Urinary metabolite quantitative trait loci in children and their interaction with dietary factors.

Human metabolism is influenced by genetic and environmental factors. Previous studies have identified over 23 loci associated with more than 26 urine metabolites levels in adults, which are known as urinary metabolite quantitative trait loci (metabQTLs). The aim of the present study is the identification for the first time of urinary metabQTLs in children and their interaction with dietary patterns. Association between genome-wide genotyping data and 44 urine metabolite levels measured by proton nuclear magnetic resonance spectroscopy was tested in 996 children from the Human Early Life Exposome project. Twelve statistically significant urine metabQTLs were identified, involving 11 unique loci and 10 different metabolites. Comparison with previous findings in adults revealed that six metabQTLs were already known, and one had been described in serum and three were involved the same locus as other reported metabQTLs but had different urinary metabolites. The remaining two metabQTLs represent novel urine metabolite-locus associations, which are reported for the first time in this study [single nucleotide polymorphism (SNP) rs12575496 for taurine, and the missense SNP rs2274870 for 3-hydroxyisobutyrate]. Moreover, it was found that urinary taurine levels were affected by the combined action of genetic variation and dietary patterns of meat intake as well as by the interaction of this SNP with beverage intake dietary patterns. Overall, we identified 12 urinary metabQTLs in children, including two novel associations. While a substantial part of the identified loci affected urinary metabolite levels both in children and in adults, the metabQTL for taurine seemed to be specific to children and interacted with dietary patterns.

Microproteins encoded by noncanonical ORFs are a major source of tumor-specific antigens in a liver cancer patient meta-cohort.

The expression of tumor-specific antigens during cancer progression can trigger an immune response against the tumor. Here, we investigate if microproteins encoded by noncanonical open reading frames (ncORFs) are a relevant source of tumor-specific antigens. We analyze RNA sequencing data from 117 hepatocellular carcinoma (HCC) tumors and matched healthy tissue together with ribosome profiling and immunopeptidomics data. Combining human leukocyte antigen-epitope binding predictions and experimental validation experiments, we conclude that around 40% of the tumor-specific antigens in HCC are likely to be derived from ncORFs, including two peptides that can trigger an immune response in humanized mice. We identify a subset of 33 tumor-specific long noncoding RNAs expressing novel cancer antigens shared by more than 10% of the HCC samples analyzed, which, when combined, cover a large proportion of the patients. The results of the study open avenues for extending the range of anticancer vaccines.

Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.

Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis.

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative beta-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric beta-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40 degrees C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first beta-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.

Manometric transduction in enzyme biosensors.

The combination of enzymatic recognition and manometric transduction is explored, using enzymes that consume or evolve a gas with low solubility in aqueous media. A design is discussed whereby change in partial pressure of a gas in the headspace is related to the turnover of analyte by the enzyme. Headspace and sample volume dimensions are considered, demonstrating the influence of flux at the air-water interface. The relative importance of diffusion and reaction for the enzyme solution is shown. When enzyme kinetics dominate, the concentration gradient is low and the overall kinetics are determined by the total amount of active enzyme, reducing either enzyme concentration or enzyme layer thickness will reduce the diffusion limitation. A Teflon-enzyme composite is presented to allow a reuseable immobilised enzyme preparation and a disc with stirring magnet identified as an efficient configuration. A glucose oxidase system was tested in the monitoring of glucose consumption during fermentation. Application to other enzyme systems is discussed.

An electrochemical method for simultaneous detection and identification of Escherichia coli, Staphylococcus aureus and Salmonella choleraesuis using a glucose oxidase-peroxidase composite biosensor.

Quantification of bacterial pollution by amperometric detection at 0.0 V of glucose consumption at a graphite-Teflon-glucose oxidase-peroxidase-ferrocene composite biosensor under flow injection conditions is reported. Using Escherichia coli as the model bacterium, the composition of the growing medium was optimized. A constant glucose concentration of 4.0 x 10(-4) M was added to the culture medium. The relative response to glucose, expressed as the ratio between the amperometric signal and the signal at incubation time t = 0 multiplied by 100, as a function of E. coli concentration, showed a typical behaviour. Limits of detection of 6.5 x 10(2) or 6.5 cfu mL(-1) were achieved after 3 or 7 h of incubation, respectively, with no pre-concentration step. The detection of bacteria did not affect the lifetime of the biosensor. The feasibility of the detection of Staphylococcus aureus and Salmonella choleraesuis throughout the glucose consumption measurement at the composite biosensor is also demonstrated. The capability of bacterial identification by evaluation of bacterial growth in the culture medium containing the antibiotics polymyxin B, vancomycin, erythromycin, bacitracin, chloramphenicol, tetracycline and ciprofloxacin, was investigated. Each micro-organism tested exhibited a different antibiotic susceptibility profile, thus suggesting the possibility of bacteria differentiation. A rapid methodology for screening of bacteria is proposed.

In-a-day electrochemical detection of coliforms in drinking water using a tyrosinase composite biosensor.

A rapid method for the detection of fecal contamination in water based on the use of a tyrosinase composite biosensor for improved amperometric detection of beta-galactosidase activity is reported. The method relies on the detection of phenol released after the hydrolysis of phenyl beta-D-galactopyranoside (PG) by beta-galactosidase. Under the optimized PG concentration and pH (4.0) values, a detection limit of 1.2x10(-3) unit of beta-galactosidase/mL-1 was obtained. The capability of the sensor for the detection of Escherichia coli was evaluated using polymyxin B sulfate to allow permeabilization of the bacteria membrane. A detection limit of 1x10(6) cfu of E. coli mL-1 was obtained with no preconcentration or pre-enrichment steps. To improve the analytical characteristics for bacteria detection, the processes involving galactosidase induction during incubation and membrane permeabilization were optimized. Using 0.25 mM isopropyl beta-D-thiogalactopyranoside for the enzyme activity induction, and 10 microg mL-1 polymyxin B sulfate as permeabilizer agent, it was possible to detect bacteria concentrations as low as 10 cfu mL-1 after 5 h of enrichment. The possibility of detecting E. coli at the required levels for drinking water quality assessment (1 cfu/100 mL) is demonstrated, the time of analysis being shorter than 6.5 h and involving a simple methodology.