Immobilization of allantoinase for the development of an optical biosensor of oxidative stress states

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase—the enzyme responsible for allantoin hydrolization in plants and lower organisms—for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor

Outcome at 2-year of treatment in first-episode psychosis patients who were enrolled in a specialized early intervention program

Treatment in early intervention services (EIS) seems superior to treatment as usual on several outcomes, but the extent of heterogeneity in response is unclear. In this study, treatment response trajectories up to 2 years in first-episode psychosis (FEP) patients enrolled in an Italian early intervention service (EIS) have been quantified. The 24-item Brief Psychiatric Rating Scale (BPRS) was used to quantify treatment response up to 2 years in 129 participants. Conditional growth modeling and latent class growth analysis were used to test changes over time in the BPRS and separation into independent classes over time. Group differences were tested on socio-demographic and clinical variables known to be related to outcome in psychosis. Scores on the BPRS showed a statistically significant decrease in overall scores across all tested models. Four trajectories were identified across 2 years. Most patients showed a progressive decrease in the BPRS scores; a scant fraction showed a more stepped decrease from very high levels of psychopathology. No potential predictor was statistically related to the time course of BPRS scores. Most patients that undergo treatment within an EIS are characterized by amelioration, but patients that have higher baseline scores of psychopathology require more intensive treatment

Protective role of vitamin B6 (PLP) against DNA damage in Drosophila models of type 2 diabetes

Growing evidence shows that improper intake of vitamin B6 increases cancer risk and several studies indicate that diabetic patients have a higher risk of developing tumors. We previously demonstrated that in Drosophila the deficiency of Pyridoxal 5' phosphate (PLP), the active form of vitamin B6, causes chromosome aberrations (CABs), one of cancer prerequisites, and increases hemolymph glucose content. Starting from these data we asked if it was possible to provide a link between the aforementioned studies. Thus, we tested the effect of low PLP levels on DNA integrity in diabetic cells. To this aim we generated two Drosophila models of type 2 diabetes, the first by impairing insulin signaling and the second by rearing flies in high sugar diet. We showed that glucose treatment induced CABs in diabetic individuals but not in controls. More interestingly, PLP deficiency caused high frequencies of CABs in both diabetic models demonstrating that hyperglycemia, combined to reduced PLP level, impairs DNA integrity. PLP-depleted diabetic cells accumulated Advanced Glycation End products (AGEs) that largely contribute to CABs as α-lipoic acid, an AGE inhibitor, rescued not only AGEs but also CABs. These data, extrapolated to humans, indicate that low PLP levels, impacting on DNA integrity, may be considered one of the possible links between diabetes and cancer

Estimating translational selection in Eukaryotic Genomes

Natural selection on codon usage is a pervasive force that acts on a large variety of prokaryotic and eukaryotic genomes. Despite this, obtaining reliable estimates of selection on codon usage has proved complicated, perhaps due to the fact that the selection coefficients involved are very small. In this work, a population genetics model is used to measure the strength of selected codon usage bias, S, in 10 eukaryotic genomes. It is shown that the strength of selection is closely linked to expression and that reliable estimates of selection coefficients can only be obtained for genes with very similar expression levels. We compare the strength of selected codon usage for orthologous genes across all 10 genomes classified according to expression categories. Fungi genomes present the largest S values (2.24–2.56), whereas multicellular invertebrate and plant genomes present more moderate values (0.61–1.91). The large mammalian genomes (human and mouse) show low S values (0.22–0.51) for the most highly expressed genes. This might not be evidence for selection in these organisms as the technique used here to estimate S does not properly account for nucleotide composition heterogeneity along such genomes. The relationship between estimated S values and empirical estimates of population size is presented here for the first time. It is shown, as theoretically expected, that population size has an important role in the operativity of translational selection

Tissue-Specific Differences in Human Transfer RNA Expression

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms

Anti-asthma medication prescribing to children in the Lombardy Region of Italy: chronic versus new users

<p>Abstract</p> <p>Background</p> <p>Although anti-asthma medications are amongst those most frequently under or over prescribed it is generally accepted that prescriptions for such agents can be used as a proxy for disease prevalence. The aims of this study were to estimate prevalence and incidence of childhood asthma in a representative Italian area by analysing three years of anti-asthmatic prescriptions and hospitalizations of subjects with chronic or first time treatment, and to underline appropriateness of therapeutic choices.</p> <p>Methods</p> <p>The analysis involved prescriptions given to 6-17 year olds between 2003 and 2005 in Italy's Lombardy Region. The youths were classified as potential asthmatics, based on the different degree of drug utilization: occasional, low or high users, and grouped as 'new onset' or 'chronic' cases based on the duration of therapy dispensed. The analysis of prescriptions and hospitalization rate of these groups provided an estimate of the 2005 asthma prevalence and incidence and allowed an estimation of the level of appropriateness of treatments.</p> <p>Results</p> <p>During 2005, the estimated incidence of potential asthmatics was 0.8% and the estimated prevalence was 3.5%. When viewed retrospectively for two years, records showed that 47% of potential asthmatics received prescriptions also during 2004 and 30% also during 2003. During the three years considered, 7.5%, 2.8%, and 1.5% of high, low, and occasional users, respectively, were hospitalized for asthma. The most important inappropriateness found was the prescription of long acting beta adrenergics as first time treatment.</p> <p>Conclusions</p> <p>This study allowed a proxy of asthma incidence, prevalence, and severity. The analyses highlighted a low compliance with the guidelines, suggesting that educational interventions are needed to obtain a more rational management of childhood asthma, especially in subjects starting therapy.</p

The Assembly of the Plasmodial PLP Synthase Complex Follows a Defined Course

Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. Methodology/Principal Findings: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. Conclusions/Significance: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows