DNA hybridization on membrane-modified carbon electrodes

The DNA-modified membrane electrode was prepared by casting a mixture of nitrocellulose (NC) with target DNA (tDNA) in organic solvent on glassy carbon electrode (GCE). Unlabeled polymerase chain reaction (PCR)-amplified human genomic sequence (628 bp) or synthetic oligodeoxynucleotides (ODNs) were used as tDNAs, creating a recognition layer. Biotinylated ODNs were used as hybridization probes to recognize specific nucleotide sequences. The hybridization events were detected via an enzyme-linked electrochemical assay involving binding of streptavidin-coupled alkaline phosphatase (SALP) to the biotin labels of the probe bound to tDNA. After the probe hybridization and SALP binding, the electrode was immersed into an electroinactive enzyme substrate (1-naphthyl phosphate). The alkaline phosphatase converted the inactive substrate into electroactive 1-naphthol that penetrated through the NC membrane to the GCE surface and was subsequently detected using an anodic voltammetric signal. The optimized method offered a good discrimination between complementary and nonspecific DNAs and yielded well-defined responses for both single-copy and repetitive tDNA sequences. In contrast to previously published methods using electrodes with mechanically attached membranes, the previously mentioned electrode is easily amenable to parallel DNA analysis. Copyright © Taylor & Francis, Inc

Effect of Short-and Long-Term High-Fat Feeding on Autophagy Flux and Lysosomal Activity in Rat Liver

Summary Autophagy-lysosomal pathway is a cellular mechanism ensuring degradation of various macromolecules like proteins or triacylglycerols (TAG). Its disruption is related to many pathological states, including liver steatosis. We compared the effect of short-and long-established steatosis on the intensity of autophagy-lysosomal pathway in rat liver. The experiments were carried out on 3-month old Wistar rats fed standard (SD) or highfat diet for 2 (HF-2) or 10 (HF-10) weeks. HF diet administered animals accumulated an increased amount of TAG in the liver (HF-2→HF-10). Autophagy flux was up-regulated in HF-2 group but nearly inhibited after 10 weeks of HF administration. The expression of autophagy related genes was up-regulated in HF-2 but normal in HF-10. In contrast, total activities of two lysosomal enzymes, lysosomal lipase (LAL) and acid phosphatase, were unaffected in HF-2 but significantly increased in HF-10 groups. mRNA expression of lysosomal enzymes was not affected by the diet. We conclude that in a state of metabolic unbalance (steatosis), autophagy machinery and lysosomal enzymes expression are regulated independently. The accumulation of TAG in the liver is associated with the increase of total LAL activity and protein expression. In contrast, the autophagy response is bi-phasic and after rapid increase it is significantly diminished. This may represent an adaptive mechanism that counteracts the excessive degradation of substrate, i.e. TAG, and eliminate over-production of potentially hazardous lipiddegradation intermediates

DNA hybridization on membrane-modified carbon electrodes

The DNA-modified membrane electrode was prepared by casting a mixture of nitrocellulose (NC) with target DNA (tDNA) in organic solvent on glassy carbon electrode (GCE). Unlabeled polymerase chain reaction (PCR)-amplified human genomic sequence (628 bp) or synthetic oligodeoxynucleotides (ODNs) were used as tDNAs, creating a recognition layer. Biotinylated ODNs were used as hybridization probes to recognize specific nucleotide sequences. The hybridization events were detected via an enzyme-linked electrochemical assay involving binding of streptavidin-coupled alkaline phosphatase (SALP) to the biotin labels of the probe bound to tDNA. After the probe hybridization and SALP binding, the electrode was immersed into an electroinactive enzyme substrate (1-naphthyl phosphate). The alkaline phosphatase converted the inactive substrate into electroactive 1-naphthol that penetrated through the NC membrane to the GCE surface and was subsequently detected using an anodic voltammetric signal. The optimized method offered a good discrimination between complementary and nonspecific DNAs and yielded well-defined responses for both single-copy and repetitive tDNA sequences. In contrast to previously published methods using electrodes with mechanically attached membranes, the previously mentioned electrode is easily amenable to parallel DNA analysis. Copyright © Taylor & Francis, Inc

DNA hybridization on membrane-modified carbon electrodes

The DNA-modified membrane electrode was prepared by casting a mixture of nitrocellulose (NC) with target DNA (tDNA) in organic solvent on glassy carbon electrode (GCE). Unlabeled polymerase chain reaction (PCR)-amplified human genomic sequence (628 bp) or synthetic oligodeoxynucleotides (ODNs) were used as tDNAs, creating a recognition layer. Biotinylated ODNs were used as hybridization probes to recognize specific nucleotide sequences. The hybridization events were detected via an enzyme-linked electrochemical assay involving binding of streptavidin-coupled alkaline phosphatase (SALP) to the biotin labels of the probe bound to tDNA. After the probe hybridization and SALP binding, the electrode was immersed into an electroinactive enzyme substrate (1-naphthyl phosphate). The alkaline phosphatase converted the inactive substrate into electroactive 1-naphthol that penetrated through the NC membrane to the GCE surface and was subsequently detected using an anodic voltammetric signal. The optimized method offered a good discrimination between complementary and nonspecific DNAs and yielded well-defined responses for both single-copy and repetitive tDNA sequences. In contrast to previously published methods using electrodes with mechanically attached membranes, the previously mentioned electrode is easily amenable to parallel DNA analysis. Copyright © Taylor & Francis, Inc

Multi-omics signatures in new-onset diabetes predict metabolic response to dietary inulin: findings from an observational study followed by an interventional trial

Abstract Aim The metabolic performance of the gut microbiota contributes to the onset of type 2 diabetes. However, targeted dietary interventions are limited by the highly variable inter-individual response. We hypothesized (1) that the composition of the complex gut microbiome and metabolome (MIME) differ across metabolic spectra (lean-obese-diabetes); (2) that specific MIME patterns could explain the differential responses to dietary inulin; and (3) that the response can be predicted based on baseline MIME signature and clinical characteristics. Method Forty-nine patients with newly diagnosed pre/diabetes (DM), 66 metabolically healthy overweight/obese (OB), and 32 healthy lean (LH) volunteers were compared in a cross-sectional case-control study integrating clinical variables, dietary intake, gut microbiome, and fecal/serum metabolomes (16 S rRNA sequencing, metabolomics profiling). Subsequently, 27 DM were recruited for a predictive study: 3 months of dietary inulin (10 g/day) intervention. Results MIME composition was different between groups. While the DM and LH groups represented opposite poles of the abundance spectrum, OB was closer to DM. Inulin supplementation was associated with an overall improvement in glycemic indices, though the response was very variable, with a shift in microbiome composition toward a more favorable profile and increased serum butyric and propionic acid concentrations. The improved glycemic outcomes of inulin treatment were dependent on better baseline glycemic status and variables related to the gut microbiota, including the abundance of certain bacterial taxa (i.e., Blautia, Eubacterium halii group, Lachnoclostridium, Ruminiclostridium, Dialister, or Phascolarctobacterium), serum concentrations of branched-chain amino acid derivatives and asparagine, and fecal concentrations of indole and several other volatile organic compounds. Conclusion We demonstrated that obesity is a stronger determinant of different MIME patterns than impaired glucose metabolism. The large inter-individual variability in the metabolic effects of dietary inulin was explained by differences in baseline glycemic status and MIME signatures. These could be further validated to personalize nutritional interventions in patients with newly diagnosed diabetes