Cell wall composition and biofilm formation of azoles-susceptible and -resistant Candida glabrata strains

In the present study, three strains of Candida glabrata have been investigated to shed light on the mechanisms involved in azole resistance during adherence and biofilm formation. In particular, a clinical isolate, susceptible to azole-based drugs, DSY562 and two different resistant mutagenic strains deriving from DSY562, SFY114 and SFY115, have been analysed with different approaches for their cell wall composition and properties. A proteomic analysis revealed that the expression of six cell wall-related proteins and biofilm formation varied between the strains. The SFY114 and SFY115 strains resulted to be less hydrophobic than the susceptible parental counterpart DSY562, on the other hand they showed a higher amount in total cell wall polysaccharides fraction in the total cell wall. Accordingly to the results obtained from the hydrophobicity and adherence assays, in the resistant strain SFY115 the biofilm formation decreased compared to the parental strain DSY562. Finally, the total glucose amount in resistant SFY115 was about halved in comparison to other strains. Taken together all these data suggest that azole drugs may affect the cell wall composition of C. glabrata, in relation to the different pathogenic behaviours

Cell wall composition and biofilm formation of azoles-susceptible and -resistant Candida glabrata strains

In the present study, three strains of Candida glabrata have been investigated to shed light on the mechanisms involved in azole resistance during adherence and biofilm formation. In particular, a clinical isolate, susceptible to azole-based drugs, DSY562 and two different resistant mutagenic strains deriving from DSY562, SFY114 and SFY115, have been analysed with different approaches for their cell wall composition and properties. A proteomic analysis revealed that the expression of six cell wall-related proteins and biofilm formation varied between the strains. The SFY114 and SFY115 strains resulted to be less hydrophobic than the susceptible parental counterpart DSY562, on the other hand they showed a higher amount in total cell wall polysaccharides fraction in the total cell wall. Accordingly to the results obtained from the hydrophobicity and adherence assays, in the resistant strain SFY115 the biofilm formation decreased compared to the parental strain DSY562. Finally, the total glucose amount in resistant SFY115 was about halved in comparison to other strains. Taken together all these data suggest that azole drugs may affect the cell wall composition of C. glabrata, in relation to the different pathogenic behaviours

Extracellular signal-regulated kinase 8 (Erk8) controls estrogen-related receptor alpha cellular localization and inhibits its transcriptional activity.

Erk8 (MAPK15) is a large MAP kinase already implicated in the regulation of the functions of different nuclear receptors and in cellular proliferation and transformation. Here, we identify ERRα as a novel Erk8-interacting protein. As a consequence of such interaction, Erk8 induces Crm1-dependent translocation of ERRα to the cytoplasm and inhibits its transcriptional activity. Also, we identify in Erk8 two LXXLL motifs, typical of agonist-bound nuclear receptor corepressors, as necessary features for this MAP kinase to interact with ERRα and to regulate its cellular localization and transcriptional activity. Ultimately, based on the well-established positive role of ERRα in mammary carcinogenesis, we demonstrate that Erk8 is able to counteract, in immortalized human mammary cells, ERRα activation induced by the EGF receptor pathway, often deregulated in breast cancer. Altogether, these results reveal a novel function for Erk8 as a bona fide ERRα corepressor, involved in the control of its cellular localization by nuclear exclusion, and suggest a key role for this MAP kinase in the biological activities of this nuclear receptor

MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8- like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target

Salivary Proteomic Analysis and Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Abstract Graft-versus-host disease (GVHD) is the major life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), developing in 35%-70% of all allo-HSCT recipients despite immunosuppressive prophylaxis. The recent application of proteomic tools that allow screening for differentially expressed or excreted proteins in body fluids could possibly identify specific biomarkers for GVHD. Whole saliva is highly attractive for noninvasive specimen collection. In the present study, we collected saliva specimens from 40 consecutives patients who underwent allo-HSCT between December 2008 and March 2011 at our institution. The specimens were analyzed by HPLC coupled to electrospray-ionization mass spectrometry. Variable expression of S100 protein family members (S100A8, S100A9, and S100A7) was detected. Fourteen of 23 patients with GVHD demonstrated the presence of S100A8, compared with only 2 patients without GVHD and 0 patients in the control group ( P = .001). S100A7 was detectable in 11 of the 23 patients with GVHD but was absent in the other 2 groups ( P = .0001). S100A9-short was detected in 20 patients with GVHD, in 9 patients without GVHD, and in 8 healthy volunteers ( P = .01) Further studies are needed to clarify the role of these proteins as a marker of GVHD or as an index of mucosal inflammation

Characterization of Cystatin B Interactome in Saliva from Healthy Elderly and Alzheimer's Disease Patients

Cystatin B is a small, multifunctional protein involved in the regulation of inflammation, innate immune response, and neuronal protection and found highly abundant in the brains of patients with Alzheimer's disease (AD). Recently, our study demonstrated a significant association between the level of salivary cystatin B and AD. Since the protein is able to establish protein-protein interaction (PPI) in different contexts and aggregation-prone proteins and the PPI networks are relevant for AD pathogenesis, and due to the relevance of finding new AD markers in peripheral biofluids, we thought it was interesting to study the possible involvement of cystatin B in PPIs in saliva and to evaluate differences and similarities between AD and age-matched elderly healthy controls (HC). For this purpose, we applied a co-immunoprecipitation procedure and a bottom-up proteomics analysis to purify, identify, and quantify cystatin B interactors. Results demonstrated for the first time the existence of a salivary cystatin B-linked multi-protein complex composed by 82 interactors and largely expressed in the body. Interactors are involved in neutrophil activation, antimicrobial activity, modulation of the cytoskeleton and extra-cellular matrix (ECM), and glucose metabolism. Preliminary quantitative data showed significantly lower levels of triosophosphate isomerase 1 and higher levels of mucin 7, BPI, and matrix Gla protein in AD with respect to HC, suggesting implications associated with AD of altered glucose metabolism, antibacterial activities, and calcification-associated processes. Data are available via ProteomeXchange with identifiers PXD039286 and PXD030679

Combined Salivary Proteome Profiling and Machine Learning Analysis Provides Insight into Molecular Signature for Autoimmune Liver Diseases Classification

Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases that target the liver and have a wide spectrum of presentation. A global overview of quantitative variations on the salivary proteome in presence of these two pathologies is investigated in this study. The acid-insoluble salivary fraction of AIH and PBC patients, and healthy controls (HCs), was analyzed using a gel-based bottom-up proteomic approach combined with a robust machine learning statistical analysis of the dataset. The abundance of Arginase, Junction plakoglobin, Desmoplakin, Hexokinase-3 and Desmocollin-1 decreased, while that of BPI fold-containing family A member 2 increased in AIHp compared to HCs; the abundance of Gelsolin, CD14, Tumor-associated calcium signal transducer 2, Clusterin, Heterogeneous nuclear ribonucleoproteins A2/B1, Cofilin-1 and BPI fold-containing family B member 2 increased in PBCp compared to HCs. The abundance of Hornerin decreased in both AIHp and PBCp with respect to HCs and provided an area under the ROC curve of 0.939. Machine learning analysis confirmed the feasibility of the salivary proteome to discriminate groups of subjects based on AIH or PBC occurrence as previously suggested by our group. The topology-based functional enrichment analysis performed on these potential salivary biomarkers highlights an enrichment of terms mostly related to the immune system, but also with a strong involvement in liver fibrosis process and with antimicrobial activity

A top-down proteomic approach reveals a salivary protein profile able to classify Parkinson's disease with respect to Alzheimer's disease patients and to healthy controls

Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin & beta;4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of & alpha;-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. & alpha;-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin & beta;4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD

Multimodality Imaging in Cardiomyopathies with Hypertrophic Phenotypes

Multimodality imaging is a comprehensive strategy to investigate left ventricular hypertrophy (LVH), providing morphologic, functional, and often clinical information to clinicians. Hypertrophic cardiomyopathy (HCM) is defined by an increased LV wall thickness not only explainable by abnormal loading conditions. In the context of HCM, multimodality imaging, by different imaging techniques, such as echocardiography, cardiac magnetic resonance, cardiac computer tomography, and cardiac nuclear imaging, provides essential information for diagnosis, sudden cardiac death stratification, and management. Furthermore, it is essential to uncover the specific cause of HCM, such as Fabry disease and cardiac amyloidosis, which can benefit of specific treatments. This review aims to elucidate the current role of multimodality imaging in adult patients with HCM