Epigenetic Inheritance Research Articles

Tissue-specific epigenetic inheritance after paternal heat exposure in male wild guinea pigs

External temperature change has been shown to modify epigenetic patterns, such as DNA methylation, which regulates gene expression. DNA methylation is heritable, and as such provides a mechanism to convey environmental information to subsequent generations. Studies on epigenetic response to temperature increase are still scarce in wild mammals, even more so studies that compare tissue-specific epigenetic responses. Here, we aim to address differential epigenetic responses on a gene and gene pathway level in two organs, liver and testis. We chose these organs, because the liver is the main metabolic and thermoregulation organ, and epigenetic modifications in testis are potentially transmitted to the F2 generation. We focused on the transmission of DNA methylation changes to naive male offspring after paternal exposure to an ambient temperature increase of 10 °C, and investigated differential methylated regions of sons sired before and after the paternal exposure using Reduced Representation Bisulfite Sequencing. We detected both a highly tissue-specific epigenetic response, reflected in genes involved in organ-specific metabolic pathways, and a more general regulation of single genes epigenetically modified in both organs. We conclude that genomes are context-specifically differentially epigenetically regulated in response to temperature increase. These findings emphasize the epigenetic relevance in cell differentiation, which is essential for the specific function(s) of complex organs, and is represented in a diverse molecular regulation of genes and gene pathways. The results also emphasize the paternal contribution to adaptive processes.

Dopamine Receptor D1 Contributes to Cocaine Epigenetic Reprogramming of Histone Modifications in Male Germ Cells.

Paternal environmental perturbations, including cocaine intake, can affect the development and behavior of the offspring through epigenetic inheritance. However, the mechanism by which cocaine alters the male germ cells epigenome is almost unexplored. Here, we report that cocaine-treated male mice showed alterations on specific histone post-translational modifications (PTMs) including increased silent chromatin marks H3K9me3 and H3K27me3 and decreased active enhancer and promoter marks H3K27ac and H3K4me3 in isolated germ cells. Also, cocaine increased H3K9ac and H4K16ac levels, involved in the replacement of histones by protamines that take place at round spermatid stage. Cocaine also altered histones H3/H4 epigenetic enzymes by increasing acetyltransferase KAT8/MOF, deacetylase SIRT1 and methyltransferase KMT1C/G9A, and decreasing deacetylases HDAC1/2 and demethylase KDM1A/LSD1 protein levels. Moreover, a pre-treatment with dopamine receptor 1 (DRD1) antagonist SCH23390 (SCH) blocked cocaine effects on H3K4me3, H3K27me3, and H4K16ac epigenetic marks. Interestingly, treatment with SCH-only was able to modify most of the histone marks tested here, pointing to a dopamine role in controlling histone PTMs in germ cells. Taken together, our data suggest a key role for DRD1 in mediating cocaine-triggered epigenetic modifications related to the silencing of gene transcription and the histone-to-protamine replacement that controls chromatin architecture of maturing sperm cells, and pinpoints a novel role of the dopaminergic system in the regulation of male germ cells reprogramming.

Oded Rechavi.

Interview with Oded Rechavi, who studies transgenerational small RNA inheritance in Caenorhabditis elegans at Tel Aviv University.

High Level of Gene Transcription at the Embryonic Stage Leads to the Suppression of Heterochromatic Trans-Inactivation in Drosophila melanogaster Adults.

In some cases, gene transfer from euchromatin to constitutive heterochromatin as a result of chromosomal rearrangement is accompanied by epigenetic inactivation of this gene (cis-inactivation). In the case of trans-inactivation, transgenes in the normal chromosome are repressed by the cis-inactivation-causing rearranged homologous chromosome. Trans-inactivation is a result of the somatic pairing of homologs and the transfer of the normal chromosomal segment to the heterochromatic compartment of the nucleus. Previously, we have shown that the degree of trans-inactivation of the UAS-eGFP reporter gene in adult flies depends on its transcription level that can be regulated by temperature using the GAL4 transcription activator and its temperature-sensitive inhibitor GAL80ts. In this paper, we investigated the epigenetic inheritance of the active/repressed state of the trans-inactivated reporter gene at different expression levels by measuring eGFP fluorescence in the individual cells of Malpighian tubules in adult flies. High expression levels at the embryonic stage protected the eGFP gene from trans-inactivation in adult flies. The activated state was inherited over the entire period of development and differentiation, while the activating effect of GAL4 was turned off.

IN-UTERO ANDROGEN EXPOSURE CHANGES NEONATAL OVARIAN GENE EXPRESSION AND MITOCHONDRIA IN A LEAN PCOS MODEL

Polycystic ovary syndrome (PCOS) affects up to 10% of women worldwide, has a strong hereditary pattern with a heterogeneous phenotype. While no candidate genes have been found, a shared disease predisposing environment, such as intrauterine hyperandrogenemia, may explain an epigenetic inheritance pattern. A lean PCOS mouse model, eliminating the obesity confounder, with maternal androgen administration prenatally, provides an opportunity to study the effect of androgens on fetal programming.

Stearoyl‐CoA desaturase 1 determines pancreatic β‐cell fate through regulation of DNA methylation pattern

Study objectiveStearoyl CoA desaturase 1 (SCD1) is a pivotal enzyme involved in saturated fatty acids metabolism that exhibits a protective role against lipotoxicity in pancreatic β‐cells. It has been shown that SCD1 deficiency contributes to impaired insulin secretion, β‐cell failure and development of type 2 diabetes (T2D). In the present study we investigated the involvement of SCD1 in regulation of DNA methylation and expression of transcription factors (TFs) responsible for maintenance of mature β‐cells function and identity.MethodsThe experiments were carried out ex vivo on pancreatic islets isolated from wild type (WT) and SCD1 knock‐out (SCD1 KO) mice, and in vitro on INS‐1E pancreatic β‐cell line, where lipotoxicity was induced by palmitic acid treatment. Morphological and functional characteristics of islets was evaluated by ELISA assays, immunofluorescence labeling and transmission electron microscopy techniques. DNA methylation and gene expression levels were assessed by bisulfite sequencing and real‐time quantitative PCR analyses, respectively.ResultsOur data demonstrated that pancreatic islets of SCD1 KO mice are characterized by different microarchitecture, secretory granules content and functional abnormalities manifested by diminished insulin secretion and greater proinsulin secretion in comparison to WT islets. On both ex vivo and in vitro models we observed significantly reduced expression of TFs crucial to maintenance of β‐cell identity such as Pdx1, MafA, Nkx6.1 and Neurod1. Interestingly, transcriptional repression of Pdx1 and MafA was related to excessive methylation of promoter regions of those TFs. In addition, we showed that downregulation of SCD1 activity/gene expression affects global DNA methylation pattern and changes methyl groups distribution within chromosomes in pancreatic β‐cells. DNA hypomethylation which occurred in SCD1‐deficient β‐cells was followed by decreased expression of DNA methyltransferase 1 (Dnmt1). We indicated that activation of AMP‐activated protein kinase (AMPK) axis influences post‐translational regulation of Dnmt1, thereby mediating the effect of SCD1 inhibition on global DNA methylation profile in β‐cells.ConclusionsTogether, our findings suggest that SCD1 is a gatekeeper that preserves epigenetic inheritance of DNA methylation pattern in β‐cells. Thus, SCD1 protects β‐cells from lipid‐derived loss of identity in methylation‐dependent manner through regulation of expression of TFs responsible for maintenance of β‐cell identity. Our results provide additional mechanistic insights toward understanding the role of SCD1 in T2D pathogenesis.Support or Funding InformationThis work was supported by grants from the National Science Center, Poland, grants no UMO‐2017/27/N/NZ3/01987 (AMD) and UMO‐2015/19/D/NZ4/03705 (JJ), and by The National Centre for Research and Development, grant no STRATEGMED 3/305813/2/NCBR/2017 (AD).

Chromatin replication and epigenetic cell memory.

Propagation of the chromatin landscape across cell divisions is central to epigenetic cell memory. Mechanistic analysis of the interplay between DNA replication, the cell cycle, and the epigenome has provided insights into replication-coupled chromatin assembly and post-replicative chromatin maintenance. These breakthroughs are critical for defining how proliferation impacts the epigenome during cell identity changes in development and disease. Here we review these findings in the broader context of epigenetic inheritance across mitotic cell division.

Two different epigenetic information channels in wild three-spined sticklebacks are involved in salinity adaptation.

Epigenetic inheritance has been proposed to contribute to adaptation and acclimation via two information channels: (i) inducible epigenetic marks that enable transgenerational plasticity and (ii) noninducible epigenetic marks resulting from random epimutations shaped by selection. We studied both postulated channels by sequencing methylomes and genomes of Baltic three-spined sticklebacks (Gasterosteus aculeatus) along a salinity cline. Wild populations differing in salinity tolerance revealed differential methylation (pop-DMS) at genes enriched for osmoregulatory processes. A two-generation experiment demonstrated that 62% of these pop-DMS were noninducible by salinity manipulation, suggesting that they are the result of either direct selection or associated genomic divergence at cis- or trans-regulatory sites. Two-thirds of the remaining inducible pop-DMS increased in similarity to patterns detected in wild populations from corresponding salinities. The level of similarity accentuated over consecutive generations, indicating a mechanism of transgenerational plasticity. While we can attribute natural DNA methylation patterns to the two information channels, their interplay with genomic variation in salinity adaptation is still unresolved.

Germline epigenetic inheritance: Challenges and opportunities for linking human paternal experience with offspring biology and health

Recently, novel experimental approaches and molecular techniques have demonstrated that a male's experiences can be transmitted through his germline via epigenetic processes. These findings suggest that paternal exposures influence phenotypic variation in unexposed progeny-a proposal that runs counter to canonical ideas about inheritance developed during the 20th century. Nevertheless, support for paternal germline epigenetic inheritance (GEI) in nonhuman mammals continues to grow and the mechanisms underlying this phenomenon are becoming clearer. To what extent do similar processes operate in humans, and if so, what are their implications for understanding human phenotypic variation, health, and evolution? Here, we review evidence for GEI in human and nonhuman mammals and evaluate these findings in relation to historical conceptions of heredity. Drawing on epidemiological data, reproductive biology, and molecular embryology, we outline developments and opportunities for the study of GEI in human populations, emphasizing the challenges that researchers in this area still face.

DNA methylation in bull spermatozoa: evolutionary impacts, interindividual variability, and contribution to the embryo

The DNA methylome of spermatozoa results from a unique epigenetic reprogramming crucial for chromatin compaction and the protection of the paternal genetic heritage. Although bull semen is widely used for artificial insemination (AI), little is known about the sperm epigenome in cattle. The purpose of this review is to synthetize recent work on the bull sperm methylome in light of the knowledge accumulated in humans and model species. We will address sperm-specific DNA methylation features and their potential evolutionary impacts, with particular emphasis on hypomethylated regions and repetitive elements. We will review recent examples of interindividual variability and intra-individual plasticity of the bull sperm methylome as related to fertility and age, respectively. Finally, we will address paternal methylome reprogramming after fertilization, as well as the mechanisms potentially involved in epigenetic inheritance, and provide some examples of disturbances that alter the dynamics of reprogramming in cattle. Because the selection of AI bulls is closely based on their genotypes, we will also discuss the complex interplay between sequence polymorphism and DNA methylation, which represents both a difficulty in addressing the role of DNA methylation in shaping phenotypes and an opportunity to better understand genome plasticity.

