Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3.

International audienceType III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a translocation of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low - but not high - doses of ADP or thrombin which depends on α(IIb)β(3) integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes

Ferric iron reduction by fermentative strain BS2 isolated from an iron-rich anoxic environment (Lake Pavin, France)

International audienceA facultatively Fe-reducing strain BS2 related to Clostridium saccarobutylicum was isolated from the anoxic zone of iron-rich Lake Pavin. A comparative study was performed on the growth, fermentative profile and heat production of BS2 on glucose in presence and absence of Fe(III). Fe(III) availability led to an optimization of glucose uptake and higher maintenance of strain BS2 in the growth media. Given the prevailing conditions (e.g., scarcity of metabolizable organic matter) in the anoxic zone of Lake Pavin, Fe(III) reduction by this facultative Fe(III) reducer may confer ecological benefits to strain BS2. Moreover, in presence of Fe(III), there was a modification of the fermentative balance that may influence the terminal-accepting processes in this layer

The Centriolar Adjunct–Appearance and Disassembly in Spermiogenesis and the Potential Impact on Fertility

During spermiogenesis, the proximal centriole forms a special microtubular structure: the centriolar adjunct. This structure appears at the spermatid stage, which is characterized by a condensed chromatin nucleus. We showed that the centriolar adjunct disappears completely in mature porcine spermatozoa. In humans, the centriolar adjunct remnants are present in a fraction of mature spermatids. For the first time, the structure of the centriolar adjunct in the cell, and its consequent impact on fertility, were examined. Ultrastructural analysis using transmission electron microscopy was performed on near 2000 spermatozoa per person, in two patients with idiopathic male sterility (IMS) and five healthy fertile donors. We measured the average length of the “proximal centriole + centriolar adjunct„ complex in sections, where it had parallel orientation in the section plane, and found that it was significantly longer in the spermatozoa of IMS patients than in the spermatozoa of healthy donors. This difference was independent of chromatin condensation deficiency, which was also observed in the spermatozoa of IMS patients. We suggest that zygote arrest may be related to an incompletely disassembled centriolar adjunct in a mature spermatozoon. Therefore, centriolar adjunct length can be potentially used as a complementary criterion for the immaturity of spermatozoa in the diagnostics of IMS patients

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

International audiencePlatelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open cana-licular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a dis-continuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion , whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and a2b1 integrin) for efficient platelet activation

Ultrasound and microbubble-assisted gene delivery in Achilles tendons: long lasting gene expression and restoration of fibromodulin KO phenotype

International audienceThe aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods

The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung diseases

International audienceThe serine proteases released by activated polymorphonuclear neutrophils (NSPs) contribute to a variety of inflammatory lung diseases, including cystic fibrosis (CF). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies (Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj, Gauthier 2009, J Biol Chem, 284,34084-34091). Thus, NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- pig model is a promising alternative to the mouse model of CF (Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani, et al. 2008, Science; 321(5897):1837-1841). We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and neutrophil extracellular traps (NETs). All the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF