Investigation of the use of histone deacetylase inhibitors for the treatment of inherited disorders of the glycolytic pathway

Histone acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) regulate gene expression by activating or repressing transcription, respectively. HDAC inhibitors (HDACIs) are a diverse class of drugs used to treat haemoglobinopathies, urea cycle disorders and several types of malignancies. Recent evidence from genome-wide as well as gene-specific epigenetic studies suggest a model whereby active genes are more likely than silent genes to be hyperacetylated and increase their transcription levels in response to HDACIs, a process underpinned by the dynamic recruitment and antagonistic activities of HATs and HDACs. Based on this model and from a therapeutic perspective, I hypothesised that the ability of HDACIs to increase expression of active genes might be relevant for diseases caused by genes that encode proteins with enzymatic function. HDACI-mediated increase in gene transcription, even in the presence of missense, disease-causing mutations, might lead to increased enzymatic activity and amelioration of the cellular and clinical phenotype. I tested this hypothesis on a group of genes involved in the glycolytic and pentose phosphate pathway (GPPP) which, when mutated, cause chronic or episodic haemolytic anaemia. Using RT-qPCR (B cell lines) and gene expression profiling (primary, in vitro generated human erythroid precursors and CD4+ T cells) I found that of the 17 GPPP genes, only Glucose-6-Phosphate Dehydrogenase (G6PD) mRNA levels increased in response to HDACIs in a time-dependent manner. Epigenetic analysis in B cells by ChIP-qPCR showed that histone hyper-acetylation and increased recruitment of HATs and HDACs underpin the selective G6PD transcriptional activation in response to HDACIs. Pharmacological and genetic assays showed that increase in G6PD transcription was also dependent on Sp1, a generic transcription factor known to recruit both HDACs and HATs. Finally, I directly tested the hypothesis that HDACIs may increase enzymatic activity in G6PD deficient cells. Using B cell lines and primary erythroid cells from patients with G6PD deficiency, I found that HDACIs induce the same epigenetic changes in the mutant as in the wild type G6PD gene; more importantly, they lead to increased levels of the mutant mRNA and protein, associated with an up to 3-fold increase in enzymatic activity. These findings are potentially of great therapeutic significance for correction of G6PD deficiency in up to 300 million individuals worldwide with the polymorphic variants of G6PD deficiency (e.g., G6PDMed and G6PDA-).Open Acces

Ultrafast Multiplexed-Allergen Detection through Advanced Fluidic Design and Monolithic Interferometric Silicon Chips

A silicon-based miniaturized sensor chip combined with an advanced microfluidic module for the simultaneous, label-free immunochemical determination of four allergens, bovine milk protein, peanut protein, soy protein, and gliadin, is presented. The sensor chip consists of an array of 10 broad-band Mach-Zehnder interferometers (BB-MZIs) monolithically integrated on silicon, along with their respective broad-band light sources. The BB-MZIs were biofunctionalized with the targeted allergens and their responses during immunoreaction were monitored by multiplexing their transmission spectra through an external miniaturized spectrometer. The assay is performed by running mixtures of calibrators or samples with the antibodies against the four allergens followed by an antispecies specific antibodies solution. Employing a fluidic module of nearly one-dimensional geometry, that provided for uniform delivery of the reagents, CV values <6% were achieved for the responses of the 10 BB-MZIs, allowing for reliable multianalyte determinations. The analysis is completed in 6.5 min, and the detection limits were 0.04 μg/mL for bovine k-casein, 1.0 μg/mL for peanut protein, 0.80 μg/mL for soy protein, and 0.10 μg/mL for gliadin. The assays were accurate (recoveries 88-118%) and repeatable (intra- and interassay CVs <7% for all four allergens). Finally, the sensor was evaluated by analyzing samples from a cleaning in place system (CIP) of a dairy industry and the results obtained were in good agreement with those received by the respective ELISAs. The analytical characteristics of the sensor combined with the short analysis time and the small chip size make the proposed system an ideal tool for on-site multianalyte determinations

Ultrafast Multiplexed-Allergen Detection through Advanced Fluidic Design and Monolithic Interferometric Silicon Chips

A silicon-based miniaturized sensor chip combined with an advanced microfluidic module for the simultaneous, label-free immunochemical determination of four allergens, bovine milk protein, peanut protein, soy protein, and gliadin, is presented. The sensor chip consists of an array of 10 broad-band Mach–Zehnder interferometers (BB-MZIs) monolithically integrated on silicon, along with their respective broad-band light sources. The BB-MZIs were biofunctionalized with the targeted allergens and their responses during immunoreaction were monitored by multiplexing their transmission spectra through an external miniaturized spectrometer. The assay is performed by running mixtures of calibrators or samples with the antibodies against the four allergens followed by an antispecies specific antibodies solution. Employing a fluidic module of nearly one-dimensional geometry, that provided for uniform delivery of the reagents, CV values <6% were achieved for the responses of the 10 BB-MZIs, allowing for reliable multianalyte determinations. The analysis is completed in 6.5 min, and the detection limits were 0.04 μg/mL for bovine k-casein, 1.0 μg/mL for peanut protein, 0.80 μg/mL for soy protein, and 0.10 μg/mL for gliadin. The assays were accurate (recoveries 88–118%) and repeatable (intra- and interassay CVs <7% for all four allergens). Finally, the sensor was evaluated by analyzing samples from a cleaning in place system (CIP) of a dairy industry and the results obtained were in good agreement with those received by the respective ELISAs. The analytical characteristics of the sensor combined with the short analysis time and the small chip size make the proposed system an ideal tool for on-site multianalyte determinations