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.     

A mass spectrometry-based targeted metabolomics strategy of human blastocoele fluid: a promising tool in fertility research

Embryo assessment is currently performed through the analysis of morphology and cleavage rate. Recent studies have sought to identify a correlation between quali-quantitative profiles of small molecules of metabolic interest and the outcome of embryo transfer. Approaches relying on both optical and non-optical spectroscopy have been proposed to non-invasively monitor the embryo culture media. However, the non-invasive approach only offers an indirect strategy to monitor embryos and a turn-around solution to bypass the limits of detection of these analytical techniques. In this paper we pave the way for direct metabolic assessment of embryos through the mass-spectrometry-based analysis of blastocoele fluid, which is withdrawn from the blastocoele cavity prior to cryostorage of blastocysts. We conclude that it is possible to detect most of the metabolites of potential interest right at the very heart of the blastocyst, without disrupting the workflow of a classic laboratory pipeline.     

A proteomic profile of washing fluid from the colorectal tract to search for potential biomarkers of colon cancer

Washing fluid (WF) from the colon rectal tract after surgical resection might represent a first step in obtaining a mixture of proteins derived from the secretion of tumoral epithelial cells potentially involved in the pathological progression of tissue. In this study, we performed a proteomic analysis of colorectal WF to search for potential biomarkers of colon cancer. The outcome of this approach might open the possibility of using WF to screen for the precancerous and early stages of colorectal cancer (CRC). Samples of WFs were obtained during surgery from 35 patients submitted to colon resection for suspicious adenocarcinoma or carcinoma, while the respective controls were obtained by washing the healthy sections. WFs were immediately centrifuged, concentrated and trichloroacetic acid (TCA) was added to obtain protein pellets. After two-dimensional gel electrophoresis (2DE), the protein patterns of malignant samples were compared with respective normal samples. Forty-one protein spots were found to be differentially expressed exhibiting ≥2 fold-change of mean value spot intensities. After mass spectrometry, these protein spots collapsed into 38 different proteins. Interestingly, 19 of the differentially expressed proteins identified in the study corresponded to those suggested as being potential biomarkers of CRC. In accordance with the literature, these proteins showed the same direction of change (up or down for all proteins). Our results suggest that WF has the potential of being a method for the exploration of clinical samples for biomarker and drug target discovery.     

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded^1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes³. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation⁴. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.     

Spatial Genetic Structure of Campanula sabatia, a Threatened Narrow Endemic Species of the Mediterranean Basin

Campanula sabatia is endemic to NW Italy (Liguria) and it was included in the European Red list of endangered species due to the heavy human pressure on its habitat. AFLP markers were used to detect the genetic diversity within and among ten populations (totalling 83 individuals) representing the range of the species. In spite of the limited distribution of this endemic taxon, high levels in percentage of polymorphic bands (PPB), gene diversity (H _S and H _T) and Shannon??s information index (I) were detected both at population (PPB?=?60?%, H _S?=?0.1853, I?=?0.2836) and at species level (PPB?=?100?%, H _T?=?0.2415, I?=?0.3871). The coefficient of genetic differentiation among population (G??_ST) was 0.1935, while the level of gene flow (N?? _m) was estimated to be 2.0832. AMOVA analysis identified a genetic variation within populations of 83?% of the total. Bayesian clustering methods assigned individuals to two geographical groups partly found within the same population, probably due to a high rate of genetic exchange among its populations. Conservation measures are suggested on the basis of the genetic diversity detected to ensure an effective protection for this endemic species.     

