Defining Interactions Between the Genome, Epigenome, and the Environment in Inflammatory Bowel Disease: Progress and Prospects.

Recent advances in our understanding of the pathogenesis of inflammatory bowel disease (IBD) have highlighted the complex interplay between the genome, the epigenome, and the environment. Despite the exciting advances in genomics that have enabled the identification of over 200 susceptibility loci, these only account for a small proportion of the disease variance and the estimated heritability in IBD. It is likely that gene-environment (GxE) interactions contribute to "missing heritability" and these may act through epigenetic mechanisms. Several environmental factors, such as the microbiome, nutrition, and tobacco smoking, induce alterations in the epigenome of children and adults, which may impact disease susceptibility. Other mechanisms for GxE interactions are also directly pertinent in early life. We discuss a model in which environmental factors imprint disease risk in a window of susceptibility during infancy that may contribute to later disease onset, whereas other elements of the exposome act later in life and contribute directly to the pathogenesis and course of the disease. Understanding the mechanisms underlying GxE interactions may provide the basis for new therapeutic targets or preventative strategies for IBD.

Periostin C-Terminal Is Intrinsically Disordered and Interacts with 143 Proteins in an In Vitro Epidermal Model of Atopic Dermatitis.

Human periostin is a 78-91 kDa matricellular protein implicated in extracellular matrix remodeling, tumor development, metastasis, and inflammatory diseases like atopic dermatitis, psoriasis, and asthma. The protein consists of six domains, including an N-terminal Cys-rich CROPT domain, four fasciclin-1 domains, and a C-terminal domain. The exons encoding the C-terminal domain may be alternatively spliced by shuffling four exons, generating ten variants of unknown function. Here, we investigate the structure and interactome of the full-length variant of the C-terminal domain with no exons spliced out. The structural analysis showed that the C-terminal domain lacked a tertiary structure and was intrinsically disordered. In addition, we show that the motif responsible for heparin-binding is in the conserved very C-terminal part of periostin. Pull-down confirmed three known interaction partners and identified an additional 140 proteins, among which nine previously have been implicated in atopic dermatitis. Based on our findings, we suggest that the C-terminal domain of periostin facilitates interactions between connective tissue components in concert with the four fasciclin domains.

Immune cell-specific smoking-related expression characteristics are revealed by re-analysis of transcriptomes from the CEDAR cohort.

Introduction: Smoking is known to affect whole-blood expression and methylation profiles. Although whole-genome methylation studies indicated that effects observed in blood may be driven by changes within leukocyte subtypes, these phenomena have not been explored using expression profiling. Material and methods: This study reanalyzed data from the Correlated Expression and Disease Association Research (CEDAR) patient cohort recruited by Momozawa et al. (E-MTAB-6667). Data from gene expression profiling of immunomagnetically sorted CD4+, CD8+, CD14+, CD15+, and CD19+ cells were processed. Differential expression analyses were conducted in each immune cell type, followed by gene ontology analysis and supplementary investigations. Results: Ninety-four differentially expressed genes were found (CD8+ n = 58, CD14+ n = 20, CD4+ n = 14, CD19+ n = 2). Two key smoking-related genes were overexpressed in specific cell types: LRRN3 (CD4+, CD8+) and MMP25 (CD8+, CD14+). In CD4+ cells smoking was associated with reduced expression of the NK cell receptor KLRB1, suggesting CD4+ subpopulation shifts and differences in interferon signaling (reduced IRF1 and IL18RAP in smokers). Key results and their integration with an immune protein-protein interaction network revealed that smoking influences integrins in CD8+ cells (ITGB7, ITGAL, ITGAM, ITGB2). C-type lectin CLEC4A was reduced in CD8+ cells and CLEC10A was increased in CD14+ cells from smokers; moreover, CLEC5A (CD8+), CLEC7A (CD8+) and CLEC9A (CD19+) were related to smoking in supplementary analyses. CD14+ cells from smokers exhibited overexpression of LDLR and the formyl peptide receptor FPR3. Conclusions: Smoking specifically alters vital immune regulation genes in lymphocyte subtypes, especially CD4+, CD8+ and CD14+ cells.

