Effects of different calorie restriction protocols on oxidative stress parameters in a transgenic mouse model of breast cancer

Aging and diseases related to aging, such as cancer, have been linked to oxidative stress. On the other hand, calorie restriction (CR) is one of the most effective interventions to slow down aging and prevent a variety of diseases such as cancer in preclinical models. CR has also been reported to modify oxidative stress. The aim of this study was to investigate the effects of different CR protocols and aging on oxidative stress parameters in the MMTV-TGF-alpha breast cancer mouse model in a cross-sectional study. Female mice were randomly enrolled in three groups: ad libitum (AL), chronic calorie restriction (CCR, 15% CR) or intermittent calorie restriction (ICR, three weeks AL followed by one week 60% CR in cyclic periods) starting at the age of 10 weeks until 81/82 weeks of age. Liver samples were analyzed for malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) levels. At week 49/50, the GSH level increased significantly in the CCR group compared to the AL and ICR-R groups which had higher mammary tumor (MT) incidence rates. Additionally, liver MDA levels in ICR groups were significantly increased, while aging led to decreased CAT and SOD activities in all CR groups. The application of different CR protocols did not have any significant effect on MDA, CAT, and SOD parameters in the liver at week 81/82. These results suggest that although GSH may interfere with MT development at the systemic level, many of the oxidative stress parameters may have more local effects on tumor development than the systemic effects

Long-term chronic caloric restriction alters miRNA profiles in the brain of ageing mice

Calorie restriction (CR) has been shown to be one of the most effective methods in alleviating the effects of ageing and age-related diseases. Although the protective effects of CR have been reported, the exact molecular mechanism still needs to be clarified. This study aims to determine differentially expressed (DE) miRNAs and altered gene pathways due to long-term chronic (CCR) and intermittent (ICR) CR in the brain of mice to understand the preventive roles of miRNAs resulting from long-term CR. Ten weeks old mice were enrolled into three different dietary groups; ad libitum, CCR or ICR, and fed until 82 weeks of age. miRNAs were analysed using GeneChip 4.1 microarray and the target of DE miRNAs was determined using miRNA target databases. Out of a total 3,163 analysed miRNAs, 55 of them were differentially expressed either by different CR protocols or by ageing. Brain samples from the CCR group had increased expression levels of mmu-miR-713 while decreasing expression levels of mmu-miR-184-3p and mmu-miR-351-5p compared to the other dietary groups. Also, current results indicated that CCR showed better preventive effects than that of ICR. Thus, CCR may perform its protective effects by modulating these specific miRNAs since they are shown to play roles in neurogenesis, chromatin and histone regulation. In conclusion, these three miRNAs could be potential targets for neurodegenerative and ageing-related diseases and may play important roles in the protective effects of CR in the brain

Multi-omics approach dissects cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy

Multi-omics profiling, in vitro and in vivo enhancer assays dissect the cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. With the identification of non-coding single nucleotide variants (SNVs) near PRDM13 and duplications overlapping a DNase I hypersensitive site (DHS) near PRDM13 or IRX1 as its underlying genetic cause, we hypothesize that NCMD is a retinal enhanceropathy. Here we aim to provide insight into the cis-regulatory mechanisms of NCMD by integrating multi-omics profiling of human retina with in vitro and in vivo enhancer assays. First, we established RegRet (http://genome.ucsc.edu/s/stvdsomp/RegRet), a genome-wide multi-omics retinal database. Next, UMI-4C profiling was performed on adult human retina to fine-map chromatin interactions of cis-regulatory elements (CREs) with the PRDM13 and IRX1 promoters. Multi-omics analysis including the UMI-4C data revealed sixteen candidate CREs (cCREs), seven for the PRDM13 and nine for the IRX1 region. Subsequently, the activity of cCREs was investigated by in vitro luciferase assays and by in vivo enhancer assays in Xenopus laevis and tropicalis. Four cCREs showed in vivo eye- and brain-specific activity in Xenopus. Furthermore, we expanded the genetic architecture of NCMD with two novel non-coding SNVs (V15, V16) with a likely effect on PRDM13 regulation. Luciferase assays showed that the non-coding SNVs that are located in the two hotspot regions of PRDM13 have an effect on cCRE activity. Interestingly, cCRE4 in which V16 is located was shown to interact with the PRDM13 promoter and demonstrated in vivo activity in Xenopus. This cCRE is active at a specific developmental stage (d103) compatible with the timepoint when retinal progenitor cells of the central retina exit mitosis. Mining of single-cell (sc) transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to emerge and begin to synapse with retinal ganglion cells. This supports the hypothesis that altered PRDM13 or IRX1 expression impairs synaptic interactions between amacrine and ganglion cells during retinogenesis. Overall, this study gained insight into the cis-regulatory mechanisms of NCMD and supports that NCMD is a retinal enhanceropathy

Multi-omics approach dissects cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy

North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy