A randomized controlled trial of folic acid intervention in pregnancy highlights a putative methylation-regulated control element at ZFP57

Table S1. Pyrosequencing and transcriptional primer sets used in this study. Pyroassay primers are given as bisulfite converted sequence. The same primers were used for both RT-PCR and RT-qPCR. (DOCX 15 kb

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na⁺ and Ca2 + influx via TRPV1 channels, which leads to mitochondrial Ca2 + accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist- induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid- sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau- type Ca2 + signals, mitochondrial Ca2 + accumulation and Na⁺- and Ca2 +-dependent membrane disorganization. Moreover, long- term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment

Transcriptional and post-transcriptional gene silencing by RNA interference in Candida albicans

RNA interference (RNAi) is an epigenetic mechanism occurring in all eukaryotic cells which uses small interfering RNAs to control gene expression. siRNAs can repress gene expression transcriptionally via chromatin remodelling, or post- transcriptionally via degradation of mRNA or inhibition of translation. Candida albicians is the most common human fungal pathogen characterised by chromosomal instability, heterozygosity and diploid nature. The RNAi mechanism is well established in other systems but not in Candida where the role of two key proteins Dicer (Dcr1) and Argonaute (Ago1) is still unknown. The aim of this project is to analyse the function of C.albicans Dcr1 and Ago1 and assess whether they have role in silencing. In other organisms, Dicer and Argonaute are not essential for cell growth and act in the same pathway. However the results of my Master project demonstrate that C. albicans Dcr1 and Ago1 act in different pathways. Firstly, I have found that Dcr1, but not Ago1, is essential for cell growth in a temperature-dependent manner. In addition, I have demonstrated that Dcr1, but not Ago1, influences the drug resistance and hyphae formation. Finally, I have demonstrated that C. albicans RNAi silences a class of genes located at the subtelomeres and that Dcr1 but not Ago1 controls this silencing process. These results suggest that the C. albicans RNAi machinery represses gene expression independently of Ago1 via a novel uncharacterised pathway

ENRICHED ENVIRONMENT INFLUENCES HORMONAL STATUS AND HIPPOCAMPAL BRAIN DERIVED NEUROTROPHIC FACTOR IN A SEX DEPENDENT MANNER

The present study is aimed at testing the hypothesis that an enriched environment (EE) induces sex-dependent changes in stress hormone release and in markers of increased brain plasticity. The focus was on hypothalamic-pituitary-adrenocortical (HPA) axis activity, plasma levels of stress hormones, gene expression of glutamate receptor subunits and concentrations of brain-derived neurotrophic factor (BDNF) in selected brain regions. Rats exposed to EE were housed in groups of 12 in large cages with various objects, which were frequently changed, for 6 weeks. Control animals were housed four per cage under standard conditions. In females the EE-induced rise in hippocampal BDNF, a neurotrophic factor associated with increased neural plasticity, was more pronounced than in males. Similar sex-specific changes were observed in BDNF concentrations in the hypothalamus. EE also significantly attenuated oxytocin and aldosterone levels only in female but not male rats. Plasma testosterone positively correlated with hippocampal BDNF in female but not male rats housed in EE. In male rats housing in EE led to enhanced levels of testosterone and adrenocorticotropic hormone (ACTH), this was not seen in females. Hippocampal glucocorticoid but not mineralocorticoid receptor levels decreased in rats housed in EE irrespective of sex. Housing conditions failed to modify mRNA levels of glutamate receptor type 1 (Glur1) and metabotropic glutamate receptor subtype 5 (mGlur5) subunits of glutamate receptors in the forebrain. Moreover, a negative association between corticosterone and BDNF was observed in both sexes. The results demonstrate that the association between hormones and changes in brain plasticity is sex related. In particular, testosterone seems to be involved in the regulatory processes related to neuroplasticity in females. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved