Focused screening to identify new adenosine kinase inhibitors

Adenosine kinase (AdK) is a key player in controlling intra- and extracellular concentrations of the signaling molecule adenosine. Extensive evidence points to an important role of AdK in several diseases, and suggests that AdK inhibition might be a promising therapeutic strategy.The development of a new AdK assay and subsequent screening of part of our focused compound library led to the identification of 12 hit compounds (hit rate of 6%) representing six new classes of non-nucleoside human AdK inhibitors. The most potent inhibitor 1 displayed a K_i value of 184 nM. Compound screening with a newly developed assay was useful and efficient for discovering novel AdK inhibitors which may serve as lead structures for developing drugs for adenosine augmentation therapy.     

Short-term fructose ingestion affects the brain independently from establishment of metabolic syndrome

Chronic fructose ingestion is linked to the global epidemic of metabolic syndrome (MetS), and poses a serious threat to brain function. We asked whether a short period (one week) of fructose ingestion potentially insufficient to establish peripheral metabolic disorder could impact brain function. We report that the fructose treatment had no effect on liver/body weight ratio, weight gain, glucose tolerance and insulin sensitivity, was sufficient to reduce several aspects of hippocampal plasticity. Fructose consumption reduced the levels of the neuronal nuclear protein NeuN, Myelin Basic Protein, and the axonal growth-associated protein 43, concomitant with a decline in hippocampal weight. A reduction in peroxisome proliferator-activated receptor gamma coactivator-1 alpha and Cytochrome c oxidase subunit II by fructose treatment is indicative of mitochondrial dysfunction. Furthermore, the GLUT5 fructose transporter was increased in the hippocampus after fructose ingestion suggesting that fructose may facilitate its own transport to brain. Fructose elevated levels of ketohexokinase in the liver but did not affect SIRT1 levels, suggesting that fructose is metabolized in the liver, without severely affecting liver function commensurable to an absence of metabolic syndrome condition. These results advocate that a short period of fructose can influence brain plasticity without a major peripheral metabolic dysfunction.     

Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis

Glutathione (GSH) and GSH‐related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)‐resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ‐resistant cells than in sensitive cells. Furthermore, silencing GSR in drug‐resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over‐expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro‐oxidant L‐buthionine ‐sulfoximine. The modulation of redox state by GSH or L‐buthionine –sulfoximine regulated GSR‐mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress‐free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment.

     

High vulnerability of the heart and liver to 3‐hydroxypalmitic acid–induced disruption of mitochondrial functions in intact cell systems

Patients affected by long‐chain 3‐hydroxyacyl‐CoA dehydrogenase (LCHAD) deficiency predominantly present severe liver and cardiac dysfunction, as well as neurological symptoms during metabolic crises, whose pathogenesis is still poorly known. In this study, we demonstrate for the first time that pathological concentrations of 3‐hydroxypalmitic acid (3HPA), the long‐chain hydroxyl fatty acid (LCHFA) that most accumulates in LCHAD deficiency, significantly decreased adenosine triphosphate‐linked and uncoupled mitochondrial respiration in intact cell systems consisting of heart fibers, cardiomyocytes, and hepatocytes, but less intense in diced forebrain. 3HPA also significantly reduced mitochondrial Ca²⁺ retention capacity and membrane potential in Ca²⁺‐loaded mitochondria more markedly in the heart and the liver, with mild or no effects in the brain, supporting a higher susceptibility of the heart and the liver to the toxic effects of this fatty acid. It is postulated that disruption of mitochondrial energy and Ca²⁺ homeostasis caused by the accumulation of LCHFA may contribute toward the severe cardiac and hepatic clinical manifestations observed in the affected patients.     

How to select a probiotic? A review and update of methods and criteria

International competition within the dairy market and increasing public awareness about the importance of functional food consumption are providing new challenges for innovation in the probiotic sector. In this context, countless references are currently dedicated to the selection and characterization of new species and more specific strains of probiotic bacteria. In general, these studies adopt basic selection criteria established by the World Health Organization (WHO), including host-associated stress resistance, epithelium adhesion ability, and antimicrobial activity. These aspects are applied to ensure that the candidate probiotic could withstand the stressful conditions of the human digestive system and exert functional proprieties. However, it cannot be assumed that these novel microbial strains are capable of offering several biological benefits attributed to probiotics. Additionally, safety-associated selection criteria, such as plasmid-associated antibiotic resistance spreading and enterotoxin production, are often neglected. This article reviews the recent developments in the processes, strategies, and methods, such as anticarcinogenic, antidepression, antianxiety, antiobesity, antidiabetic, immunostimulatory, and cholesterol-lowering assessments, to select probiotic strains with the ultimate objective of assisting future probiotic microbe evaluation studies.     

