Colchicine inhibits ROS generation in response to glycoprotein VI stimulation.

Colchicine inhibits coronary and cerebrovascular events in patients with coronary artery disease (CAD), and although known to have anti-inflammatory properties, its mechanisms of action are incompletely understood. In this study, we investigated the effects of colchicine on platelet activation with a particular focus on its effects on activation via the collagen glycoprotein (GP)VI receptor, P2Y12 receptor, and procoagulant platelet formation. Therapeutic concentrations of colchicine in vitro (equivalent to plasma levels) significantly decreased platelet aggregation in whole blood and in platelet rich plasma in response to collagen (multiplate aggregometry) and reduced reactive oxygen species (ROS) generation (H2DCF-DA, flow cytometry) in response to GPVI stimulation with collagen related peptide-XL (CRP-XL, GPVI specific agonist). Other platelet activation pathways including P-selectin expression, GPIIb/IIIa conformational change and procoagulant platelet formation (GSAO+/CD62P+) (flow cytometry) were inhibited with higher concentrations of colchicine known to inhibit microtubule depolymerization. Pathway specific mechanisms of action of colchicine on platelets, including modulation of the GPVI receptor pathway at low concentrations, may contribute to its protective role in CAD.

Genome-wide analysis of Saccharomyces cerevisiae identifies cellular processes affecting intracellular aggregation of Alzheimer's amyloid-ß42: importance of lipid homeostasis.

Amyloid-ß (Aß)-containing plaques are a major neuropathological feature of Alzheimer's disease (AD). The two major isoforms of Aß peptide associated with AD are Aß40 and Aß42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aß42 peptides is an early event in AD progression. Improved understanding of cellular processes affecting Aß42 aggregation may have implications for development of therapeutic strategies. Aß42 fused to green fluorescent protein (Aß42-GFP) was expressed in ∼4600 mutants of a Saccharomyces cerevisiae genome-wide deletion library to identify proteins and cellular processes affecting intracellular Aß42 aggregation by assessing the fluorescence of Aß42-GFP. This screening identified 110 mutants exhibiting intense Aß42-GFP-associated fluorescence. Four major cellular processes were overrepresented in the data set, including phospholipid homeostasis. Disruption of phosphatidylcholine, phosphatidylserine, and/or phosphatidylethanolamine metabolism had a major effect on intracellular Aß42 aggregation and localization. Confocal microscopy indicated that Aß42-GFP localization in the phospholipid mutants was juxtaposed to the nucleus, most likely associated with the endoplasmic reticulum (ER)/ER membrane. These data provide a genome-wide indication of cellular processes that affect intracellular Aß42-GFP aggregation and may have important implications for understanding cellular mechanisms affecting intracellular Aß42 aggregation and AD disease progression.

Comparison of capillary versus aspiration technique in endoscopic ultrasound-guided fine-needle aspiration: A preliminary report.

INTRODUCTION: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is widely used to diagnose pancreatic malignancies. Different EUS-FNA techniques have been described to improve sample quality. Recently, a new technique, using capillarity, has been proposed. AIM: To assess the quality of cytological samples, comparing two different FNA techniques, in order to optimize tissue acquisition. METHODS: All consecutive patients with solid pancreatic lesions, requiring an EUS-FNA, were included in the study between July and September 2013. All procedures were done under deep sedation. FNA was performed using a 25 gauge needle, using both capillary and aspiration technique. Patients were randomized to undergo firstly one or the other technique. Samples were evaluated "on site" by expert cytotechnologist. An expert cytopathologist, blinded for the technique used, reviewed the slides, for final diagnosis and assessed sampling quality. Quality of samples was evaluated through the assessment of the amount of blood, cellularity, tumoral versus normal cells ratio and adequacy for final diagnosis. Data were analyzed with Student's t-test and Chi-square test, assuming a significant P value of 0.05. RESULTS: A total of 30 consecutive patients (19 M, mean age 67.8 years) with an EUS finding of pancreatic solid lesion were included in the study. Cytological final diagnosis was adenocarcinoma in 25/30 (83.3%) cases, neuroendocrine tumor 1/30 (3.3%), intraductal papillary mucosal neoplasms with high-grade dysplasia 2/30 (6.7%), gastrointestinal stromal tumor in 1/30 (3.3%) and negative for malignant cells 1/30 (3.3%). The difference between the overall blood amount score per technique was not statistically significant (P = 0.61) as well as the cellularity score (P = 0.08). In 13/30 patients (43%) the two techniques reported concordant T/N ratio. In 6/30 patients (20%) final diagnosis was achieved only by capillary obtained smears. In 1/30 patients (3.3%) the diagnosis was done with aspiration. In the remnant, the ratio between the two techniques was similar. Adequacy was reached in 24/30 (80%) with aspiration and 29/30 (97%) with capillary technique (P = 0.04). CONCLUSIONS: Aspiration and capillary sampling techniques provided similar results in cellularity and blood amount. However, adequacy rate was significantly superior in capillary technique. Furthermore, in 20% of cases, final diagnosis was achieved only with capillary samples.

Strategy for protein isoform identification from expressed sequence tags and its application to peptide mass fingerprinting.

Expressed Sequence Tags (ESTs) are an invaluable resource for protein identification and characterisation in proteomics. They allow proteins to be identified in the absence of genome sequence data. When EST sequences are used for protein identification, they are usually first processed into contigs to reduce redundancy and generate longer sequences from the overlapping ESTs. However, the process of generating contigs may accidentally group biologically meaningful isoforms together. Here we report means of discovering isoforms in EST sequences and how to use this information in the framework of protein identification and characterisation with peptide mass fingerprinting. We illustrate our strategies with examples from the dbEST database as well as protein isoforms from two-dimensional polyacrylamide gels.

Proteomic analysis of a developmentally regulated secretory vesicle.

Secretion of spore coat proteins from the prespore secretory vesicles (PSVs) in Dictyostelium discoideum is a signal mediated event that underlies terminal cell differentiation, and represents an important case of developmentally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the subsequent secretion of their cargo, we purified this organelle from prespore cells. Analysis of protein extracts of highly purified PSVs indicated that, in addition to the cargo of structural spore coat proteins, many more proteins are associated with the PSVs. Their identification is paramount to the understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire, previously uncharacterized, organelle, with the goal of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enzymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membrane (actin binding proteins coactosin and profilin 1). In addition, the results suggest that proteins can play multiple roles in the cell, and that protein function can be dictated in part by subcellular localization. The identification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of developmentally regulated secretion.

Towards an automated approach for protein identification in proteome projects.

The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.

Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis.

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.

[Conventional and single-use burs. Cavity preparation comparison]. / Frese convenzionali e monouso. Comparazione in preparazioni di cavita.

Our work's aim, was to value the wear of conventional and monouse burs, while preparing the cavity. Usually the conventional burs give better technical efficiency, but the monouse ones represent a notable progress both in technology and prevention.

Bilateral accessory digastric muscles.

Four aberrant muscles occurring in the submandibular region are described. Accessory digastric muscles were found lying in the submental triangle. All four muscles were attached to the mylohyoid raphe. On one side the anterior muscle ran to the mandible while the posterior muscle reached the hyoid bone. Three of these muscles (the exception being that attached to the mandible) fused with the ipsilateral anterior belly of digastric thus presenting an unusual bilateral arrangement.