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

The extreme hyper-reactivity of Cys94 in lysozyme avoids its amorphous aggregation

Many proteins provided with disulfde bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfdes are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfdes. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pKa of its sulfydryl group (6.6/7.1) and a productive complex with GSSG (KD=0.3mM), causes a fast glutathionylation of this residue (t1/2=3s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research felds for Alzheimer’s and Parkinson diseases

The unusual properties of lactoferrin during its nascent phase

Lactoferrin, a multifunctional iron-binding protein containing 16 disulfides, is actively studied for its antibacterial and anti-carcinogenic properties. However, scarce information is nowadays available about its oxidative folding starting from the reduced and unfolded status. This study discovers unusual properties when this protein is examined in its reduced molten globule-like conformation. Using kinetic, CD and fluorescence analyses together with mass spectrometry, we found that a few cysteines display astonishing hyper-reactivity toward different thiol reagents. In details, four cysteines (i.e. 668, 64, 512 and 424) display thousands of times higher reactivity toward GSSG but normal against other natural disulfides. The formation of these four mixed-disulfides with glutathione probably represents the first step of its folding in vivo. A widespread low pKa decreases the reactivity of other 14 cysteines toward GSSG limiting their involvement in the early phase of the oxidative folding. The origin of this hyper-reactivity was due to transient lactoferrin-GSSG complex, as supported by fluorescence experiments. Lactoferrin represents another disulfide containing protein in addition to albumin, lysozyme, ribonuclease, chymotrypsinogen, and trypsinogen which shows cysteines with an extraordinary and specific hyper-reactivity toward GSSG confirming the discovery of a fascinating new feature of proteins in their nascent phase

Antenatal Thymosin β4: a New Tool for Accelerating Fetal Development in Preterms? Thymosin Beta-4: a Breakthrough in the "Physiological" Regenerative Medicine in Preterm Newborns

To prevent the health risks related to prematurity, multiple drugs have been introduced in clinical practice in recent years. This paper focuses on a new "physiological" regenerative approach to be started in the perinatal period, particularly on very low birth weight preterm infants. This new preventive approach underlined the necessity to start regenerative medicine very early after birth, a period in which kidney, brain, pancreas, and lung stem cells maintain their proliferative and differentiating abilities. Among the multiple factors proposed in the literature as potential growth promoters for preterm neonates, thymosin beta-4 (Tβ4) has been indicated as one of the most important candidates for regenerative medicine

Responsiveness to 6-n-Propylthiouracil (PROP) Is Associated with Salivary Levels of Two Specific Basic Proline-Rich Proteins in Humans

Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the ‘taster’ variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait

Gene expression deregulation by KRAS G12D and G12V in a BRAF V600E context

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>and <it>BRAF </it>mutations appear of relevance in the genesis and progression of several solid tumor types but the co-occurrence and interaction of these mutations have not yet been fully elucidated. Using a microsatellite stable (MSS) colorectal cancer (CRC) cell line (Colo741) having mutated <it>BRAF </it>and <it>KRAS</it>^<it>WT</it>, we also aimed to investigate the <it>KRAS-BRAF </it>interaction. Gene expression profiles for control <it>KRAS</it>^<it>WT</it>, <it>KRAS</it>^{<it>G</it>12<it>V </it>}and <it>KRAS</it>^{<it>G</it>12<it>D </it>}transfected cells were obtained after cell clone selection and RT-PCR screening. Extensive qPCR was performed to confirm microarray data.</p> <p>Results</p> <p>We found that the <it>KRAS</it>^{<it>G</it>12<it>V </it>}state deregulated several genes associated to cell cycle, apoptosis and nitrogen metabolism. These findings indicated a reduced survival and proliferation with respect to the <it>KRAS</it>^{<it>WT </it>}state. The <it>KRAS</it>^{<it>G</it>12<it>D </it>}state was, instead, characterized by several other distinct functional changes as for example those related to chromatin organization and cell-cell adhesion without affecting apoptosis related genes.</p> <p>Conclusion</p> <p>These data predict that the G12D mutation may be more likely selected in a <it>BRAF </it>mutated context. At the same time, the presence of the <it>KRAS</it>^{<it>G</it>12<it>V </it>}mutation in the cells escaping apoptosis and inducing angiogenesis via IL8 may confer a more aggressive phenotype. The present results get along with the observations that CRCs with G12V are associated with a worse prognosis with respect to the WT and G12D states and may help identifying novel CRC pathways and biomarkers of clinical relevance.</p

Cervical mucus proteome in endometriosis

Additional file 1: Table S1. Identified proteins in CM in the group of controls and in patients affected by endometriosis

Quantitative analysis of the seminal plasma proteome in secondary hypogonadism

In the grey zone of testosterone levels between 8 and 12 nmol/L, the usefulness of therapy is controversial; as such, markers of tissue action of androgens may be helpful in adjusting clinical decisions. To better understand the effect of the hypothalamic-pituitary-testicular axis on male accessory secretion, we performed a proteomic quantitative analysis of seminal plasma in patients with secondary hypogonadism, before and after testosterone replacement therapy (TRT). Ten male patients with postsurgical hypogonadotrophic hypogonadism were enrolled in this study, and five of these patients were evaluated after testosterone treatment. Ten men with proven fertility were selected as a control group. An aliquot of seminal plasma from each individual was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLC-nano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer. The label-free quantitative analysis was performed via Precursor Ions Area Detector Node. Eleven proteins were identified as decreased in hypogonadic patients versus controls, which are primarily included in hydrolase activity and protein binding activity. The comparison of the proteome before and after TRT comes about within the discovery of six increased proteins. This is the primary application of quantitative proteomics pointed to uncover a cluster of proteins reflecting an impairment not only of spermatogenesis but of the epididymal and prostate epithelial cell secretory function in male hypogonadism. The identified proteins might represent putative clinical markers valuable within the follow-up of patients with distinctive grades of male hypogonadism

Salivary biomarkers and proteomics: Future diagnostic and clinical utilities = Biomarkers e proteomica salivari: Prospettive future cliniche e diagnostiche

Saliva testing is a non-invasive and inexpensive test that can serve as a source of information useful for diagnosis of disease. As we enter the era of genomic technologies and –omic research, collection of saliva has increased. Recent proteomic platforms have analysed the human salivary proteome and characterised about 3000 differentially expressed proteins and peptides: in saliva, more than 90% of proteins in weight are derived from the secretion of three couples of “major” glands; all the other components are derived from minor glands, gingival crevicular fluid, mucosal exudates and oral microflora. The most common aim of proteomic analysis is to discriminate between physiological and pathological conditions. A proteomic protocol to analyze the whole saliva proteome is not currently available. It is possible distinguish two type of proteomic platforms: top-down proteomics investigates intact naturally-occurring structure of a protein under examination; bottom-up proteomics analyses peptide fragments after pre-digestion (typically with trypsin). Because of this heterogeneity, many different biomarkers may be proposed for the same pathology. The salivary proteome has been characterised in several diseases: oral squamous cell carcinoma and oral leukoplakia, chronic graft-versus-host disease Sjögren’s syndrome and other autoimmune disorders such as SAPHO, schizophrenia and bipolar disorder, and genetic diseases like Down’s Syndrome and Wilson disease. The results of research reported herein suggest that in the near future human saliva will be a relevant diagnostic fluid for clinical diagnosis and prognosis