Ocean acidification weakens the structural integrity of coralline algae

The uptake of anthropogenic emission of carbon dioxide is resulting in a lowering of the carbonate saturation state and a drop in ocean pH. Understanding how marine calcifying organisms such as coralline algae may acclimatize to ocean acidification is important to understand their survival over the coming century. We present the first long‐term perturbation experiment on the cold‐water coralline algae, which are important marine calcifiers in the benthic ecosystems particularly at the higher latitudes. Lithothamnion glaciale, after three months incubation, continued to calcify even in undersaturated conditions with a significant trend towards lower growth rates with increasing pCO₂. However, the major changes in the ultra‐structure occur by 589 μatm (i.e. in saturated waters). Finite element models of the algae grown at these heightened levels show an increase in the total strain energy of nearly an order of magnitude and an uneven distribution of the stress inside the skeleton when subjected to similar loads as algae grown at ambient levels. This weakening of the structure is likely to reduce the ability of the alga to resist boring by predators and wave energy with severe consequences to the benthic community structure in the immediate future (50 years).     

Identification of cancer stem cells from human glioblastomas: growth and differentiation capabilities and CD133/prominin-1 expression

CD133 can be a marker of tumorigenic CSCs (cancer stem cells) in human GBM (glioblastoma multiforme), although tumorigenic CD133-negative CSCs have been also isolated. Additional evidence indicates that CSCs from GBM exhibit different phenotypes, with increasing interest in the potential significance of the different CSCs with respect to diagnosis, prognosis and the development of novel targets for treatment. We have analysed the expression of CD133 in freshly isolated cells from 15 human GBM specimens. Only 4 of them contained cells positive for AC133 by FACS analysis, and all of them yielded distinct CSC lines, whereas only 6 CSC lines were obtained from the other 11 GBMs. Of these 10 CSCs lines, we further characterized 6 CSC lines. Three CSCs grew as fast-growing neurospheres with higher clonogenic ability, whereas the remaining 3 grew as slow-growing semi-adherent spheres of lower clonogenicity. In addition, the former CSC lines displayed better differentiation capabilities than the latter ones. PCR and Western blot analysis showed that all 6 GBM CSC lines expressed CD133/prominin-1, suggesting that cells negative by FACS analysis may actually represent cells expressing low levels of CD133 undetected by FACS. Nevertheless, all the 6 CSC lines were tumorigenic in nude mice. In conclusion, CSCs from human primary GBMs show different phenotypes and variable levels of CD133 expression, but these parameters did not directly correlate with the tumorigenic potential.     

Kernel texture and hordoindoline patterns in barley (Hordeum vulgare)

Hordoindolines, the tryptophan-rich polypeptides affecting grain hardness in barley, appeared as three pairs of polypeptides in the acidic polyacrylamide gel electrophoresis (A-PAGE) and two-dimensional A-PAGE?×?SDS-PAGE patterns of starch-granule proteins from 18 barley cultivars. On capillary RP-HPLC/nESI-MS/MS spectrometry, one pair of polypeptides was found to correspond to hordoindoline A (HINA), one to hordoindoline B1 (HINB1) and one to hordoindoline B2 (HINB2), the two polypeptides of each pair deriving from post-translational cleavage of a native hordoindoline at different positions at the N-terminus and/or C-terminus. Amongst the barley cultivars analyzed, cvs Hart and Sundance, which were claimed to be unique in lacking the Hina gene coding for HINA, revealed similar Hina coding sequences and accumulated hordoindoline HINA on their starch granules. The amount of total hordoindolines (HINA?+?HINB1?+?HINB2) on the starch granules, as quantified by densitometric scanning of A-PAGE gels, was comparable with that of puroindolines (PINA?+?PINB) in soft-textured wheat. By contrast, the amount of B-type hordoindolines (HINB1 and HINB2 combined) was 50?% lower than that of PINB, suggesting that the absence of barley cultivars with soft kernels is likely due to the reduced amount of B-type hordoindolines accumulated on the starch granules. Approximately 22 and 27?% of the phenotypic variation for kernel hardness in 56 barley cultivars analyzed by the Single Kernel Characterization System (SKCS) were explained by differences in kernel weight and B-type hordoindoline level, respectively. By contrast, the outer husk of barley grain showed no effect on the SKCS index.