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Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). Entamoeba histolytica , the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from E. histolytica . Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting K M and k cat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of EhFADS by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that EhFADS gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in E. histolytica , the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from E. histolytica for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against E. histolytica infections.

Gastroesophageal reflux disease (GERD) is commonly encountered in clinical practice in Sri Lanka. However, its prevalence in Sri Lanka is unknown. Our objective was to study the island-wide prevalence of GERD symptoms in Sri Lanka and its associated factors. A total of 1200 individuals aged 18-70 years (male: female 1: 1.16, mean age 42.7 years [SD 14.4 years]). were recruited from all 25 districts of the country, using stratified random sampling. An interviewer-administered, country-validated questionnaire was used to assess the GERD symptom prevalence and associated factors. Weight, height, waist, and hip circumference were measured. Heartburn and/or regurgitation at least once a week, an internationally used criterion for probable GERD was used to diagnose GERD. In this study, GERD symptom prevalence was 25.3% (male 42.1% and female 57.9%). Factors independently associated with GERD were inadequate sleep, snacking at midnight, sleeping within two hours of consuming a meal, skipping breakfast, increased mental stress, and certain medications used such as statins, and antihypertensive medications (p<0.001, univariate and logistic regression analysis). 38.4% of the study population have been using medication for heartburn and regurgitation in the past 3 months and 19.8% were on proton pump inhibitors. To conclude, the prevalence of GERD symptoms in Sri Lanka (25.3%) is higher than its estimated global prevalence of 13.8%. Several meal-related lifestyle habits, mental stress, and the use of some medications are significantly associated with GERD, indicating the importance of lifestyle modification and stress reduction in its management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wickramasinghe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Supragastric belching (SGB) and aerophagia are behavioral disorders characterized by air induced esophageal distension. SGB is known to be associated with Gastro Esophageal Reflux Disease (GERD). Low Mean Nocturnal Baseline Impedance (MNBI) values support GERD diagnosis. We aimed to assess if chronic esophageal distension by air affects the esophageal mucosa integrity by assessing changes in MNBI., Methods: In a single-center database study, we searched retrospectively for patients with a diagnosis of pathological SGB (n = 146) or aerophagia (n = 34) based on impedance-pH reflux monitoring. During the examined period, patients with a conclusive negative diagnosis of SGB and no evidence of aerophagia were used as a control cohort (n = 191). MNBI at 3, 5, and 17 cm over Lower Esophageal Sphincter (LES) was evaluated. GERD was diagnosed if acid exposure time (AET) >6%. All impedance studies of included patients were prospectively reevaluated., Results: GERD was diagnosed in 31.7% patients with SGB, a rate not different in comparison to patients without SGB (30.8%, p = 0.906). MNBI at 3 and 5 cm above the LES was significantly decreased among patients with SGB. SGB was not correlated with MNBI at 3 cm over the LES, (p: 0.086 OR: 1.000 95% CI: 0.999-1.001) when using multivariate analysis. Moreover no difference was spotted as far as MNBI at 3, 5, and 17 cm over the LES is concerned among patients with or without aerophagia., Conclusion: Even if patients with SGB do show lower MNBI values, esophageal distention due to excessive air movement does not directly lead to impairment of esophageal mucosa integrity., (© 2023 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

DNA polymerases synthesize DNA from deoxyribonucleotides in a semiconservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are 2 genome-containing organelles, mitochondria, and plastids, which were derived from an alphaproteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNA polymerases that localize and work in them to maintain their genomes. The evolution of organellar DNA polymerases has yet to be fully understood because of 2 unsettled issues. First, the diversity of organellar DNA polymerases has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNA polymerases that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNA polymerases known to date show no phylogenetic affinity to those of the extant alphaproteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNA polymerase sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNA polymerases were further