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A genomics and systems biology approach to explore the molecular signature and functional consequences of long-term, structured fasting in humans

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

Dietary intake shapes the growth of organisms, enables organ development and maintenance, has an impact on the function of the immune system and ultimately influences health and lifespan. Although diet has an enormous impact on human health, scientific consensus about how what we eat affects our biology remains largely elusive. However, there is consensus that our diet has a much more pervasive role than previously thought in modulating molecular mechanisms governing susceptibility to diseases such as type 2 diabetes, cardiovascular disease and cancer. The biological mechanisms linking diet to disease however remain unclear. To address the complex biological impact of diet, we have established the FastBio project, an unconventional, 'humans-as-model-organisms' approach through which we compare the molecular and functional effects of a highly structured dietary regime, specified by the Eastern Orthodox Christian Church (EOCC), to the unstructured diet followed by the general population in Greece.

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.
Exceeding our initial target of 400 individuals, we recruited 411 FastBio participants. Participants were from the greater area of Thessaloniki and were invited to participate at two specific timepoints. Sample collection took place at a single sampling centre and we achieved 95% recall rate, i.e. 390 of the initial 411 participants were present at the second sampling timepoint. Biological material (blood, plasma, fecal material, and peripheral blood mononuclear cells (PBMCs)) was collected and handled under conditions ensuring highest possible quality. We also collected data on anthropometric traits (height, weight, waist and hip circumference), physiological traits (systolic and diastolic blood pressure, pulse, metabolic rate, fat and muscle distribution) and on ~40 blood biomarkers (e.g. LDL, HDL, triglycerides). We also recorded detailed information on medical history, dietary intake and lifestyle. The degree of sample completeness of the collection is noteworthy reaching 99.6% and all material was collected as outlined in the project's ethics protocol. Following sample collection, we focused on establishing and organizing a biobank type of collection at BSRC Alexander Fleming (HI) with multiple biological samples and linked information for each participant.

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.
A key strength of the FastBio project, that contributes to progress beyond the state of the art, is that it comprises an unconventional, human systems biology project. This stems from the fact that we are investigating a biological system and its function as a whole (dietary intake and its molecular consequences) rather than as separate components (the impact of separate nutrients). Furthermore, this is being done in the context of the system's embedded genetic variation, given that participants are from the greater area of Thessaloniki. Systems biology experiments involve introducing a perturbation to a stable system and monitoring the system's response, through integration of response data and formulation of mathematical models that describe the system. In this sense, it is rare for such studies to be performed on humans since human population samples that can be viewed as a stable system are uncommon, given the plethora of genetic, environmental and behavioural factors that introduce variation to the system. In the case of the FastBio project, the unstructured diet of the general population represents the stable system and the structured diet of fasting individuals represents the perturbation to the system. Furthermore, dynamic assays in cell culture exploring how the system responds to various stimuli, will inform functional pathways influenced by dietary intake. Expected results include uncovering of molecular mechanisms that are influenced and modulated by nutrient intake. This may involve known pathways, but with a yet unknown link to diet. Importantly it will likely also involve unknown pathways and molecular mechanisms that contribute to disease susceptibility and pathogenesis through their interaction with the nutrient environment. The project therefore will lead to novel insights regarding the potent signalling nature of nutrients and can ultimately yield results of high translational value.
Logo of FastBio project
FastBio involves integration of data across multiple biological levels to address the impact of diet
FastBio explores the molecular impact of dietary intake in humans. Credit: Getty Images/Ikon Images
PBMCs were isolated from blood to study the impact of nutrient environment in primary cell culture
Extracted RNA from blood was sequenced to explore gene expression patterns
FastBio sample collection involved drawing of blood from participants