Functional classification of protein structures by local structure matching in graph representation

As a result of high‐throughput protein structure initiatives, over 14,400 protein structures have been solved by Structural Genomics (SG) centers and participating research groups. While the totality of SG data represents a tremendous contribution to genomics and structural biology, reliable functional information for these proteins is generally lacking. Better functional predictions for SG proteins will add substantial value to the structural information already obtained. Our method described herein, Graph Representation of Active Sites for Prediction of Function (GRASP‐Func), predicts quickly and accurately the biochemical function of proteins by representing residues at the predicted local active site as graphs rather than in Cartesian coordinates. We compare the GRASP‐Func method to our previously reported method, Structurally Aligned Local Sites of Activity (SALSA), using the Ribulose Phosphate Binding Barrel (RPBB), 6‐Hairpin Glycosidase (6‐HG), and Concanavalin A‐like Lectins/Glucanase (CAL/G) superfamilies as test cases. In each of the superfamilies, SALSA and the much faster method GRASP‐Func yield similar correct classification of previously characterized proteins, providing a validated benchmark for the new method. In addition, we analyzed SG proteins using our SALSA and GRASP‐Func methods to predict function. Forty‐one SG proteins in the RPBB superfamily, nine SG proteins in the 6‐HG superfamily, and one SG protein in the CAL/G superfamily were successfully classified into one of the functional families in their respective superfamily by both methods. This improved, faster, validated computational method can yield more reliable predictions of function that can be used for a wide variety of applications by the community.     

Determinants of bryophyte species composition and diversity on the Great Alvar of Öland,Sweden

Factors driving the species richness and distribution of bryophytes are poorly studied and not well understood, particularly in grasslands. We analysed the occurrence of bryophyte species and variation in species richness across 674 plots (0.5?m?×?0.5?m) in alvar vegetation (grassland on limestone pavement with thin or no soil) on Öland (Sweden) in relation to substrate characteristics and chemistry, inundation frequency, grazing pressure and geographical variables. We found 148 taxa, including 11 nationally red-listed ones. Species richness per plot was significantly associated with substrate type, positively associated with pH and grazing intensity, but negatively associated with soil depth. However, richness of species typical of, or restricted to, alvar habitats responded differently to richness of species more common in other habitats. Typical alvar species were favoured by high pH, shallow soil and low phosphate availability, while generalists preferred relatively low pH, higher phosphate availability and organic or mull soil types. Distance from the alvar margin had only weak effects. Concerning the effects on individual species and community composition, inundation frequency and pH were found to have the largest effects, although other factors (substrate type, soil depth, bare soil, bare stone, phosphate availability and grazing pressure) were more important for some individual species, stressing the importance of microsite variability and variability in management for regional species richness. From a conservation perspective, it is concluded that grazing is generally positive whilst factors increasing phosphate availability may disadvantage the typical alvar species, and proximity to the alvar margin is not a major problem.     

Identification of phosphate granules occurring in seedling tissue of two palm species (Phoenix dactylifera and Washingtonia filifera)

Haustoria of two palm species, Phoenix dactylifera L. (date) and Washingtonia filifera (Lindl.) Wendl were carefully dissected from seeds, and the ultrastructural characteristics of the large, electron-opaque granules present in the cells of this tissue were compared using standard aldehyde and OsO₄ fixations. In Washingtonia, the granules were smaller than those in date and were more variable in size and presence of non-opaque inclusions. All granules appeared to be membrane bounded although they often filled the bounded space. No protein, lipid, carbohydrate or tannins were found in the granules by standard staining procedures. The granules stained positively with two different metallic-phosphate stains which contained either bismuth or lead. Energy dispersive X-ray microprobe analysis, done on aldehyde-fixed granules and those stained with both phosphate stains, confirmed the fact that phosphorus and calcium were present in the granules. The granules also bound the metallic stains as expected. All procedures consistently confirmed the presence of phosphate in the granules. The data are most consistent with the hypothesis that the granules are composed of polyphosphate.Abbreviations and symbols EDAX energy-dispersive X-ray microanalysis - K K shell peak - K K shell peak - L L shell peak - L L shell peak - M M shell peak