Accuracy of automated and manual systems for susceptibility testing of Pseudomonas aeruginosa to piperacillin and piperacillin-tazobactam

This study aimed to evaluate the accuracy of routine systems (Vitek2 cards AST-N022 and AST-N026; Kirby Bauer; Etest) for susceptibility testing of Pseudomonas aeruginosa to piperacillin and piperacillin-tazobactam. Vitek2 (card AST-N022) showed the worst performance; the other three methods (Vitek2 card AST-N026, Kirby-Bauer and E-test) performed comparably but never fulfilled the minimal standard proposed by FDA.     

Identification of QTL for resistance to plum pox virus strains M and D in Lito and Harcot apricot cultivars

Sharka is a severe apricot viral disease caused by the plum pox virus (PPV) and is responsible for large crop losses in many countries. Among the known PPV strains, both PPV-D (Dideron) and PPV-M (Marcus) are virulent in apricot, the latter being the most threatening. An F1 apricot progeny derived from Lito, described in the literature as resistant, crossed to the susceptible selection BO81604311 (San Castrese × Reale di Imola) was used to study the genetic control of resistance to PPV. A population of 118 individuals was phenotyped by inoculating both PPV-D and PPV-M strains in replicated seedlings and scored for 3 years. An additional set of 231 seedlings from the same cross was also phenotyped for 2 years. SSR-based linkage maps were used for quantitative trait locus (QTL) analysis. A major QTL of resistance to both PPV-M and PPV-D strains was found in the top half of the Lito linkage group 1, where a QTL was previously described in Stark Earli-Orange, the donor of Lito resistance. The LOD score was considerably enhanced when the recovery of plants from infection was taken into account. The results obtained in Lito were compared with those observed in a second apricot cross progeny (Harcot × Reale di Imola) in which QTL of resistance to sharka were also mapped in the same linkage group 1 for both PPV strains. Several models of resistance to sharka disease are discussed considering the segregation frequencies, the QTL alignment in the two maps and the information gathered from the literature.     

p66Shc, a multifaceted protein linking Erk signalling, glucose metabolism, and oxidative stress

p66Shc, a 66?kDa proto-oncogene Src collagen homologue (Shc) adaptor protein, is classically known as a signalling protein implicated in receptor tyrosine kinase signal transduction. The p66Shc isoform exerts a physiologically relevant, inhibitory signalling effect on the Erk pathway in skeletal muscle myoblasts, which is necessary for actin cytoskeleton polymerization and normal glucose transport responses. More recently, p66Shc has been also identified as a sensor of oxidative stress-induced apoptosis and as a longevity protein in mammals, actions which require Ser36 phosphorylation of the protein and consequent accumulation of intracellular reactive oxygen species. Oxidative stress plays a key role in dysfunction of several organs and tissues, and this is of interest in metabolic diseases such as type 2 diabetes. Thus changes in p66Shc expression and/or function may play an important role in the pathogenesis of type 2 diabetes and potentially serve as an effective target for its treatment.     

The surprising composition of the salivary proteome of preterm human newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β(4) and β(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.     

Evidence for a stromal-epithelial “lactate shuttle” in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT1, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A1, SLC16A3, monocarboxylic acid transporter     

Caveolin-2-deficient mice show increased sensitivity to endotoxemia

Caveolin proteins are structural components of caveolae and are involved in the regulation of many biological processes. Recent studies have shown that caveolin-1 modulates inflammatory responses and is important for sepsis development. In the present study, we show that caveolin-1 and caveolin-2 have opposite roles in lipopolysaccharide (LPS)-induced sepsis using caveolin-deficient (Cav-1^-/- and Cav-2^-/-) mice for each of these proteins. While Cav-1^-/- mice displayed delayed mortality following challenge with LPS, Cav-2^-/- mice were more sensitive to LPS compared to wild-type (WT). With Cav-2^-/- mice, this effect was associated with increased intestinal injury and increased intestinal permeability. This negative outcome was also correlated with enhanced expression of iNOS in intestinal epithelial cells, and enhanced production of nitric oxide (NO). By contrast, Cav-1^-/- mice demonstrated a decrease in iNOS expression with decreased NO production, but no alteration in intestinal permeability. The differential expression of iNOS was associated with a significant increase in STAT-1 activation in these mice. Intestinal cells of Cav-2^-/- mice showed increased phosphorylation of STAT-1 at tyrosine 701 compared to wild-type. However, Cav-1^-/- mice-derived intestinal cells showed decreased levels of phosphorylation of STAT-1 at tyrosine 701. Since caveolin-2 is almost completely absent in Cav-1^-/- mice, we conclude that it is not just the absence of caveolin-2 that is responsible for the observed effects, but that the balance between caveolin-1 and caveolin-2 is important for iNOS expression and ultimately for sepsis outcome.Key words: caveolin, sepsis, nitric oxide, lipopolysaccharide, permeability, endotoxemia, inflammation     

Proteomic analysis of saliva: a unique tool to distinguish primary Sjögren's syndrome from secondary Sjögren's syndrome and other sicca syndromes

Introduction

A growing interest has arisen in salivary proteomics as a tool for the identification of biomarkers for primary Sjögren's syndrome (pSS). Nonetheless, only a limited number of preclinical validation studies have been performed, limiting the possibility of translating proteomic results into clinical practice. The primary aim of this study was to refine the diagnostic power of a panel of candidate salivary biomarkers described in pSS with respect to both healthy volunteers and pathological controls. We also explored the pathogenetic function of the detected putative biomarkers both in the local exocrinopathy and in the systemic inflammatory processes of SS.

Methods

One hundred and eighty patients were included in the study overall. In the first "exploratory phase", we enrolled 40 females with pSS, 40 sex- and age-matched healthy volunteers, 10 patients with sicca non-SS and 15 secondary SS (sSS) patients. The testing cohort of the second "challenge phase" of the study was represented by 75 unselected, consecutive subjects: 19 pSS, 21 healthy volunteers, 10 sicca non-SS and 25 sSS patients. Salivary proteomic analysis was performed combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA) were employed to validate 2DE results. Ingenuity Pathway Analysis (IPA) Knowledge base was adopted to associate candidate biomarkers in a signalling pathogenetic network.

Results

A total of 28, 6, 7 and 12 protein spots were found to be significantly different in pSS samples with respect to healthy volunteers, non-SS sicca syndrome, SSc-sSS and rheumatoid arthritis-sSS, leading to the identification of 15 differently expressed proteins. Among them, α-amylases precursor, carbonic anhydrase VI, β-2 microglobulin, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), epidermal fatty acid binding protein (E-FABP) and immunoglobulin k light chain (IGK-light chain) apparently showed the most significant differences in pSS when compared to healthy volunteers and non-SS pathological controls. On the other hand, as expected, pSS and sSS salivary profiles shared a great number of similarities.

Conclusions

This study demonstrated that salivary fluid might represent a novel ideal milieu for the detection of a diagnostic panel of candidate biomarkers for pSS, and to gain an insight into the pathogenetic processes underlying glandular and systemic autoimmune disorders.