Isolation and initial structure-functional characterization of endogenous tRNA-derived stress-induced RNAs

ABSTRACT Recent transcriptome-wide studies have identified a diverse pool of transfer RNA (tRNA)-derived RNAs or tRNA-derived fragments (tRFs). Some of these RNAs have been demonstrated to be functional and involved in multiple biological processes ranging from the regulation of gene expression to transgenerational epigenetic inheritance. Post-transcriptional maturation of tRNAs includes various processing events including extensive decoration by various RNA modifications, which are required for correct tRNA folding and stability. Moreover, tRNA modifications determine the pattern and specificity of tRNA cleavage. The major drawbacks of many studies in the field of tRFs are that most of them used synthetic RNAs that closely mimic endogenous tRFs in their sequence, yet lack RNA modification that is found in vivo. Here, we developed a simple method to isolate tRNA-derived stress-induced RNAs (tiRNAs), a specific subset of tRFs. Our approach is scalable, cost-effective and relies on the purification of individual tiRNAs based on a sequence-specific RNA/DNA isolation technique using DNA probes. Our method facilitates functional studies of tiRNAs by addressing how physiological RNA modifications within tRNA fragments affect their biological activities. Here, we report pilot functional studies on selected endogenous tiRNAs, namely tiRNAAla and tiRNAGly. We show that natural 5ʹ-tiRNAAla molecules assemble into G-quadruplex structures, and endogenous 5ʹ-tiRNAGly possesses translation inhibition activity.

Remember where you came from: ABA insensitivity is epigenetically inherited in mesophyll, but not seeds

Plants transmit their experiences of environmental conditions to their progeny through epigenetic inheritance, improving their progeny’s fitness under prevailing conditions. Though ABA is known to regulate epigenetic-modification genes, no strong phenotypic link between those genes and intergenerational “memory” has been shown. Previously, we demonstrated that mesophyll insensitivity to ABA (FBPase::abi1-1{fa} transgenic plants) results in a range of developmental phenotypes, including early growth vigor and early flowering (i.e., stress-escape behavior). Here, we show that null plants, used as controls (segregates of FBPase::abi1 that are homozygote descendants of a heterozygous transgenic plant, but do not contain the transformed abi1-1 gene) phenotypically resembled their FBPase::abi1-1 parents. However, in germination and early seedling development assays, null segregants resembled WT plants. These FBPase::abi1-1 null segregants mesophyll-related phenotypes were reproducible and stable for at least three generations. These results suggest that the heritability of stress response is linked to ABA’s epigenetic regulatory effect through ABI1 and mesophyll-related traits. The discrepancy between the epigenetic heritability of seed and mesophyll-related traits is an example of the complexity of epigenetic regulation, which is both gene and process-specific, and may be attributed to the fine-tuning of tradeoffs between flowering time, growth rate and levels of risk that allow annual plants to optimize their fitness in uncertain environments.

Does variable epigenetic inheritance fuel plant evolution?

Epigenetic changes influence gene expression and contribute to the modulation of biological processes in response to the environment. Transgenerational epigenetic changes in gene expression have been described in many eukaryotes. However, plants appear to have a stronger propensity for inheriting novel epialleles. This mini-review discusses how plant traits, such as meristematic growth, totipotency, and incomplete epigenetic erasure in gametes promote epiallele inheritance. Additionally, we highlight how plant biology may be inherently tailored to reap the benefits of epigenetic metastability. Importantly, environmentally triggered small RNA expression and subsequent epigenetic changes may allow immobile plants to adapt themselves, and possibly their progeny, to thrive in local environments. The change of epigenetic states through the passage of generations has ramifications for evolution in the natural and agricultural world. In populations containing little genetic diversity, such as elite crop germplasm or habitually self-reproducing species, epigenetics may provide an important source of heritable phenotypic variation. Basic understanding of the processes that direct epigenetic shifts in the genome may allow for breeding or bioengineering for improved plant traits that do not require changes to DNA sequence.

Intergenerational epigenetic inheritance in reef-building corals

The perception that the inheritance of phenotypic traits operates solely through genetic means is slowly being eroded: epigenetic mechanisms have been shown to induce heritable changes in gene activity in plants1,2 and metazoans1,3. Inheritance of DNA methylation patterns provides a potential pathway for environmentally induced phenotypes to contribute to evolution of species and populations1–5. However, in basal metazoans, it is unknown whether inheritance of CpG methylation patterns occurs across the genome (as in plants) or as rare exceptions (as in mammals)4. Here, we show that DNA methylation patterns in a reef-building coral are determined by genotype and developmental stage, as well as by parental environment. Transmission of CpG methylation from adults to their sperm and larvae demonstrates genome-wide inheritance. Variation in the hypermethylation of genes in adults and their sperm from distinct environments suggests intergenerational acclimatization to local temperature and salinity. Furthermore, genotype-independent adjustments of methylation levels in stress-related genes were strongly correlated with offspring survival rates under heat stress. These findings support a role of DNA methylation in the intergenerational inheritance of traits in corals, which could extend to enhancing their capacity to adapt to climate change. Intergenerational inheritance of traits in corals can help species survive environmental change. Examination of intergenerational DNA methylation profiles in a reef-building coral shows there to be genome-wide inheritance, with the potential for adaptive capacity to environmental stressors.

