Specific DNMT3C flanking sequence preferences facilitate methylation of young murine retrotransposons.

The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline. Using specialized assay systems, we investigate the flanking sequence preferences of DNMT3C and observe characteristic preferences for cytosine at the -2 and -1 flank that are unique among DNMT3 enzymes. We identify two amino acids in the catalytic domain of DNMT3C (C543 and V547) that are responsible for the DNMT3C-specific flanking sequence preferences and evolutionary conserved in muroids. Reanalysis of published data shows that DNMT3C flanking preferences are consistent with genome-wide methylation patterns in mouse ES cells only expressing DNMT3C. Strikingly, we show that CpG sites with the preferred flanking sequences of DNMT3C are enriched in murine retrotransposons that were previously identified as DNMT3C targets. Finally, we demonstrate experimentally that DNMT3C has elevated methylation activity on substrates derived from these biological targets. Our data show that DNMT3C flanking sequence preferences match the sequences of young murine retrotransposons which facilitates their methylation. By this, our data provide mechanistic insights into the molecular co-evolution of repeat elements and (epi)genetic defense systems dedicated to maintain genomic stability in mammals.

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Vascular smooth muscle cell mechanotransduction through serum and glucocorticoid inducible kinase-1 promotes interleukin-6 production and macrophage accumulation in murine hypertension.

Objective: The objective of this investigation was to demonstrate that in vivo induction of hypertension (HTN) and in vitro cyclic stretch of aortic vascular smooth muscle cells (VSMCs) can cause serum and glucocorticoid-inducible kinase (SGK-1)-dependent production of cytokines to promote macrophage accumulation that may promote vascular pathology. Methods: HTN was induced in C57Bl/6 mice with angiotensin II infusion (1.46 mg/kg/day × 21 days) with or without systemic infusion of EMD638683 (2.5 mg/kg/day × 21 days), a selective SGK-1 inhibitor. Systolic blood pressure was recorded. Abdominal aortas were harvested to quantify SGK-1 activity (pSGK-1/SGK-1) by immunoblot. Flow cytometry quantified the abundance of CD11b+/F480+ cells (macrophages). Plasma interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was assessed by enzyme-linked immunosorbent assay. Aortic VSMCs from wild-type mice were subjected to 12% biaxial cyclic stretch (Stretch) for 3 or 12 hours with or without EMD638683 (10 µM) and with or without SGK-1 small interfering RNA with subsequent quantitative polymerase chain reaction for IL-6 and MCP-1 expression. IL-6 and MCP-1 in culture media were analyzed by enzyme-linked immunosorbent assay. Aortic VSMCs from SGK-1flox+/+ mice were transfected with Cre-Adenovirus to knockdown SGK-1 (SGK-1KD VSMCs) and underwent parallel tension experimentation. Computational modeling was used to simulate VSMC signaling. Statistical analysis included analysis of variance with significance at a P value of <.05. Results: SGK-1 activity, abundance of CD11b+/F4-80+ cells, and plasma IL-6 were increased in the abdominal aorta of mice with HTN and significantly reduced by treatment with EMD638683. This outcome mirrored the increased abundance of IL-6 in media from Stretch C57Bl/6 VSMCs and attenuation of the effect with EMD638683 or SGK-1 small interfering RNA. C57Bl/6 VSMCs also responded to Stretch with increased MCP-1 expression and secretion into the culture media. Further supporting the integral role of mechanical signaling through SGK-1, target gene expression and cytokine secretion was unchanged in SGK-1KD VSMCs with Stretch, and computer modeling confirmed SGK-1 as an intersecting node of signaling owing to mechanical strain and angiotensin II. Conclusions: Mechanical activation of SGK-1 in aortic VSMCs can promote inflammatory signaling and increased macrophage abundance, therefore this kinase warrants further exploration as a pharmacotherapeutic target to abrogate hypertensive vascular pathology.

Transient naive reprogramming corrects hiPS cells functionally and epigenetically.

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.

A mammalian DNA methylation landscape.

A study of 348 species offers clues into the diversity of mammalian life spans.

DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation.

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences.

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability.

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.

Reporting on patient and public involvement (PPI) in research publications: using the GRIPP2 checklists with lay co-researchers.

BACKGROUND: Patient and public involvement (PPI) in health and social care research is considered important internationally, with increasing evidence that PPI improves the quality, relevance and outcomes of research. There has been a growth in research publications that describe PPI in the research process, but the frequency and detail of PPI reporting varies considerably. This paper reports on a collaborative study that aimed to describe the extent of PPI in publications from research funded by the Collaboration for Leadership in Applied Health Research and Care (CLAHRC) in the East of England (EoE), part of the National Institute of Health Research (NIHR) in England (2014-2019). METHODS: A descriptive study of all research publications (1st January 2014 to 31st October 2017) funded by the NIHR CLAHRC EoE. Members of the Public Involvement in Research group (PIRg), at the University of Hertfordshire, were actively involved, with four PIRg co-researchers. We used an internationally recognised reporting checklist for PPI called the GRIPP2 (Guidance for Reporting Involvement of Patients and the Public, Version 2) to guide the reviewing process. RESULTS: Out of 148 research papers identified, 16 (14%) reported some aspect of PPI activity and were included for review. Ten of the publications (63%) acknowledged the contributions of PPI individuals and/or groups and five had PPI co-authors. There was considerable variation in the PPI reported in the publications, with some 'missed opportunities' to provide detail of PPI undertaken. The perspectives of the co-researchers shaped the reporting of the results from this study. The co-researchers found the GRIPP2-SF (short form) to be useful, but the GRIPP2-LF (long form) was considered over complicated and not user-friendly. CONCLUSIONS: This is one of the first studies to involve lay co-researchers in the review of PPI reporting using the GRIPP2 reporting checklists (GRIPP2-SF and GRIPP2-LF). We make recommendations for a revised version of the GRIPP2-SF, with clearer instructions and three additional sections to record whether PPI is reported in the abstract or key words, in the acknowledgements section, and whether there are PPI co-authors. We also recommend the provision of training and support for patient and public peer reviewers.

Involving patients, family carers and members of the public in research is known as patient and public involvement, or PPI. In health and social care research, PPI is considered important by many, including patients and research funders. Increasingly, research publications include an account of how members of the public have been involved in research being reported. But often there is not much detail regarding who was involved, what they did, and what difference their involvement made.In our study, we reviewed publications from research funded by an organisation called the Collaboration for Leadership in Applied Health Research and Care (CLAHRC) in the East of England (EoE), to understand how members of the public had been involved. We also wanted to see how lay co-researchers found the use of a checklist developed to assess the reporting of PPI: the 'Guidance for Reporting Involvement of Patients and the Public, Version 2' (GRIPP2).Members of a Public Involvement in Research group (PIRg), based at the University of Hertfordshire, were actively involved in the study, with four PIRg members becoming co-researchers. The GRIPP2 has a long form and short form, and we used these to review 16 research publications.We found great variation in the PPI activities reported in the publications. The lay co-researchers thought the GRIPP2 short form was useful, but the long form was considered over complicated and not user-friendly. We recommend a revised version of the GRIPP2 short form, with clearer instructions, additional sections and training and support for patient and public reviewers.

The emergence of the brain non-CpG methylation system in vertebrates.

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.

Developmental remodelling of non-CG methylation at satellite DNA repeats.

In vertebrates, DNA methylation predominantly occurs at CG dinucleotides however, widespread non-CG methylation (mCH) has been reported in mammalian embryonic stem cells and in the brain. In mammals, mCH is found at CAC trinucleotides in the nervous system, where it is associated with transcriptional repression, and at CAG trinucleotides in embryonic stem cells, where it positively correlates with transcription. Moreover, CAC methylation appears to be a conserved feature of adult vertebrate brains. Unlike any of those methylation signatures, here we describe a novel form of mCH that occurs in the TGCT context within zebrafish mosaic satellite repeats. TGCT methylation is inherited from both male and female gametes, remodelled during mid-blastula transition, and re-established during gastrulation in all embryonic layers. Moreover, we identify DNA methyltransferase 3ba (Dnmt3ba) as the primary enzyme responsible for the deposition of this mCH mark. Finally, we observe that TGCT-methylated repeats are specifically associated with H3K9me3-marked heterochromatin suggestive of a functional interplay between these two gene-regulatory marks. Altogether, this work provides insight into a novel form of vertebrate mCH and highlights the substrate diversity of vertebrate DNA methyltransferases.

Towards understanding the origin of animal development.

Almost all animals undergo embryonic development, going from a single-celled zygote to a complex multicellular adult. We know that the patterning and morphogenetic processes involved in development are deeply conserved within the animal kingdom. However, the origins of these developmental processes are just beginning to be unveiled. Here, we focus on how the protist lineages sister to animals are reshaping our view of animal development. Most intriguingly, many of these protistan lineages display transient multicellular structures, which are governed by similar morphogenetic and gene regulatory processes as animal development. We discuss here two potential alternative scenarios to explain the origin of animal embryonic development: either it originated concomitantly at the onset of animals or it evolved from morphogenetic processes already present in their unicellular ancestors. We propose that an integrative study of several unicellular taxa closely related to animals will allow a more refined picture of how the last common ancestor of animals underwent embryonic development.

Enteric neurons increase maternal food intake during reproduction.

Reproduction induces increased food intake across females of many animal species1-4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.

Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri.

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits, such as nervous systems, arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here, we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system which, with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range, allows exploration of genomic changes both within sponges and in early animal evolution.

Genomic adaptations to aquatic and aerial life in mayflies and the origin of insect wings.

The evolution of winged insects revolutionized terrestrial ecosystems and led to the largest animal radiation on Earth. However, we still have an incomplete picture of the genomic changes that underlay this diversification. Mayflies, as one of the sister groups of all other winged insects, are key to understanding this radiation. Here, we describe the genome of the mayfly Cloeon dipterum and its gene expression throughout its aquatic and aerial life cycle and specific organs. We discover an expansion of odorant-binding-protein genes, some expressed specifically in breathing gills of aquatic nymphs, suggesting a novel sensory role for this organ. In contrast, flying adults use an enlarged opsin set in a sexually dimorphic manner, with some expressed only in males. Finally, we identify a set of wing-associated genes deeply conserved in the pterygote insects and find transcriptomic similarities between gills and wings, suggesting a common genetic program. Globally, this comprehensive genomic and transcriptomic study uncovers the genetic basis of key evolutionary adaptations in mayflies and winged insects.

Evolution of DNA Methylome Diversity in Eukaryotes.

Cytosine DNA methylation (5mC) is a widespread base modification in eukaryotic genomes with critical roles in transcriptional regulation. In recent years, our understanding of 5mC has changed because of advances in 5mC detection techniques that allow mapping of this mark on the whole genome scale. Profiling DNA methylomes from organisms across the eukaryotic tree of life has reshaped our views on the evolution of 5mC. In this review, we explore the macroevolution of 5mC in major eukaryotic groups, and then focus on recent advances made in animals. Genomic 5mC patterns as well as the mechanisms of 5mC deposition tend to be evolutionary labile across large phylogenetic distances; however, some common patterns are starting to emerge. Within the animal kingdom, 5mC diversity has proven to be much greater than anticipated. For example, a previously held common view that genome hypermethylation is a trait exclusive to vertebrates has recently been challenged. Also, data from genome-wide studies are starting to yield insights into the potential roles of 5mC in invertebrate cis regulation. Here we provide an evolutionary perspective of both the well-known and enigmatic roles of 5mC across the eukaryotic tree of life.

Convergent evolution of a vertebrate-like methylome in a marine sponge.

Vertebrates have highly methylated genomes at CpG positions, whereas invertebrates have sparsely methylated genomes. This increase in methylation content is considered a major regulatory innovation of vertebrate genomes. However, here we report that a sponge, proposed as the potential sister group to the rest of animals, has a highly methylated genome. Despite major differences in genome size and architecture, we find similarities between the independent acquisitions of the hypermethylated state. Both lineages show genome-wide CpG depletion, conserved strong transcription factor methyl-sensitivity and developmental methylation dynamics at 5-hydroxymethylcytosine enriched regions. Together, our findings trace back patterns associated with DNA methylation in vertebrates to the early steps of animal evolution. Thus, the sponge methylome challenges previous hypotheses concerning the uniqueness of vertebrate genome hypermethylation and its implications for regulatory complexity.

Origin and evolution of eukaryotic transcription factors.

Transcription factors (TFs) have a central role in genome regulation directing gene transcription through binding specific DNA sequences. Eukaryotic genomes encode a large diversity of TF classes, each defined by unique DNA-interaction domains. Recent advances in genome sequencing and phylogenetic placement of diverse eukaryotic and archaeal species are re-defining the evolutionary history of eukaryotic TFs. The emerging view from a comparative genomics perspective is that the Last Eukaryotic Common Ancestor (LECA) had an extensive repertoire of TFs, most of which represent eukaryotic evolutionary novelties. This burst of TF innovation coincides with the emergence of genomic nuclear segregation and complex chromatin organization.

Capture of a functionally active methyl-CpG binding domain by an arthropod retrotransposon family.

The repressive capacity of cytosine DNA methylation is mediated by recruitment of silencing complexes by methyl-CpG binding domain (MBD) proteins. Despite MBD proteins being associated with silencing, we discovered that a family of arthropod Copia retrotransposons have incorporated a host-derived MBD. We functionally show how retrotransposon-encoded MBDs preferentially bind to CpG-dense methylated regions, which correspond to transposable element regions of the host genome, in the myriapod Strigamia maritima Consistently, young MBD-encoding Copia retrotransposons (CopiaMBD) accumulate in regions with higher CpG densities than other LTR-retrotransposons also present in the genome. This would suggest that retrotransposons use MBDs to integrate into heterochromatic regions in Strigamia, avoiding potentially harmful insertions into host genes. In contrast, CopiaMBD insertions in the spider Stegodyphus dumicola genome disproportionately accumulate in methylated gene bodies compared with other spider LTR-retrotransposons. Given that transposons are not actively targeted by DNA methylation in the spider genome, this distribution bias would also support a role for MBDs in the integration process. Together, these data show that retrotransposons can co-opt host-derived epigenome readers, potentially harnessing the host epigenome landscape to advantageously tune the retrotransposition process.

[Perforación de colon sigmoides secundaria a endometriosis. Reporte de un caso]. / Perforation of the sigmoid colon secondary to endometriosis. Case report.

ANTECEDENTES: La endometriosis es la presencia de glándulas endometriales o estroma viable fuera de la cavidad uterina, que afecta aproximadamente al 2-10% de las mujeres en edad reproductiva1. Es común la afección de estructuras pélvicas, incluyendo el intestino. La perforación del colon por endometriosis es muy rara y representa una urgencia quirúrgica. CASO CLÍNICO: Mujer de 28 años con cuadro de dolor abdominal en fosa iliaca derecha y hueco pélvico, fiebre y náuseas. Se realiza laparotomía exploradora con hallazgo de perforación de colon sigmoides, que requiere resección de la lesión y colostomía terminal, encontrando como diagnóstico definitivo endometriosis. CONCLUSIÓN: La perforación de intestino o de colon es una complicación poco frecuente, pero de gravedad, que debemos tener siempre presente como sospecha ante un cuadro de abdomen agudo en una paciente en edad fértil y con antecedentes de haber presentado sintomatología gastrointestinal intermitente. BACKGROUND: Endometriosis is the presence of endometrial glands or viable stroma outside the uterine cavity, which affects approximately 2-10% of women of reproductive age 1. Pelvic structures, including the bowel, are commonly affected. Perforation of the colon by endometriosis is very rare and represents a surgical emergency. CLINICAL CASE: A 28-year-old female patient with abdominal pain in the right iliac fossa and pelvic cavity, fever and nausea, exploratory laparotomy is performed with the discovery of sigmoid perforation of the colon, requiring resection of the lesion and terminal colostomy, finding as definitive diagnosis endometriosis. CONCLUSION: Bowel or colon perforation is a rare but serious complication, which should always be kept in mind as a suspicion of acute abdomen in a female patient of reproductive age and with a history of intermittent gastrointestinal symptoms.