Lipid Peroxidation Products HNE and ONE Promote and Stabilize Alpha-Synuclein Oligomers by Chemical Modifications.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

Quantitating denaturation by formic acid: imperfect repeats are essential to the stability of the functional amyloid protein FapC.

Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of mFA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and mFA+pH We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid.

Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

Waiting in the wings: RUNX3 reveals hidden depths of immune regulation with potential implications for inflammatory bowel disease.

BACKGROUND: Complex interactions between the environment and the mucosal immune system underlie inflammatory bowel disease (IBD). The involved cytokine signalling pathways are modulated by a number of transcription factors, one of which is runt-related transcription factor 3 (RUNX3). OBJECTIVE: To systematically review the immune roles of RUNX3 in immune regulation, with a focus on the context of IBD. METHODS: Relevant articles and reviews were identified through a Scopus search in April 2020. Information was categorized by immune cell types, analysed and synthesized. IBD transcriptome data sets and FANTOM5 regulatory networks were processed in order to complement the literature review. RESULTS: The available evidence on the immune roles of RUNX3 allowed for its description in twelve cell types: intraepithelial lymphocyte, Th1, Th2, Th17, Treg, double-positive T, cytotoxic T, B, dendritic, innate lymphoid, natural killer and macrophages. In the gut, the activity of RUNX3 is multifaceted and context-dependent: it may promote homeostasis or exacerbated reactions via cytokine signalling and regulation of receptor expression. RUNX3 is mostly engaged in pathways involving ThPOK, T-bet, IFN-γ, TGF-ß/IL-2Rß, GATA/CBF-ß, SMAD/p300 and a number of miRNAs. RUNX3 targets relevant to IBD may include RAG1, OSM and IL-17B. Moreover, in IBD RUNX3 expression correlates positively with GZMM, and negatively with IFNAR1, whereas in controls, it strongly associates with TGFBR3. CONCLUSIONS: Dysregulation of RUNX3, mostly in the form of deficiency, likely contributes to IBD pathogenesis. More clinical research is needed to examine RUNX3 in IBD.

Automated Bowel Sound Analysis: An Overview.

Despite technological progress, we lack a consensus on the method of conducting automated bowel sound (BS) analysis and, consequently, BS tools have not become available to doctors. We aimed to briefly review the literature on BS recording and analysis, with an emphasis on the broad range of analytical approaches. Scientific journals and conference materials were researched with a specific set of terms (Scopus, MEDLINE, IEEE) to find reports on BS. The research articles identified were analyzed in the context of main research directions at a number of centers globally. Automated BS analysis methods were already well developed by the early 2000s. Accuracy of 90% and higher had been achieved with various analytical approaches, including wavelet transformations, multi-layer perceptrons, independent component analysis and autoregressive-moving-average models. Clinical research on BS has exposed their important potential in the non-invasive diagnosis of irritable bowel syndrome, in surgery, and for the investigation of gastrointestinal motility. The most recent advances are linked to the application of artificial intelligence and the development of dedicated BS devices. BS research is technologically mature, but lacks uniform methodology, an international forum for discussion and an open platform for data exchange. A common ground is needed as a starting point. The next key development will be the release of freely available benchmark datasets with labels confirmed by human experts.

Analysis of Gastrointestinal Acoustic Activity Using Deep Neural Networks.