examined experimentally. The results presented here suggest that the diversity of organellar DNA polymerases has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed 2 mitochondrial DNA polymerases, POP, and a candidate of the direct descendant of the proto-mitochondrial DNA polymerase I, rdxPolA, identified in this study., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Background: Mechanisms underlying perception of dysphagia and chest pain have not been completely elucidated, although oesophageal mucosal afferent nerves might play an important role., Aims: To evaluate the relationship between oesophageal mucosal afferent nerves and the severity of dysphagia and chest pain in oesophageal motility disorders., Methods: We prospectively recruited patients with oesophageal motility disorders having dysphagia and/or chest pain from whom oesophageal biopsies were obtained. High-resolution manometry classified patients into disorders of oesophagogastric junction (OGJ) outflow and disorders of peristalsis. Symptom severity was assessed using validated questionnaires including Brief Oesophageal Dysphagia Questionnaire (BEDQ). Immunohistochemistry was performed on oesophageal biopsies to evaluate the location of calcitonin gene-related peptide (CGRP)-immunoreactive mucosal afferent nerves. Findings were compared to existing data from 10 asymptomatic healthy volunteers., Results: Of 79 patients, 61 patients had disorders of OGJ outflow and 18 had disorders of peristalsis. CGRP-immunoreactive mucosal nerves were more superficially located in the mucosa of patients with oesophageal motility disorders compared to healthy volunteers. Within disorders of OGJ outflow, the location of CGRP-immunoreactive nerves negatively correlated with BEDQ score both in the proximal (ρ = -0.567, p < 0.001) and distal oesophagus (ρ = -0.396, p = 0.003). In the proximal oesophagus, strong chest pain was associated with more superficially located mucosal nerves than weak chest pain (p = 0.04). Multivariate analysis showed superficial nerves in the proximal oesophagus was independently associated with severe dysphagia in disorders of OGJ outflow (p = 0.008)., Conclusions: Superficial location of mucosal nerves in the proximal oesophagus might contribute to symptoms, especially severe dysphagia, in disorders of OGJ outflow., (© 2023 John Wiley & Sons Ltd.)

Most species belonging to the diplomonad genera, Trepomonas and Hexamita , are considered to have secondarily adapted to free-living lifestyles from the parasitic ancestor. Here, we report the annotated transcriptome data of Trepomonas sp. NIES-1444 and Hexamita sp. NIES-1440, the analysis of which will provide insights into the lifestyle transitions., Competing Interests: The authors declare no conflict of interest.

Background: Reflux hypersensitivity (RH) is characterized by normal esophageal exposure to acid and positive correlation of symptoms to reflux episodes. Positivity of Symptomatic Index (SI) and/or Symptom Association Probability (SAP) is used diagnostically, though experts support that concordance of both is needed. We evaluated differences among patients with RH and concordance of SI/SAP or not., Methods: Patients with typical reflux symptoms without previous GERD diagnosis, submitted simultaneously to Ph-Impedance off PPI and high resolution manometry were included. Self-response to PPI was evaluated. Patients showing SI and/or SAP positivity were considered having RH and further classified to definite RH if both SI/SAP were positive or indefinite if only one positive., Key Results: Totally 2659 patients (M/F: 35.6%/64.7%, mean age: 45 ± 14) were included. Final diagnosis was; FH: 21.8%, RH: 29.3% (definite: 14.3%/indefinite: 15%), GERD: 36% and inconclusive GERD: 12.9%. Patients with definite RH showed increased total reflux time, total number of reflux episodes and length of hiatus hernia, and also numerically but not statistically significant increased rates of PPI responsiveness versus indefinite RH and decreased mean nocturnal baseline impedance. Moreover, they showed significantly increased rate of PPI response versus patients with functional heartburn (FH)., Conclusion & Inferences: Some PPI responsiveness is frequent among patients with RH as also with FH, and cannot discriminate those entities clinically when diagnosing RH using SI and/or SAP positive criterion. Patients with RH and SI/SAP concordance differ from patients without. Implementation of a strict RH definition (both SI and SAP positive) can better distinguish RH from FH and should be used in the future., (© 2023 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Background/aims: Stress is a known associated factor for gastroesophageal reflux disease (GERD). However, the dynamics between stress and GERD are not fully studied, especially in Sri Lanka. Our objective was to assess it., Methods: For this cross-sectional descriptive study, 1200 individuals (age ranged 18-70 years, mean 42.7 years [SD 14.4 