may play a role in association with traditional orthopedic therapies.
Theoretically, Aspergillus spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the reasons are not clear. This aspect ...underlines the lack of a good strategy for the cultivation and isolation of Aspergillus spp. In order to develop a complete analytical method to detect Aspergillus in clinical and pharmaceutical samples, we investigated the growth performance of two blood culture systems versus the pharmacopeia standard method. At <72 h, all test systems showed comparable sensitivity, about 1−2 conidia. However, the subculture analysis showed a suboptimal recovery for the methods, despite the positive growth and the visualization of the “Aspergillus balls” in the culture media. To investigate this issue, we studied three different subculture approaches: (i) the use of a sterile subculture unit, (ii) the use of a sterile subculture unit and the collection of a larger aliquot (100 µL), following vigorous agitation of the vials, and (iii) to decapsulate the bottle, withdrawing and centrifuging the sample, and aliquot the pellet onto SDA plates. Our results showed that only the third procedure recovered Aspergillus from all positive culture bottles. This work confirmed that our strategy is a valid and faster method to culture and isolate Aspergillus spp. from blood culture bottles.
A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological ...functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.
The optimal surgical treatment of intra-articular calcaneal fractures (IACF) is still under debate. In the literature, results are based on clinical or radiographical findings. Few studies have ...evaluated the effect of patient expectations on patient-reported outcomes after surgery and little is known about outcomes directly reported by the patient who experienced it. Patient reported outcome measures (PROMs) may represent a viable and useful tool for evaluating the efficacy of the procedure and can be considered as an indicators of health-care quality. The aim of this study is to evaluate PROMs after minimally invasive reduction and percutaneous Kirschner-wires fixation for IACF, and to compare PROMs to pre-operative and last follow-up radiographic findings.
33 consecutive patients with IACF treated with minimally invasive reduction and percutaneous K-wires fixation were included. Data collection included demographics, pre-operative and last available Böhler and Gissane angle X-rays, foot function index (FFI), and foot and ankle outcome score (FAOS).
At a mean follow up of 36.7 months, the mean FFI score was 24.3 ± 19.9 and the mean FAOS score was 68 ± 24.8. Patients with better Gissane angle showed better activity limitations FFI subscores. Moreover, worse pre-operative Gissane and Böhler angle were significantly associated with a worse total FAOS score and subscores.
Minimally invasive reduction and percutaneous K-wires fixation provided satisfactory PROMs. Despite these results, prospective randomized studies are required to confirm the validity and reliability of PROMs in evaluating different treatments.
ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ...ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used "surface-spanning enrichment with click-sugars (SUSPECS)" proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding.
Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic ...process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.
Receptor-mediated endocytosis provides a mechanism for the selective uptake of specific molecules thereby controlling the composition of the extracellular environment and biological processes. The ...low-density lipoprotein receptor-related protein 1 (LRP1) is a widely expressed endocytic receptor that regulates cellular events by modulating the levels of numerous extracellular molecules via rapid endocytic removal. LRP1 also participates in signalling pathways through this modulation as well as in the interaction with membrane receptors and cytoplasmic adaptor proteins. LRP1 single nucleotide polymorphisms are associated with several diseases and conditions such as migraines, aortic aneurysms, cardiopulmonary dysfunction, corneal clouding, and bone dysmorphology and mineral density. Studies using Lrp1 knockout mice revealed a critical, non-redundant and tissue-specific role of LRP1 in regulating various physiological events. However, exactly how LRP1 functions to regulate so many distinct and specific processes is still not fully clear. Our recent proteomics studies have identified more than 300 secreted proteins that either directly interact with LRP1 or are modulated by LRP1 in various tissues. This review will highlight the remarkable ability of this receptor to regulate secreted molecules in a tissue-specific manner and discuss potential mechanisms underpinning such specificity. Uncovering the depth of these “hidden” specific interactions modulated by LRP1 will provide novel insights into a dynamic and complex extracellular environment that is involved in diverse biological and pathological processes.
Introduction
Ankle osteoarthritis is more commonly posttraumatic. Consequently, dealing with hardware removal is quite frequent when performing a total ankle arthroplasty (TAA). The purpose of this ...study is to compare outcomes regarding either a staged or concurrent hardware removal when performing TAA.
Materials and methods
275 consecutive patients with TAA previously treated with internal fixation were retrospectively reviewed. Finally, 57 patients were enrolled based on exclusion criteria, and were differentiated into two groups considering the timing of hardware removal (staged—group A vs concurrent—group B) to compare: neurovascular and wound complications, time to recover full weight bearing, scar-tissue esthetic, and surgical time. Moreover, a subgroup comparison considering the surgical approach (single approach, minor additional approach, major additional approach) was performed between the group A and group B.
Results
No statistically significant difference other that longer surgical time (
p
< 0.05) was observed between group A and group B. When considering surgical approach subgroups, statistically
s
ignificant higher surgical wound complications and revision rate were reported in group B (concurrent) major additional approach subgroup, and a statistically significant shorter time to full weight bearing was reported in group A (staged) major additional approach subgroup.
Conclusions
When performing TAA requiring hardware removal, no clear superiority of staged over concurrent hardware removal was observed. However, when considering a subgroup of patients requiring a separate major incision, a staged approach has shown reduced surgical time, less risk of wound complications, and shorter recovery to full weight bearing.
Level of evidence
III.
PDF
