Periodic Reporting for period 2 - FastBio (A genomics and systems biology approach to explore the molecular signature and functional consequences of long-term, structured fasting in humans)
Reporting period: 2018-12-01 to 2021-03-31
Individuals who follow the EOCC fasting regime abstain from meat, dairy products and eggs for a total of 180-200 days annually, in a highly structured, temporal manner initiated during childhood. Traditionally, fasting included the consumption of vegetables, legumes, nuts, fruits, olives, bread, snails and seafood. Unstructured diet involves the diet followed by the general, non-fasting population. Our underlying hypothesis is that given that fasting individuals follow the EOCC regime for many years, starting from childhood, this leaves a distinct biological imprint on their cells. We aimed to study 200 individuals from each dietary group to investigate the molecular and functional impact of dietary intake. We implemented a two timepoint sampling strategy inviting all participants to participate during a fasting and a non-fasting timepoint (defined by the fasting group) to capture acute and long-term effects of dietary intake.
This approach enables us to address the biological signatures of structured vs. unstructured diet through three objectives. First, we are investigating the effects of the two dietary regimes, and of genetic variation, on higher-level phenotypes including anthropometric, physiological, and blood biomarker traits sampled at two timpoints. Second, we are carrying out a comprehensive set of omics assays (transcriptomics, epigenomics, metabolomics/proteomics and investigation of the gut microbiome), and will associate omics phenotypes with underlying genetic variation. Our aim is to examine each biological level separately, but importantly, to integrate data across biological levels to uncover complex molecular signatures linked to diet. Third, we are interrogating the functional consequences of dietary regimes at the cellular level through cell culture. It has been shown that immune system cells in blood (including T-cells and monocytes) capture signatures of the nutrient environment. Furthermore, it has also been shown that under specific nutrient environments, cells become more resistant to stress. We are currently addressing the in vitro behaviour of cells from fasting and non-fasting participants.
Objective 1: FastBio sample collection was conducted with strict inclusion and exclusion criteria in order to obtain dietary groups (fasting, non-fasting) made up of similar numbers of female and male participants and with similar age distributions. We are currently analysing data for a total of 411 individuals, 200 fasting (108 female) and 211 non-fasting (116 female). Initial analyses of anthropometric, biochemical and physiological data, comparing data across timepoints, but also across dietary groups for a given timepoint, have been performed. We have very recently received data on genetic variation and are preparing analyses of phenotypic traits and of underlying genetic variation.
Objective 2: This objective comprises the bulk of the project and we are currently in various stages of data generation and processing for selected omics assays. DNA was extracted from blood and was genotyped at over 560,000 markers for the total of 411 participants. Generated data have been received recently and are being prepared for downstream analyses, including association with higher level (e.g. biomarkers) and omics phenotypes (e.g. gene expression). Extracted DNA was also used to investigate epigenetic signatures (DNA methylation) in a first subset of fasting and non-fasting participants at both timepoints. We expect to receive these data in the next few coming weeks. RNA was extracted from blood and following selection of molecules coding for proteins, is being sequenced to capture gene expression patterns in whole blood from participants at both sampling timepoints. We are currently receiving batches of RNA-Seq data and are performing initial quality control. Final batches are expected towards the end of the year. Regarding analyses of the gut microbiome, given the impact of extraction method on the detected composition of gut microbial communities, we are currently optimizing extraction protocols using test samples prior to processing FastBio samples. We are also performing extractions of microbial DNA from a known control (community standard) and have performed pilot 16S rRNA sequencing in-house to understand how extraction method and parameters affect findings. Furthermore, although our initial aim was to study the plasma metabolome, given rapid progress in the field of proteomics and HI in-house infrastructure and expertise, we are testing a small pilot subset of our samples using targeted and untargeted proteomics approaches. Additionally, as a complementary approach to 16S rRNA sequencing, we have run a test metaproteomics experiment on the in-house platform. Preliminary data are currently being explored in collaboration with HI facility scientists.
Objective 3: During sample collection, PBMCs at both sampling timepoints were isolated for a subset of 50 participants and were stored in liquid nitrogen. We are currently optimizing cell culture protocols and conditions on test samples in order not to waste precious biological material collected. We are determining rates of survival and subsequent growth and are conducting cell sorting to determine numbers of T cells and monocytes that can be obtained from each sample. This will help us determine the type and number of assays (e.g. primary cell culture) we can perform with the FastBio material.