Pediatric Brain Tumors: Signatures from the Intact Proteome

The present investigation aimed to explore the intact proteome of tissues of pediatric brain tumors of different WHO grades and localizations, including medulloblastoma, pilocytic astrocytoma, and glioblastoma, in comparison with the available data on ependymoma, to contribute to the understanding of the molecular mechanisms underlying the onset and progression of these pathologies. Tissues have been homogenized in acidic water–acetonitrile solutions containing proteases inhibitors and analyzed by LC–high resolution MS for proteomic characterization and label-free relative quantitation. Tandem MS spectra have been analyzed by either manual inspection or software elaboration, followed by experimental/theoretical MS fragmentation data comparison by bioinformatic tools. Statistically significant differences in protein/peptide levels between the different tumor histotypes have been evaluated by ANOVA test and Tukey’s post-hoc test, considering a p-value > 0.05 as significant. Together with intact protein and peptide chains, in the range of molecular mass of 1.3–22.8 kDa, several naturally occurring fragments from major proteins, peptides, and proteoforms have been also identified, some exhibiting proper biological activities. Protein and peptide sequencing allowed for the identification of different post-translational modifications, with acetylations, oxidations, citrullinations, deamidations, and C-terminal truncations being the most frequently characterized. C-terminal truncations, lacking from two to four amino acid residues, particularly characterizing the β-thymosin peptides and ubiquitin, showed a different modulation in the diverse tumors studied. With respect to the other tumors, medulloblastoma, the most frequent malignant brain tumor of the pediatric age, was characterized by higher levels of thymosin β4 and β10 peptides, the latter and its des-IS form particularly marking this histotype. The distribution pattern of the C-terminal truncated forms was also different in glioblastoma, particularly underlying gender differences, according to the definition of male and female glioblastoma as biologically distinct diseases. Glioblastoma was also distinguished for the peculiar identification of the truncated form of the α-hemoglobin chain, lacking the C-terminal arginine, and exhibiting oxygen-binding and vasoconstrictive properties different from the intact form. The proteomic characterization of the undigested proteome, following the top-down approach, was challenging to originally investigate the post-translational events that differently characterize pediatric brain tumors. This study provides a contribution to elucidate the molecular profiles of the solid tumors most frequently affecting the pediatric age, and which are characterized by different grades of aggressiveness and localization.     

Influence of meat quality and NaCl percentage on glutathione peroxidase activity and values for acid-reactive substances of raw and dry-cured Longissimus dorsi

Fresh and dry-cured porcine loins (Longissimus dorsi) were analysed for glutathione peroxidase (GSHPx) activity and acid reactive substances (TBARS) in order to assess the influence of meat quality and salt (NaCl) concentration on oxidative stability. The results showed lower GSHPx activity and higher TBA levels in normal meat than in PSE meat indicating a higher oxidative stress in normal meat quality. GSHPx remained active at the end of the curing process. Higher salt concentration led to lower enzyme activity and TBARS values prompted the thought of a double role of NaCl as an enzyme inhibitor and as an antioxidant molecule     

Competitive Effects of Trichloroethylene on Cr(VI) Removal by Zero-Valent Iron

Eleven columns were set up under various groundwater geochemistry conditions to investigate the competitive effect of trichloroethylene (TCE) on hexavalent chromium [Cr(VI)] removal by zero-valent iron (Fe0). They were found to be electron competitors in the redox reactions. In the presence of TCE, the Cr(VI) removal capacities of Fe0 were decreased by about 40% when compared with their respective Cr(VI) removal capacities with identical groundwater geochemistry but without TCE. The specific reaction rate constant (kSA) of TCE was decreased by about 50% when Cr(VI) was singly applied. The kSA of TCE was further decreased by 75% in the presence of both Cr(VI) and carbonate. However, there was no apparent effect on the kSA of TCE when Cr(VI), hardness and carbonate were all present. It revealed that TCE was a stronger electron competitor of Cr(VI) and the degradation of TCE became more favorable when both hardness and carbonate were present. This suggests that the passivated precipitates formed on the Fe0 surface in the presence of both hardness and carbonate may significantly affect the Cr(VI) removal by Fe0 but has insignificant effect on the TCE removal.     

Increased globotriaosylceramide in familial dysautonomia

Familial Dysautonomia (FD) is an autosomal recessive disease of unknown etiology, occurring primarily in Ashkenazi Jews. Patients are neurologically impaired, with deficits primarily in autonomic and sensory functions. The biochemical and genetic defects have remained elusive, precluding carrier detection and prenatal diagnosis. High-performance liquid chromatography data indicated up to a threefold increase in the neutral glycosphingolipid globotriaosylceramide in Dysautonomic fibroblasts and lymphoblasts. Total ganglioside values, measured by colorimetric, fluorometric or specific sodium borohydride incorporation, were decreased. Affected fibroblasts exhibited a range of pleomorphic phenotypes, such that the usual swirl-like confluent growth pattern of normal fibroblasts was distorted to varying degrees, suggesting abnormalities in the FD plasma membrane, possibly affecting cell-cell contacts. The glycosphingolipid increase could not be accounted for on the basis of markedly decreased α-galactosidase activity, as in Fabry's disease, where patients also display decreased autonomic function.     

