A comparative study of phenols composition,antioxidant, and antifungal potency of leaves extract from five Moroccan Vitis vinifera L. varieties

Vitis vinifera (L) crops, covers an area of about 54,000 ha corresponding to a production of 230,000 t of grapes, representing 5.5% of the national perennial corps. However, this plant is sensitive to various ravages and diseases, among others, the cryptogrammic by fungi. The degrading enzymes of the cell wall are among the factors of pathogenicity the more important than fungi use to colonize their host plants. In this study, the first step was devoted to the antioxidant activity and phenols composition (phenylalanine-ammonia-lyase [PAL], tyrosine-ammonia-lyase [TAL], flavonoids, and tannins) as well as high-performance liquid chromatography analysis in five vineyard leaves extract, which are “Superior, Early sweet and Flame” (Allochthonous), “Abbou and Adari” (endemic). In the second step, the lytic activities “pectinase, polygalacturonase, xylanase, mannanase, glucanase, and cellulase” were investigated among six epiphytic fungus to compare their hydrolytic aggressiveness. Subsequently, as a third part, phenols antifungal potency and Trichoderma biocontrol were displayed in the study. The phenolic compounds observed in the attributes leaves were different among varieties (p < .05), and show that the endemic variety “Abbou” leads the phytochemical amount, which has the highest quantities of PAL, and TAL, as well as the DPPH and FRAP antioxidant activity, while the introduced variety show a higher amount in total phenol and flavonoids. The polygalacturonase, mannanase, and pectinase activities are probably the main factors responsible for the aggressiveness of the studied fungus. Phenols potency showed a good mycelial inhibition with 82.09% as compared to the biocontrol (64.45%). Given the efficacy of the phytochemical control in this study, the phenols could be a part of integrated management strategies for sustainable control of vineyard disease.     

Towards stable diagnostic setups in clinical proteomics: Absolute quantitation of peptide biomarkers using MALDI‐TOF‐MS

In routine clinical diagnostics, peptide biomarkers are most commonly quantified using immunological techniques but these methods often lack sensitivity and/or specificity. Hence, quantitative mass spectrometry detection is desirable as an alternative diagnostic tool. To date, quantitative mass spectrometry is mostly based on ESI‐MS coupled to LC, requiring highly sophisticated instrumentation and knowledge and is time consuming and expensive. In contrast, MALDI‐TOF‐MS is a very simple, sensitive and rapid method for the detection of peptide biomarkers. However, the infeasibility of absolute quantification has been a tremendous handicap to the use of MS in stable clinical diagnostics. Here, we describe the development of a technical platform based on ClinProt particles and heavy‐isotope internal peptide standards for the fast and reliable preparation of samples. This combines the advantages of MALDI‐TOF as a read‐out system with absolute quantitation of peptide biomarkers. As a proof‐of‐concept, this platform was successfully employed for the absolute determination of the concentration of the highly abundant serum peptide des‐Ala‐Fibrinopeptide A in 45 serum samples from healthy donors. Such technology essentially contributes to the development of a stable MALDI‐TOF‐MS‐based clinical assay.     

6-phosphofructokinase from Pichia pastoris: purification,kinetic and molecular characterization of the enzyme

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.     

Proteomic analysis to improve adequacy of hemo- and peritoneal dialysis: Removal of small and high molecular weight proteins with high- and low-flux filters or a peritoneal membrane

The reduction of morbidity and mortality in patients undergoing hemo- or peritoneal dialysis is strongly related to an efficient and selective clearance of uremic toxins. We used proteomics methods to analyze and further characterize the dialytic removal of still undefined middle and high molecular weigh proteins as a basis for further improvement of dialysis assessment. Dialysates from 26 hemodialysis patients treated with different types of low- (F6HPS?) and high-flux (FX80?, APS650?, FX60?) filters as well as peritoneal fluids from 10 continuous ambulatory peritoneal dialysis (CAPD) patients were analyzed by SELDI-TOF and 2-DE. The protein patterns showed selective differences in the proteins cleared depending on the dialysis method used and the filter membrane. While SELDI analyses of dialysates from the F6HPS revealed almost no protein clearance, high-flux filters and CAPD dialysates showed protein release of different molecular weight ranges. Furthermore, 2-DE and MS analysis identified 48 different proteins from the dialysate of high-flux filters and 21 from peritoneal dialysis fluids. In F6HPS dialysates, however, only few proteins could be identified.     

Whole cell profiling and identification of galectin-1 as a potential marker of renal cell carcinoma

For most cancers, the patient's prognosis improves dramatically if the disease is detected at an early stage. Although advancements in imaging technology have improved early detection, many cancers remain undetected until it is too late for curative intervention. We have established, for the first time, expression difference mapping analysis of whole cell proteins from renal cell carcinoma (RCC) cell lines using ProteinChip technology. A total of 20 different RCC cell lines were cultured in vitro directly on ProteinChip arrays for 24 h. Direct MS analysis of proteins from the attached cells showed identical protein profiles by all analysed RCC lines. Comparative on‐chip analysis of isolated malignant cells from native tumour specimens revealed protein patterns highly similar to those from the continuous RCC lines. However, cultured primary cortex cells showed specific protein differences. Differential protein profiling of isolated cytosolic and enriched membrane fractions from the RCC lines revealed that the protein pattern of the membrane proteins included or were identical to those of the entire cells. Proteomics analysis of the chip‐binding membrane fractions allowed the identification of three forms of galectin‐1 as potential RCC marker. ProteinChip analysis with a bound‐specific antibody certified that galectin‐1 could be an RCC marker. Immunostaining methods confirmed the overexpression of galectin‐1 in renal carcinoma in comparison to healthy tissue.     

Metabolomic Profiling of Blood-Derived Microvesicles in Breast Cancer Patients

Malignant cells differ from benign ones in their metabolome and it is largely unknown whether this difference is reflected in the metabolic profile of their microvesicles (MV), which are secreted into the blood of cancer patients. Here, they are present together with MV from the various blood and endothelial cells. Harvesting MV from 78 breast cancer patients (BC) and 30 controls, we characterized the whole blood MV metabolome using targeted and untargeted mass spectrometry. Especially (lyso)-phosphatidylcholines and sphingomyelins were detected in a relevant abundance. Eight metabolites showed a significant discriminatory power between BC and controls. High concentrations of lysoPCaC26:0 and PCaaC38:5 were associated with shorter overall survival. Comparing BC subtype-specific metabolome profiles, 24 metabolites were differentially expressed between luminal A and luminal B. Pathway analysis revealed alterations in the glycerophospholipid metabolism for the whole cancer cohort and in the ether lipid metabolism for the molecular subtype luminal B. Although this mixture of blood-derived MV contains only a minor number of tumor MV, a combination of metabolites was identified that distinguished between BC and controls as well as between molecular subtypes, and was predictive for overall survival. This suggests that these metabolites represent promising biomarkers and, moreover, that they may be functionally relevant for tumor progression.     

Urine proteomics in kidney and urogenital diseases: Moving towards clinical applications

To date, multiple biomarker discovery studies in urine have been conducted. Nevertheless, the rate of progression of these biomarkers to qualification and even more clinical application is extremely low. The scope of this article is to provide an overview of main clinically relevant proteomic findings from urine focusing on kidney diseases, bladder and prostate cancers. In addition, approaches for promoting the use of urine in clinical proteomics including potential means to facilitate the validation of existing promising findings (biomarker candidates identified from previous studies) and to increase the chances for success for the identification of new biomarkers are discussed.