Proteomic Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved in Vesicular Traffic and Nutritional Adaptation

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation

Comparison of Oxford Cognitive Screen and Montreal Cognitive Assessment feasibility in the stroke unit setting. A pilot study

Background: : Cognitive status evaluation is not routine in the acute stroke setting and there is no consensus on which neuropsychological tool is more feasible and informative. The aim of this pilot study was to compare the feasibility and acceptability of two brief cognitive tests, the Montreal Cognitive Assessment (MoCA) and the Oxford Cognitive Screen (OCS), in acute stroke, with a focus on patients' experience, administration time, and the cognitive data obtained. Methods: : Patients with a diagnosis of ischemic or hemorrhagic stroke or of transient ischemic attack admitted to two stroke units were included. The sample consisted of 34 participants (mean age ±SD 71.1 ± 16.1 years, 25 males). Within five days of onset, patients were evaluated by means of the MoCA and OCS by a trained neuropsychologist. Results: Both tests were feasible in the stroke unit setting and had a high level of acceptability by patients. MoCA test was fully completed by 25 patients, OCS by 21 patients. The OCS administration time was longer than that of the MoCA. However, OCS was perceived less demanding than MoCA by patients. Twenty patients completed both the MoCA and the OCS entirely, and only 2 of them did not show any impairment in both tests. Seventeen patients showed at least an impaired domain on the OCS and 15 patients presented with a MoCA global score below cut-off for cognitive impairment. Conclusions: Our preliminary study did not show a superiority of the OCS over the widely used MoCA, and suggests the need for further validation in larger samples of stroke patients, exploring tests accuracy in detecting cognitive post-stroke impairment

Cleavage of chromogranin A N-terminal domain by plasmin provides a new mechanism for regulating cell adhesion

It has been proposed that chromogranin A (CgA), a protein secreted by many normal and neoplastic neuroendocrine cells, can play a role as a positive or a negative modulator of cell adhesion. The mechanisms that regulate these extracellular functions of CgA are unknown. We show here that plasmin can regulate the anti/pro-adhesive activity of CgA by proteolytic cleavage of the N-terminal domain. Limited proteolytic processing decreased its anti-adhesive activity and induced pro-adhesive effects in fibronectin or serum-dependent fibroblast adhesion assays. Cleavage of Lys(77)-Lys(78) dibasic site in CgA(1-115) was relatively rapid and associated with an increase of pro-adhesive effect. In contrast, antibodies against the region 53-90 enhanced the anti-adhesive activity of CgA and CgA(1-115). Structure-activity relationship studies showed that the conserved region 47-64 (RILSILRHQNLLKELQDL) is critical for both pro- and anti-adhesive activity. These findings suggest that CgA might work on one hand as a negative modulator of cell adhesion and on the other hand as a precursor of positive modulators, the latter requiring proteolytic processing for activation. Given the importance of plasminogen activation in tissue invasion and remodeling, the interplay between CgA and plasmin could provide a novel mechanism for regulating fibroblast adhesion and function in neuroendocrine tumors

Structure-activity relationships of chromogranin A in cell adhesion. Identification of an adhesion site for fibroblasts and smooth muscle cells.

Previous studies showed that chromogranin A (CgA), a glycoprotein stored and co-released with various hormones by neuroendocrine cells and neurons, can modulate cell adhesion. We have investigated the structure-activity relationships of CgA using fibroblasts and coronary artery smooth muscle cells in adhesion assays. A recombinant CgA fragment 1-78 and a peptide 7-57 containing reduced and alkylated cysteines (Cys(17) and Cys(38)) induced cell adhesion after adsorption onto solid phases at 50-100 nm. Peptides lacking the disulfide loop region, including residues 47-68, 39-59, and 39-68, induced cell adhesion, either bound to solid phases at 200-400 nm or added to the liquid phase at 5-10 microm, whereas peptide 60-68 was inactive, suggesting that residues 47-57 are important for activity. The effect of CgA-(1-78) was blocked by anti-CgA antibodies against epitopes including residues Arg(53), His(54), and Leu(57). Substitutions of residues His(54), Gln(55), and Asn(56) with alanine decreased the cell adhesion activity of peptide 47-68. These results suggest that the region 47-57 (RILSILRHQNL) contains a cell adhesion site and that the disulfide bridge is not necessary for the proadhesive activity. The ability of soluble peptides to elicit proadhesive effects suggests an indirect mechanism. The high sequence conservation and accessibility to antibodies suggest that this region is important for the physiological role of CgA

Trends in the Postmortem Diagnosis of Opportunistic Invasive Fungal Infections in Patients With AIDS

Abstract We retrospectively evaluated autopsy-proven invasive fungal infections (IFIs) in patients with AIDS who died between 1984 and 2002. IFIs were identified in 297 (18.2%) of 1,630 autopsies. Their prevalence significantly decreased over time (from 25.0% in 1984–1988 to 15% in 1998–2002; P = .004), mainly owing to a significant decrease in pneumocystosis (P = .017) and cryptococcosis (P = .003), whereas the prevalence of aspergillosis and histoplasmosis remained relatively stable and of candidiasis and zygomycosis tended to increase in the last years (P = .028 and P = .042, respectively). IFIs were suspected or confirmed during life in only 46.8% of the cases; this proportion did not vary significantly over time (P = .320). The infections contributed to the deaths of 103 patients (34.7%), and their global impact on mortality was 6.3%. Of fatal cases, 38 (36.9%) were characterized by missed antemortem diagnoses, 17 (45%) of which met Goldman criteria for class I errors. The epidemiology of IFIs in patients with AIDS is evolving and not completely mirrored by clinical diagnoses or current diagnostic methods. Our results confirm the valuable role of autopsy data, even with highly effective therapies and advanced technologies

Unsupervised neural networks as a support tool for pathology diagnosis in MALDI-MSI experiments:A case study on thyroid biopsies

Artificial intelligence is getting a foothold in medicine for disease screening and diagnosis. While typical machine learning methods require large labeled datasets for training and validation, their application is limited in clinical fields since ground truth information can hardly be obtained on a sizeable cohort of patients. Unsupervised neural networks - such as Self-Organizing Maps (SOMs) - represent an alternative approach to identifying hidden patterns in biomedical data. Here we investigate the feasibility of SOMs for the identification of malignant and non-malignant regions in liquid biopsies of thyroid nodules, on a patient-specific basis. MALDI-ToF (Matrix Assisted Laser Desorption Ionization -Time of Flight) mass spectrometry-imaging (MSI) was used to measure the spectral profile of bioptic samples. SOMs were then applied for the analysis of MALDI-MSI data of individual patients' samples, also testing various pre-processing and agglomerative clustering methods to investigate their impact on SOMs' discrimination efficacy. The final clustering was compared against the sample's probability to be malignant, hyperplastic or related to Hashimoto thyroiditis as quantified by multinomial regression with LASSO. Our results show that SOMs are effective in separating the areas of a sample containing benign cells from those containing malignant cells. Moreover, they allow to overlap the different areas of cytological glass slides with the corresponding proteomic profile image, and inspect the specific weight of every cellular component in bioptic samples. We envision that this approach could represent an effective means to assist pathologists in diagnostic tasks, avoiding the need to manually annotate cytological images and the effort in creating labeled datasets

Tumor size, stage and grade alterations of urinary peptidome in RCC

Background: Several promising biomarkers have been found for RCC, but none of them has been used in clinical practice for predicting tumour progression. The most widely used features for predicting tumour aggressiveness still remain the cancer stage, size and grade. Therefore, the aim of our study is to investigate the urinary peptidome to search and identify peptides whose concentrations in urine are linked to tumour growth measure and clinical data. Methods: A proteomic approach applied to ccRCC urinary peptidome (n = 117) based on prefractionation with activated magnetic beads followed by MALDI-TOF profiling was used. A systematic correlation study was performed on urinary peptide profiles obtained from MS analysis. Peptide identity was obtained by LC-ESI-MS/MS. Results: Fifteen, twenty-six and five peptides showed a statistically significant alteration of their urinary concentration according to tumour size, pT and grade, respectively. Furthermore, 15 and 9 signals were observed to have urinary levels statistically modified in patients at different pT or grade values, even at very early stages. Among them, C1RL, A1AGx, ZAG2G, PGBM, MMP23, GP162, ADA19, G3P, RSPH3, DREB, NOTC2 SAFB2 and CC168 were identified. Conclusions: We identified several peptides whose urinary abundance varied according to tumour size, stage and grade. Among them, several play a possible role in tumorigenesis, progression and aggressiveness. These results could be a useful starting point for future studies aimed at verifying their possible use in the managements of RCC patients