The Epigenetics of the Endocannabinoid System.

The endocannabinoid system (ES) is a cell-signalling system widely distributed in biological tissues that includes endogenous ligands, receptors, and biosynthetic and hydrolysing machineries. The impairment of the ES has been associated to several pathological conditions like behavioural, neurological, or metabolic disorders and infertility, suggesting that the modulation of this system may be critical for the maintenance of health status and disease treatment. Lifestyle and environmental factors can exert long-term effects on gene expression without any change in the nucleotide sequence of DNA, affecting health maintenance and influencing both disease load and resistance. This potentially reversible “epigenetic” modulation of gene expression occurs through the chemical modification of DNA and histone protein tails or the specific production of regulatory non-coding RNA (ncRNA). Recent findings demonstrate the epigenetic modulation of the ES in biological tissues; in the same way, endocannabinoids, phytocannabinoids, and cannabinoid receptor agonists and antagonists induce widespread or gene-specific epigenetic changes with the possibility of trans-generational epigenetic inheritance in the offspring explained by the transmission of deregulated epigenetic marks in the gametes. Therefore, this review provides an update on the epigenetics of the ES, with particular attention on the emerging role in reproduction and fertility.

The Role of Epigenetics in Plant Breeding Understanding Heritable Changes beyond DNA Sequence

Epigenetics, the study of heritable changes in gene expression without alterations in the DNA sequence, has become a pivotal area in plant breeding, offering novel strategies for crop improvement. This review examines the role of epigenetics in enhancing plant traits and resilience. It explores the mechanisms of epigenetic regulation, including DNA methylation, histone modification, and small RNA interference, and their applications in developing crops with improved stress tolerance, enhanced trait quality, and better adaptation to environmental changes. The integration of epigenetics with traditional breeding methods and genomic technologies provides a powerful framework for advancing plant breeding. Despite the promising benefits, challenges such as the complexity of epigenetic regulation and inheritance patterns need to be addressed. The review underscores the potential of epigenetic approaches to revolutionize plant breeding and contribute to sustainable agricultural practices.

Longitudinal genetic studies of cognitive characteristics.

The present review describes longitudinal studies of cognitive traits and functions determining the causes of their variations and their possible correction to prevent cognitive impairment. The present study reviews the involvement of such environmental factors as nutrition, prenatal maternal stress, social isolation and others in cognitive functioning. The role of epigenetic factors in the implementation of environmental effects in cognitive characteristics is revealed. Considering the epigenome significance, several studies were focused on the design of substances affecting methylation and histone modification, which can be used for the treatment of cognitive disorders. The appropriate correction of epigenetic factors related to environmental differences in cognitive abilities requires to determine the mechanisms of chromatin modifications and variations in DNA methylation. Transposons representing stress-sensitive DNA elements appeared to mediate the environmental influence on epigenetic modifications. They can explain the mechanism of transgenerational transfer of information on cognitive abilities. Recently, large-scale meta-analyses based on the results of studies, which identified genetic associations with various cognitive traits, were carried out. As a result, the role of genes actively expressed in the brain, such as BDNF, COMT, CADM2, CYP2D6, APBA1, CHRNA7, PDE1C, PDE4B, and PDE4D in cognitive abilities was revealed. The association between cognitive functioning and genes, which have been previously involved in developing psychiatric disorders (MEF2C, CYP2D6, FAM109B, SEPT3, NAGA, TCF20, NDUFA6 genes), was revealed, thus indicating the role of the similar mechanisms of genetic and neural networks in both normal cognition and cognitive impairment. An important role in both processes belongs to common epigenetic factors. The genes involved in DNA methylation (DNMT1, DNMT3B, and FTO), histone modifications (CREBBP, CUL4B, EHMT1, EP300, EZH2, HLCS, HUWE1, KAT6B, KMT2A, KMT2D, KMT2C, NSD1, WHSC1, and UBE2A) and chromatin remodeling (ACTB, ARID1A, ARID1B, ATRX, CHD2, CHD7, CHD8, SMARCA2, SMARCA4, SMARCB1, SMARCE1, SRCAP, and SS18L1) are associated with increased risk of psychiatric diseases with cognitive deficiency together with normal cognitive functioning. The data on the correlation between transgenerational epigenetic inheritance of cognitive abilities and the insert of transposable elements in intergenic regions is discussed. Transposons regulate genes functioning in the brain due to the processing of their transcripts into non-coding RNAs. The content, quantity and arrangement of transposable elements in human genome, which do not affect changes in nucleotide sequences of protein encoding genes, but affect their expression, can be transmitted to the next generation.

Developmental origins of health and disease (DOHaD) and paternal origins of health and disease (POHaD). Multigenerational inheritance

This review addresses the concept of ontogenetic origin of health and disease (DOHaD) and the new concept of paternal origin of health and disease (POHaD). These concepts are based on scientific evidence that environmental factors impacting mother or father can play a role in reprogramming the health of their off springs throughout their life span. Moreover, the changes that have arisen can be transmitted through generations via diverse epigenetic mechanisms. Terms, such as epigenetics (a kind of “driver” for these concepts), epigenetic inheritance (including Intergenerational Inheritance – from generation to the next generation and Transgenerational Inheritance – through generations), epigenetic changes caused by the environment (Environmental Epigenetics) are discussed. Vulnerable periods towards epigenetic changes (Windows of Susceptibility) that occur in male germ cells responsible for epigenetic inheritance are considered. Epigenetic epidemiological studies in the field of reproductology are described; their advantages and disadvantages are discussed. These studies can serve as the basis for obtaining new knowledge about the causes of epigenetic variations in germ cells that occur in health and upon exposure to environmental factors as well as the inherited phenotypic outcomes.

Delineation of a Human Mendelian Disorder of the DNA Demethylation Machinery: TET3 Deficiency

Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms.

Symposium summary: Epigenetic inheritance-impact for biology and society 26-28 August 2019, Zurich, Switzerland.

The concept of epigenetic inheritance proposes a new and unconventional way to think about heredity in health and disease, at the interface between genetics and the environment. Epigenetic inheritance is a form of biological inheritance not encoded in the DNA sequence itself but mediated by epigenetic factors. Because epigenetic factors can be modulated by the environment, they can relay this information to the genome and modify its activity consequentially. If epigenetic changes induced by environmental exposure are present in the germline and persist in germ cells during development until conception, they have the potential to transfer the traces of ancestral exposure to the progeny.This form of heredity relates to the extremely important question of nature versus nurture and how much of our own make-up is genetically or epigenetically determined, a question that remains largely unresolved. Because it questions the dominant dogma of genetics and brings a paradigm shift in sciences, it has to creating strong bridges between disciplines and provide solid causal evidence to be firmly established.The second edition of a conference fully dedicated to epigenetic inheritance was held in August 2019 in Zurich, Switzerland. This symposium titled ‘Epigenetic inheritance: impact for biology and society’ (http://www.epigenetic-inheritance-zurich.ethz.ch), gathered experts in the field of epigenetic inheritance to discuss the concept and pertinent findings, exchange views and expertise about models and methods, and address challenges raised by this new discipline. The symposium offered a mix of invited lectures and short talks selected from abstracts, poster sessions and a workshop ‘Meet the experts: Q&A’. A tour of a local omics facility the Functional Genomics Center Zurich was also offered to interested participants. Additional comments and impressions were shared by attendees on Twitter #eisz19 during and after the symposium. This summary provides an overview of the different sessions and talks and describes the main findings presented.