years], 46.1% males), were recruited using stratified random cluster sampling from all 25 districts of Sri Lanka. An interviewer-administered questionnaire, which included a country-validated GERD symptom screening tool, and the Perceived Stress Scale (PSS), was used to assess GERD symptoms and stress. Probable GERD was defined as those having heartburn and/ or regurgitation at least once per week which is on par with globally accepted criteria. Those who did not fulfill these criteria were considered as controls., Results: PSS score was higher in those with probable GERD (mean 13.75 [standard deviation (SD) 6.87]) than in controls (mean 10.93 [SD 6.80]), (p <0.001, Mann-Whitney U test). The adjusted odds ratio for GERD symptoms was 1.96 times higher (95% confidence interval 1.50-2.55) in the moderate to high-stress level compared to the low-stress level participants. PSS score correlated significantly with the GERD screening tool score (R 0.242, p <0.001). Heartburn, regurgitation, chest pain, cough, and burping were significantly frequent in those with moderate to high-stress levels (p <0.001). Those with higher stress scores were more likely to use acid-lowering drugs (p = 0.006)., Conclusions: Individuals exposed to higher levels of stress are more likely to have GERD symptoms. Therefore, stress reduction should be an important part of GERD symptom management., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wickramasinghe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The esophagogastric junction (EGJ) is classified into 3 anatomical subtypes according to lower esophageal sphincter-crural diaphragm (LES-CD) separation. We aimed to assess their relationship to esophageal motility, reflux characteristics, and symptom perception., Methods: We analyzed data from 1740 consecutive patients with typical reflux symptoms, who underwent high resolution manometry and a 24-h pH-impedance study during a 13-year period. A diagnosis of gastroesophageal reflux disease (GERD) was made if acid exposure time (AET) was >6%. EGJ types were classified as 1, 2, or 3, if LES-CD separation was up to 1 cm, 1-3 cm, or ≥3 cm, respectively., Results: EGJ type distribution was 72.2%, 22.1% and 5.7%, for types 1, 2 and 3, respectively. GERD was diagnosed in 31.2% and was more common among patients with EGJ type 2/3 vs. 1 (P<0.001). Length of LES-CD separation significantly correlated with AET and number of reflux episodes. Patients with type 2 or 3 EGJ more often showed ineffective or absent peristalsis compared with type 1 (P=0.008 and P<0.001 respectively). In the multivariate analysis, EGJ type 2/3 correlated with AET (P=0.001) and reflux episodes (P=0.041) but not with positive symptomatic markers or with ineffective/absent peristalsis., Conclusions: Our study confirms that EGJ anatomical morphology is a strong risk factor for GERD and correlates with both AET and the number of reflux events, though the length of separation is more important than the type. The multivariate analysis revealed that EGJ type 2 or 3 was not correlated with symptom perception or esophageal hypomotility., Competing Interests: Conflict of Interest: None, (Copyright: © Hellenic Society of Gastroenterology.)

Aim: Low mean nocturnal baseline impedance (MNBI) values support gastroesophageal reflux disease (GERD) diagnosis. Recent data denote that age and obesity may affect MNBI. We aimed to evaluate diagnostic MNBI cutoffs as also the effect of aging and body mass index (BMI) on MNBI., Methods: In total 311 patients (M/F: 139/172, mean age: 47 ± 13) referred for typical GERD symptoms that have undertaken both high-resolution manometry (HRM) and pH-Impedance studies off PPI were evaluated. MNBI at 3, 5, and 17 cm over lower esophageal sphincter (LES) were evaluated. GERD was diagnosed if acid exposure time (AET) >6%., Results: Mean BMI was 26.6 ± 5.9 kg/cm 2 . GERD was diagnosed in 39.2% and 13.5% had inconclusive GERD. MNBI was correlated to patients' age, BMI, AET, and the length of LES-CD separation and at 3 cm also to the total number of reflux and LES hypotension. In the multivariate analysis MNBI at 3 and 5 cm was independently correlated only to age, BMI, and AET. Patients with definite GERD showed lower MNBI at 3 cm compared with inconclusive GERD though both showed lower values when compared with GERD absence. At 3 cm MNBI ability for diagnosing GERD was good (0.815, p < 0.001 95% CI: 0.766-0.863) with an optimal cutoff point of 1281 Ohm., Conclusion: According to our study findings age and BMI affect independently lower esophageal MNBI values in patients evaluated for GERD. MNBI significantly aids toward GERD diagnosis though in a real-life setting MNBI values much lower than the one previously proposed should be used., (© 2023 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role., Results: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication., Conclusions: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa., (© 2023. The Author(s).)