Automated bowel sound (BS) analysis methods were already well developed by the early 2000s. Accuracy of ~90% had been achieved by several teams using various analytical approaches. Clinical research on BS had revealed their high potential in the non-invasive investigation of irritable bowel syndrome to study gastrointestinal motility and in a surgical setting. This article proposes a novel methodology for the analysis of BS using hybrid convolutional and recursive neural networks. It is one of the first methods of using deep learning to be widely explored. We have developed an experimental pipeline and evaluated our results with a new dataset collected using a device with a dedicated contact microphone. Data have been collected at night-time, which is the most interesting period from a neurogastroenterological point of view. Previous works had ignored this period and instead kept brief records only during the day. Our algorithm can detect bowel sounds with an accuracy >93%. Moreover, we have achieved a very high specificity (>97%), crucial in diagnosis. The results have been checked with a medical professional, and they successfully support clinical diagnosis. We have developed a client-server system allowing medical practitioners to upload the recordings from their patients and have them analyzed online. This system is available online. Although BS research is technologically mature, it still lacks a uniform methodology, an international forum for discussion, and an open platform for data exchange, and therefore it is not commonly used. Our server could provide a starting point for establishing a common framework in BS research.

Co-crystallization of an organic solid and a tetraaryladamantane at room temperature.

Tetraaryladamantanes have proven useful as chaperones for the co-crystallization of small molecules that do not readily crystallize by themselves. The co-crystals are often useful for structure elucidation. Usually, the small molecules are encapsulated in the crystal lattice of the aryladamantane that forms during rapid thermal crystallization. Thus far, co-crystallization has been limited to liquids as guest molecules. Here we report the co-crystal structures of phenol, which is solid at room temperature, with both 1,3,5,7-tetrakis(2,4-dimethoxyphenyl)adamantane (TDA) and 1,3,5,7-tetrakis(2,4-diethoxyphenyl)adamantane (TEO). The co-crystals were obtained from solutions in dichloromethane by slow evaporation or diffusion. The implications for generating other co-crystals of two solids are briefly discussed.

Fecal Fat Concentration and Excretion in the First 2 Years of Life: a Cross-Sectional Study.

OBJECTIVES: Data on pancreatic exocrine secretion in the youngest children are scarce. The aim of the study was to determine the range of normal values for fecal fat concentration (FFC) and fecal fat excretion (FFE) in infants and toddlers up to 2 years of age. METHODS: A total of 160 subjects aged 1 to 24 months (8 groups of 20: aged 1-3, 4-6 months, etc) were included in the study. In all children, FFC (%) and FFE (g/day) were assessed in 3-day stool collection. RESULTS: FFC correlated with age (r = -0.50, P < 0.0001). Of infants aged 1 to 3 months 65% had FFC higher than the <5% norm proposed for older children. The values of 90th/95th FFC percentile ranged from 9.7/13.0% at 1 month to 3.1/3.2% at 24 months. FFE did not differ between age groups. It was, however, higher than 4 and 3 g/day in 7.5% and 15.0% infants, respectively. The first detailed nomogram for the assessment of FFC and FFE in children aged 1 to 24 months was created. CONCLUSIONS: Healthy infants may have higher FFC and FFE than older children. We provide reference values, which should allow for both precise and facile FFC and FFE interpretation in clinical practice.

H-index in medicine is driven by original research.

AIM: To investigate the contribution of selected types of articles to h-indices of medical researchers. METHODS: We used the Web of Science to export the publication records of various members from 26 scientific medical societies (13 European, 13 North American) associated with 13 medical specialties. Those included were presidents (n=26), heads of randomly chosen committees (n=52), and randomly selected members of those committees (n=52). Publications contributing to h-index were categorized as research articles, reviews, guidelines, meta-analyses, or other published work. RESULTS: Overall, 3259 items authored by 129 scholars were analyzed. The median h-index was 19.5. The median contribution of research articles to h-index was 84.4%. Researchers in the upper h-index tercile (≥28.5) had a larger share of research articles that contributed to h-index in comparison with those in the lower h-index tercile (≤12.5) (median 87.3% [1st-3rd quartile: 80.0%-93.1%] vs 80.0% [50.0%-88.9%], P=0.015). We observed an analogous difference with regard to guidelines (1.1% [0%-3.7%] vs 0% [0%-0%], P=0.007). CONCLUSIONS: Original research drives h-indices in medicine. Although guidelines contribute to h-indices in medicine, their influence is low. The specific role of randomized controlled trials in building h-index in medicine remains to be assessed.