Structures and stabilities of small DNA dumbbells with Watson-Crick and Hoogsteen base pairs

The structures and stabilities of cyclic DNA octamers of different sequences have been studied by NMR and CD spectroscopy and by restrained molecular dynamics. At low oligonucleotide concentrations, some of these molecules form stable monomeric structures consisting of a short stem of two base pairs connected by two mini-loops of two residues. To our knowledge, these dumbbell-like structures are the smallest observed to date. The relative stabilities of these cyclic dumbbells have been established by studying their melting transitions. Dumbbells made up purely of GC stems are more stable than those consisting purely of AT base pairs. The order of the base pairs closing the loops also has an important effect on the stabilities of these structures. The NMR data indicate that there are significant differences between the solution structures of dumbbells with G-C base pairs in the stem compared to those with A-T base pairs. In the case of dumbbells with G-C base pairs, the residues in the stem form a short segment of a BDNA helix stabilized by two Watson-Crick base pairs. In contrast, in the case of d, the stem is formed by two A-T base pairs with the glycosidic angles of the adenine bases in a syn conformation, most probably forming Hoogsteen base pairs. Although the conformations of the loop residues are not very well defined, the thymine residues at the first position of the loop are observed to fold back into the minor groove of the stem.     

Multi‐phosphorylation of the Intrinsically Disordered Unique Domain of c‐Src Studied by In‐Cell and Real‐Time NMR Spectroscopy

Intrinsically disordered regions (IDRs) are preferred sites for post‐translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase–phosphatase networks. Here we used real‐time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the “unique domain” of c‐Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin‐dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP‐dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK‐dependent sites, thus suggesting indirect effects of kinase–phosphatase networks. This study provides a proof‐of‐concept of the use of real‐time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.     

Identification of New N‐Acylhomoserine Lactone Signalling Compounds of Dinoroseobacter shibae DFL‐12T by Overexpression of luxI Genes

Bacteria of the Roseobacter clade are widespread in the ocean and occur in many different habitats. In the genome of Dinoroseobacter shibae DFL‐12, luxI homologous genes that encode synthases responsible for the formation of N‐acylhomoserine lactones (AHLs) have been described. These compounds are known autoinducers that regulate several biological traits—namely, flagella formation and cell differentiation—in D. shibae through quorum sensing. The AHLs produced by D. shibae mainly consisted of N‐octadecadienoylhomoserine lactone (C18:2‐AHL) and N‐octadecenoylhomoserine lactone (C18:1‐HSL). In the wild type these AHLs are synthesized only in low abundance. The luxI genes were therefore expressed in Escherichia coli; this resulted in the formation of AHLs mostly different from those found in the D. shibae wild type. A luxI₁‐deficient mutant of D. shibae was then reprovided with an overexpressed luxI₁ gene. This strain produced large amounts of C18:2‐AHL and C18:1‐AHL, allowing full characterization of these compounds by mass spectrometric techniques and derivatization. Synthesis of the proposed structures confirmed that the major compound is (2E,11Z)‐N‐octadeca‐2,11‐dienoylhomoserine lactone ( 6 , C18:2‐HSL), accompanied by (Z)‐N‐octadec‐11‐enoylhomoserine lactone ( 5 , C18:1‐HSL). AHL 6 has not been reported before from other organisms and contains an unusual 2E double bond.     

Mathematical Modeling of Drying of Liquid/Solid Slurries in Steady State One-Dimensional Flow

ABSTRACT

A mathematical model of simultaneous mass, heat and momentum transfer for two-phase flow of a gas and a solid/liquid slurry was developed. The model was applied to calculation of the drying process of coal-water slurry droplets in a gas medium in a steady one-dimensional flow. The model was based on the well-known two-stage drying process for slurry droplets. After the first period of drying, in which the evaporation rate is controlled by the gas phase resistance, the evaporating liquid diffuses through the porous shell (crust) and then, by convection, into the gas medium. Inside the dry external crust of the drop, a wet central core forms, which shrinks as evaporation proceeds. The temperature of the slurry droplet rises. The process ends when the temperature of the dry outer crust reaches the coal ignition temperature in the case of combustion or when the moisture of the particle reaches the final required moisture. The developed model was based on one-dimensional balance equations of mass, energy and momentum for the liquid/solid and gas phases. The system of governing equations was represented by first-order differential equations and solved simultaneously. The numerical solution of the governing equations was obtained using Gear's method. The model permitted calculation     

Failure of silver nanowire transparent electrodes under current flow

Silver nanowire transparent electrodes have received much attention as a replacement for indium tin oxide, particularly in organic solar cells. In this paper, we show that when silver nanowire electrodes conduct current at levels encountered in organic solar cells, the electrodes can fail in as little as 2 days. Electrode failure is caused by Joule heating which causes the nanowires to breakup and thus create an electrical discontinuity in the nanowire film. More heat is created, and thus failure occurs sooner, in more resistive electrodes and at higher current densities. Suggestions to improve the stability of silver nanowire electrodes are given.