Background: Supragastric belching (SGB) is a phenomenon where air is rapidly sucked from the pharynx into the esophagus and immediately expelled through abdominal straining. It is considered a behavior disorder and is increasingly recognized not only in patients with excessive belching, but also in those with reflux-like symptoms. Increased prevalence of esophageal hypomotility and increased acid exposure were previously reported in small cohorts of SGB patients. We aimed to clarify the impact of SGB on motility, reflux, and acid exposure in a large cohort of SGB patients., Methods: In a single-center database study, we searched for patients with pathological SGB. MII-pH and Manometry tracings were manually re-evaluated in all patients. Demographic, clinical, motility, reflux, and SGB-related data were gathered., Key Results: Three hundred and forty-eight patients were included. Heartburn, belching, and regurgitation were the most common symptoms. Ineffective esophageal motility (IEM) was found in 27% of patients. SGB related to 47% of all reflux and to 53.6% of acid reflux events, and accounted for 27.3% of acid exposure time (AET). In those with severe SGB, 62% of acid reflux events and 46% of AET were SGB-related., Conclusions & Inferences: Supragastric belching is common, associated with higher incidence of IEM and is responsible for almost a third of esophageal acid burden. The impact of SGB is proportional to its severity. Diagnosis of SGB should be sought in patients with excessive belching and in patients with refractory reflux symptoms. Recognizing SGB and treating patients with behavioral therapy may alleviate acid exposure and improve quality of life., (© 2022 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought. Abbreviations : APEAR: ATG8-PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Dinoflagellates possess plastids that are diverse in both pigmentation and evolutionary background. One of the plastid types found in dinoflagellates is pigmented with chlorophylls a and b (Chl a + b ) and originated from the endosymbionts belonging to a small group of green algae, Pedinophyceae. The Chl a + b -containing plastids have been found in three distantly related dinoflagellates Lepidodinium spp., strain MGD, and strain TGD, and were proposed to be derived from separate partnerships between a dinoflagellate (host) and a pedinophycean green alga (endosymbiont). Prior to this study, a plastid genome sequence was only available for L. chlorophorum , which was reported to bear the features that were not found in that of the pedinophycean green alga Pedinomonas minor , a putative close relative of the endosymbiont that gave rise to the current Chl a + b -containing plastid. In this study, we sequenced the plastid genomes of strains MGD and TGD to compare with those of L. chlorophorum as well as pedinophycean green algae. The mapping of the RNA-seq reads on the corresponding plastid genome identified RNA editing on plastid gene transcripts in the three dinoflagellates. Further, the comparative plastid genomics revealed that the plastid genomes of the three dinoflagellates achieved several features, which are not found in or much less obvious than the pedinophycean plastid genomes determined to date, in parallel., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Matsuo, Morita, Nakayama, Yazaki, Sarai, Takahashi, Iwataki and Inagaki.)

By clarifying the phylogenetic positions of 'orphan' protists (unicellular micro-eukaryotes with no affinity to extant lineages), we may uncover the novel affiliation between two (or more) major lineages in eukaryotes. Microheliella maris was an orphan protist, which failed to be placed within the previously described lineages by pioneering phylogenetic analyses. In this study, we analysed a 319-gene alignment and demonstrated that M. maris represents a basal lineage of one of the major eukaryotic lineages, Cryptista. We here propose a new clade name 'Pancryptista' for Cryptista plus M. maris . The 319-gene analyses also indicated that M. maris is a key taxon to recover the monophyly of Archaeplastida and the sister relationship between Archaeplastida and Pancryptista, which is collectively called 'CAM clade' here. Significantly, Cryptophyceae tend to be attracted to Rhodophyta depending on the taxon sampling (ex., in the absence of M. maris and Rhodelphidia) and the particular phylogenetic 'signal' most likely hindered the stable recovery of the monophyly of Archaeplastida in previous studies.