Increased Soluble VCAM-1 and Normal P-Selectin in Cystic Fibrosis: a Cross-Sectional Study.

PURPOSE: As life expectancy in cystic fibrosis (CF) increases, questions regarding its potential impact on cardiovascular health arise. Soluble vascular cell adhesion molecule 1 (sVCAM-1), P-selectin (sP-selectin) are proposed as biomarkers of cardiovascular disease. We aimed to: compare their concentrations in clinically stable CF patients and healthy subjects (HS) and verify whether they independently correlate with CF characteristics. METHODS: Serum sVCAM-1 and sP-selectin levels were measured using ELISA. CF was characterized using: forced expiratory volume in 1 s, exocrine pancreatic and CF-related liver disease status, Pseudomonas aeruginosa colonization, serum high-sensitivity C-reactive protein, and body mass index (BMI). CFTR genotypes were classified as severe (classes I and II) or other. RESULTS: 108 CF patients and 51 healthy subjects volunteered for the study. In the CF group BMI was lower (median [IQR]: 20.5 kg/m2 [18.4-22.2] vs. 21.6 kg/m2 [19.9-23.4], p = 0.02) and hsCRP levels were higher (3.6 mg/L [1.1-7.1] vs. 0.5 mg/dL [0.3-1.0], p < 10-10). While sVCAM-1 concentrations were greater in CF patients (1018 ng/mL [851-1279] vs. 861 ng/mL [806-979], p < 10-4), sP-selectin levels did not differ (155 ng/mL [129-188] vs. 156 ng/mL [144-177], p = 0.48). None of the multivariable regression models was valid for the prediction of sVCAM-1 and sP-selectin in CF. CONCLUSIONS: We found higher sVCAM-1 concentrations in CF patients than in healthy subjects, which were not explained by CF characteristics. Further research is required to check whether sVCAM-1 is a marker of microangiopathy in CF.

Determinants of Serum Glycerophospholipid Fatty Acids in Cystic Fibrosis.

The etiology of altered blood fatty acid (FA) composition in cystic fibrosis (CF) is understood only partially. We aimed to investigate the determinants of serum glycerophospholipids' FAs in CF with regard to the highest number of FAs and in the largest cohort to date. The study comprised 172 CF patients and 30 healthy subjects (HS). We assessed Fas' profile (gas chromatography/mass spectrometry), CF transmembrane conductance regulator (CFTR) genotype, spirometry, fecal elastase-1, body height and weight Z-scores, liver disease, diabetes and colonization by Pseudomonas aeruginosa. The amounts of saturated FAs (C14:0, C16:0) and monounsaturated FAs (C16:1n-7, C18:1n-9, C20:1n-9, C20:3n-9) were significantly higher in CF patients than in HS. C18:3n-6, C20:3n-6 and C22:4n-6 levels were also higher in CF, but C18:2n-6, C20:2n-6 and C20:4n-6, as well as C22:6n-3, were lower. In a multiple regression analysis, levels of seven FAs were predicted by various sets of factors that included age, genotype, forced expiratory volume in one second, pancreatic status and diabetes. FA composition abnormalities are highly prevalent in CF patients. They seem to be caused by both metabolic disturbances and independent clinical risk factors. Further research into the influence of CFTR mutations on fat metabolism and desaturases' activity is warranted.

Comprehensive Genomic Profiling Identifies a Subset of Crizotinib-Responsive ALK-Rearranged Non-Small Cell Lung Cancer Not Detected by Fluorescence In Situ Hybridization.

INTRODUCTION: For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. MATERIALS AND METHODS: Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. RESULTS: A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. CONCLUSION: Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. IMPLICATIONS FOR PRACTICE: Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.