Characterization of anti-NF-κB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system

RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-κB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-κB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-κB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-κB p502 and p652, respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-κB p652 in vitro

Succinate Dehydrogenase Loss in Familial Paraganglioma: Biochemistry, Genetics, and Epigenetics

It is counterintuitive that metabolic defects reducing ATP production can cause, rather than protect from, cancer. Yet this is precisely the case for familial paraganglioma, a form of neuroendocrine malignancy caused by loss of succinate dehydrogenase in the tricarboxylic acid cycle. Here we review biochemical, genetic, and epigenetic considerations in succinate dehydrogenase loss and present leading models and mysteries associated with this fascinating and important tumor

Modeling dioxygenase enzyme kinetics in familial paraganglioma

Hypoxia inducible factors (HIFs) play vital roles in cellular maintenance of oxygen homeostasis. These transcription factors are responsible for the expression of genes involved in angiogenesis, metabolism, and cell proliferation. Here, we generate a detailed mathematical model for the enzyme kinetics of α-ketoglutarate-dependent HIF prolyl 4-hydroxylase domain (PHD) dioxygenases to simulate our in vitro data showing synergistic PHD inhibition by succinate and hypoxia in experimental models of succinate dehydrogenase loss, which phenocopy familial paraganglioma. Our mathematical model confirms the inhibitory synergy of succinate and hypoxia under physiologically-relevant conditions. In agreement with our experimental data, the model predicts that HIF1α is not stabilized under atmospheric oxygen concentrations, as observed. Further, the model confirms that addition of α-ketoglutarate can reverse PHD inhibition by succinate and hypoxia in SDH-deficient cells

Oxygen concentration controls epigenetic effects in models of familial paraganglioma.

Familial paraganglioma (PGL) is a rare neuroendocrine cancer associated with defects in the genes encoding the subunits of succinate dehydrogenase (SDH), a tricarboxylic acid (TCA) cycle enzyme. For unknown reasons, a higher prevalence of PGL has been reported for humans living at higher altitude, with increased disease aggressiveness and morbidity. In this study, we evaluate the effects of oxygen on epigenetic changes due to succinate accumulation in three SDH loss cell culture models. We test the hypothesis that the mechanism of α-ketoglutarate (α-KG)-dependent dioxygenase enzymes explains the inhibitory synergy of hypoxia and succinate accumulation. We confirm that SDH loss leads to profound succinate accumulation. We further show that hypoxia and succinate accumulation synergistically inhibit α-KG-dependent dioxygenases leading to increased stabilization of transcription factor HIF1α, HIF2α, and hypermethylation of histones and DNA. Increasing oxygen suppresses succinate inhibition of α-KG-dependent dioxygenases. This result provides a possible explanation for the association between hypoxia and PGL, and suggests hyperoxia as a potential novel therapy

Neuromodulation Therapy for Chemotherapy-Induced Peripheral Neuropathy: A Systematic Review

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and painful condition in patients who have received chemotherapy. The role of neuromodulation therapy in treating pain and improving neurological function in CIPN remains unclear and warrants evidence appraisal. In compliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we performed a systematic review to assess change in pain intensity and neurological function after implementation of any neuromodulation intervention for CIPN. Neuromodulation interventions consisted of dorsal column spinal cord stimulation (SCS), dorsal root ganglion stimulation (DRG-S), or peripheral nerve stimulation (PNS). In total, 15 studies utilized SCS (16 participants), 7 studies utilized DRG-S (7 participants), and 1 study utilized PNS (50 participants). Per the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) criteria, there was very low-quality GRADE evidence supporting that dorsal column SCS, DRG-S, and PNS are associated with a reduction in pain severity from CIPN. Results on changes in neurological function remained equivocal due to mixed study findings on thermal sensory thresholds and touch sensation or discrimination. Future prospective, well-powered, and comparative studies assessing neuromodulation for CIPN are warranted

Characterization of anti-NF-iB RNA aptamerbinding

specificity in vitro and in the yeast three-hybrid syste

HIF1α and H3K9me2 accumulation and 5-hydroxy-methyl-2’-deoxycytidine (5hmdC) depletion in PGL specimens compared to controls.

<p>Normal ganglia 1 (NG1), normal ganglia 2 (NG2) and IDH-mutant (IDH). Sporadic PGL (Spo. PGL). A. HIF1α staining. B. HIF2α staining. C. H3K9me2 staining. Arrows indicate H3K9me2 staining in nuclei of neurons or chief cells. D. 5hmdC staining. Arrows indicate 5hmdC staining in the nuclei of neurons and chief cells.</p

Oxygen dependence of SDHB knockdown and SDHC knockout on cytosine methylation in genomic DNA.

<p>A. 5-methylcytosine levels by HPLC-MS for SDHB knockdown cells exposed to different oxygen concentrations. Data are representative of at least three independent experiments. B. 5-methylcytosine levels by HPLC-MS for SDHC knockout iMEFs exposed to different oxygen concentration. Data (mean ± standard deviation) are representative of at least three independent experiments. Statistical significance by T-test (*P<0.05 and **P<0.01) is indicated.</p

Characterization SDH loss models of PGL: HEK293 SDHB knockdown cells and <i>SDHC</i> conditional knockout iMEFs.

<p>A. Western blot analysis of HEK293 cells transduced with SDHB silencing lentiviruses (shRNA1 or shRNA2) or control vector (scrambled; scr.) and iMEFs treated with TAM for 7 d. β-actin was used as a loading control. B. PCR genotyping with primers for <i>SDHC</i> floxed (fl) allele before and after CRE recombination (rec). C. SDH enzyme assay in lysates from the indicated HEK293 cells and iMEFs (standard deviation reflects triplicates). Data are mean ± standard deviation. Statistical significance by T-test (*P<0.05 and **P<0.01) is indicated. D. Relative metabolite levels in the indicated whole-cell lysates measured by GC/MS analysis. Data are normalized to cell number and the respective control value.</p

A Comprehensive Review of the Genetic and Epigenetic Contributions to the Development of Fibromyalgia

This narrative review summarizes the current knowledge of the genetic and epigenetic contributions to the development of fibromyalgia (FM). Although there is no single gene that results in the development of FM, this study reveals that certain polymorphisms in genes involved in the catecholaminergic pathway, the serotonergic pathway, pain processing, oxidative stress, and inflammation may influence susceptibility to FM and the severity of its symptoms. Furthermore, epigenetic changes at the DNA level may lead to the development of FM. Likewise, microRNAs may impact the expression of certain proteins that lead to the worsening of FM-associated symptoms