MALDI-TOF MS proteomic phenotyping of filamentous and other fungi from clinical origin

Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance.     

Cholesterol metabolism differs after statin therapy according to the type of hyperlipemia

AimNon-cholesterol sterols reflect cholesterol metabolism. Statins reduce cholesterol synthesis usually with a rise in cholesterol absorption. Common hyperlipemias have shown different patterns of cholesterol metabolism. We evaluated whether cholesterol absorption and synthesis may differ after statin therapy in primary hyperlipemias.Main methodsWe determined lipid profile, apoprotein B and serum sterols (lathosterol, sitosterol, campesterol by gas chromatography/mass spectrometry) before and after statins in 80 untreated hyperlipemic patients, 40 with polygenic hypercholesterolemia (PH) and 40 with familial combined hyperlipemia (FCH).Key findingsAt baseline in FCH lathosterol was significantly higher while campesterol and sitosterol were significantly lower than in PH. After statins, the reduction in LDL-C did not significantly differ between the two groups; in PH there was a significant decrease of lathosterol from 96.1 to 52.6 102 μmol/mmol cholesterol (p = 0.0001) with no significant modifications in campesterol and sitosterol; on the opposite, in FCH lathosterol decreased from 117 to 43 102 μmol/mmol cholesterol (p = 0.0001) and campesterol and sitosterol significantly increased from 38 to 48 102 μmol/mmol cholesterol (p = 0.0001), and from 75 to 86 102 μmol/mmol cholesterol, (p = 0.022), respectively. After statin therapy only in FCH Δ-LDL-C showed a significant inverse correlation with Δ-sitosterol and with Δ-campesterol.SignificancePrimary hyperlipemias show different patterns of response to statins: in PH LDL reduction appears completely “synthesis inhibition” dependent, while in FCH LDL decrease appears to be synthesis dependent, partially limited by absorption increase. Studying cholesterol metabolism before and after hypolipemic therapy might be useful in identifying the best tailored treatment.     

Toxigenic Clostridium difficile PCR ribotypes from wastewater treatment plants in southern Switzerland

The occurrence of Clostridium difficile in nine wastewater treatment plants in the Ticino Canton (southern Switzerland) was investigated. The samples were collected from raw sewage influents and from treated effluents. Forty-seven out of 55 characterized C. difficile strains belonged to 13 different reference PCR ribotypes (009, 010, 014, 015, 039, 052, 053, 066, 070, 078, 101, 106, and 117), whereas 8 strains did not match any of those available in our libraries. The most frequently isolated ribotype (40%) was 078, isolated from six wastewater treatment plants, whereas ribotype 066, a toxigenic emerging ribotype isolated from patients admitted to hospitals in Europe and Switzerland, was isolated from the outgoing effluent of one plant. The majority of the isolates (85%) were toxigenic. Forty-nine percent of them produced toxin A, toxin B, and the binary toxin (toxigenic profile A(+) B(+) CDT(+)), whereas 51% showed the profile A(+) B(+) CDT(-). Interestingly, eight ribotypes (010, 014, 015, 039, 066, 078, 101, and 106) were among the riboprofiles isolated from symptomatic patients admitted to the hospitals of the Ticino Canton in 2010. Despite the limitation of sampling, this study highlights that toxigenic ribotypes of C. difficile involved in human infections may occur in both incoming and outgoing biological wastewater treatment plants. Such a finding raises concern about the possible contamination of water bodies that receive wastewater treatment plant effluents and about the safe reuse of treated wastewater.     

Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.     

Mitochondrial calcium homeostasis as potential target for mitochondrial medicine

Mitochondria are crucial in different intracellular pathways of signal transduction. Mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions, ranging from energy production to cell death. The fine modulation of mitochondrial calcium (Ca(2+)) homeostasis plays a fundamental role in many of the processes involving this organelle. When mitochondrial Ca(2+) homeostasis is compromised, different pathological conditions can occur, depending on the cell type involved. Recent data have shed light on the molecular identity of the main proteins involved in the handling of mitochondrial Ca(2+) traffic, opening fascinating and ambitious new avenues for mitochondria-based pharmacological strategies.     

Cerebrospinal fluid top-down proteomics evidenced the potential biomarker role of LVV- and VV-hemorphin-7 in posterior cranial fossa pediatric brain tumors

Posterior cranial fossa is the most frequent location of pediatric brain tumors. Its diagnosis is currently performed by postsurgery histopathology and the identification of biomarkers in cerebrospinal fluid (CSF) could provide a less invasive tool. Patient CSF was collected during surgery before the tumor removal (PRE-CSF) and 6 days after the resection (POST-CSF) and analyzed by top down LC-MS proteomics for comparison. The PRE-CSFs generally exhibited a less complex LC-MS profile than the relative POST-CSFs suggesting a suppressive role of the tumor toward proteins and peptides production or release. Particularly, a panel of peptides, identified as alpha- and beta-hemoglobin chains fragments, were generally absent in the PRE-CSF and present in the POST ones independently from contaminant blood hemoglobin. Among them, the LVV- and VV-hemorphin-7 showed the most repeatable trend and with a few remarkable exceptions: their unusual absence in POST surgery CSF was in fact interestingly correlated to the presence of tumor in the patient despite surgery due to metastases or to subtotal resection. These results ascribed a relevant biological role to LVV- and VV-h7 peptides in the disease and a strong potential as biomarkers. Their analysis in POST surgery CSF could be used to predict patient prognosis.     

Is cancer a metabolic rebellion against host aging?: In the quest for immortality,tumor cells try to save themselves by boosting mitochondrial metabolism

Aging drives large systemic reductions in oxidative mitochondrial function, shifting the entire body metabolically toward aerobic glycolysis, a.k.a, the Warburg effect. Aging is also one of the most significant risk factors for the development of human cancers, including breast tumors. How are these two findings connected? One simplistic idea is that cancer cells rebel against the aging process by increasing their capacity for oxidative mitochondrial metabolism (OXPHOS). Then, local and systemic aerobic glycolysis in the aging host would provide energy-rich mitochondrial fuels (such as L-lactate and ketones) to directly “fuel” tumor cell growth and metastasis. This would establish a type of parasite-host relationship or “two-compartment tumor metabolism,” with glycolytic/oxidative metabolic coupling. The cancer cells (“the seeds”) would flourish in this nutrient-rich microenvironment (“the soil”), which has been fertilized by host aging. In this scenario, cancer cells are only trying to save themselves from the consequences of aging by engineering a metabolic mutiny, through the amplification of mitochondrial metabolism. We discuss the recent findings of Drs. Ron DePinho (MD Anderson) and Craig Thomspson (Sloan-Kettering) that are also consistent with this new hypothesis, linking cancer progression with metabolic aging. Using data mining and bioinformatics approaches, we also provide key evidence of a role for PGC1a/NRF1 signaling in the pathogenesis of (1) two-compartment tumor metabolism and (2) mitochondrial biogenesis in human breast cancer cells.Key words: aging, mitochondria, cancer metabolism, autophagy, mitophagy, aerobic glycolysis, oxidative phosphorylation, Metformin, drug resistance, chemoresistance, Warburg effect, metabolic compartments, parasite, PGC1a, PGC1b, NRF1, two-compartment tumor metabolism     

Metabolic effects of the iodothyronine functional analogue TRC150094 on the liver and skeletal muscle of high-fat diet fed overweight rats: an integrated proteomic study

A novel functional iodothyronine analogue, TRC150094, which has a much lower potency toward thyroid hormone receptor (α1/β1) activation than triiodothyronine, has been shown to be effective at reducing adiposity in rats simultaneously receiving a high-fat diet (HFD). Here, by combining metabolic, functional and proteomic analysis, we studied how the hepatic and skeletal muscle phenotypes might respond to TRC150094 treatment in HFD-fed overweight rats. Drug treatment increased both the liver and skeletal muscle mitochondrial oxidative capacities without altering mitochondrial efficiency. Coherently, in terms of individual respiratory in-gel activity, blue-native analysis revealed an increased activity of complex V in the liver and of complexes II and V in tibialis muscle in TCR150094-treated animals. Subsequently, the identification of differentially expressed proteins and the analysis of their interrelations gave an integrated view of the phenotypic/metabolic adaptations occurring in the liver and muscle proteomes during drug treatment. TRC150094 significantly altered the expression of several proteins involved in key liver metabolic pathways, including amino acid and nitrogen metabolism, and fructose and mannose metabolism. The canonical pathways most strongly influenced by TRC150094 in tibialis muscle included glycolysis and gluconeogenesis, amino acid, fructose and mannose metabolism, and cell signaling. The phenotypic/metabolic influence of TRC150094 on the liver and skeletal muscle of HFD-fed overweight rats suggests the potential clinical application of this iodothyronine analogue in ameliorating metabolic risk parameters altered by diet regimens.