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background & Aims: Reflux hypersensitivity (RH), a functional esophageal disorder, is detected in 14%-20% of patients who present with typical esophageal symptoms. As many as 40% of patients with RH do not respond to treatment with pain modulators or proton pump inhibitors (PPIs); behavior disorders might contribute to lack of treatment efficacy. We aimed to assess the prevalence of behavioral disorders and their effects on typical reflux symptoms in patients with RH., Methods: We performed a retrospective study of 542 patients with PPI-refractory esophageal symptoms (heartburn, regurgitation, or chest pain) or with symptoms that responded to PPI therapy, evaluated for anti-reflux surgery from January 2016 through August 2019 at a single center in London, United Kingdom. We collected data on symptoms, motility, and impedance-pH monitoring and assigned patients to categories of RH (n = 116), functional heartburn (n = 126), or non-erosive reflux disease (n = 300)., Results: Of the 116 patients with a diagnosis of RH, 59 had only hypersensitivity, whereas 57 patients (49.2%) had either excessive supragastric belching (SGB, 39.7%), based on 24-hour impedance-pH monitoring, or rumination (9.5%), based on postprandial manometry combined with impedance. The prevalence of SGB and rumination in patients with RH was significantly higher than in patients with functional heartburn (22%; P < .001). Patients with RH and rumination were significantly younger (P = .005) and had the largest number of non-acid reflux episodes (P = .023). In patients with RH with SGB, SGB episodes were associated with 40.6% of marked reflux symptoms (heartburn, regurgitation, or chest pain), based on impedance-pH monitoring. In patients with RH and rumination, 40% of reflux-related symptoms (mostly regurgitation) were due to possible rumination episodes., Conclusions: Almost half of patients with a diagnosis of RH have behavior disorders, including excessive SGB or rumination. Episodes of SGB or rumination are associated with typical reflux symptoms. Segregation of patients with diagnosis of RH into those with vs without behavioral disorders might have important therapeutic implications., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Paraesophageal hernias (PEH) can be associated with obstructive symptoms, but high-resolution manometry (HRM) characteristics have not been described in detail., Methods: HRM studies of confirmed PEH patients (n = 60, 66.3 ± 1.5 years, 76.7% F), axial hernias (n = 125, 56.1 ± 1.1 years, 58.4% F), and healthy controls (n = 20, 27.9 ± 0.7 years, 45% F) were analyzed. Axial hernias (type 1) were compared to PEH subtyped as isolated PEH (type 2), PEH with axial hernia (type 3), PEH with herniated abdominal organs (type 4), and unknown. Distal contractile integral (DCI), esophageal length, lower esophageal sphincter pressures (LESP), EGJ contractile integral (EGJ-CI), and integrated relaxation pressure (IRP) were extracted. Intra-luminal pressures were measured proximal (intrabolus pressure, IBP) and distal (intragastric pressure, IGP) to EGJ. Univariate and multivariate analyses were performed to characterize esophageal and EGJ motor physiology in PEH., Key Results: PEH patients had LESP and EGJ-CI similar to controls; axial hernia patients had lower LESP. While IRP was within normal limits, PEH had elevated IBP and IGP, and shorter esophageal length compared to axial hernia and normal controls (P ≤ .0001 across groups). Short esophageal length was consistent across PEH subtypes (P = .06). On multivariate regression analysis, IBP remained an independent predictor of PEH (P < .0001). Within PEH subtypes, gastric pressure was higher when axial HH was also present, in contrast to isolated PEH (P = .03); other metrics did not differ., Conclusions & Inferences: Luminal pressure increase both upstream and downstream of the EGJ on HRM likely represents obstructive phenomena in PEH, identification of which may support suspicion of PEH., (© 2020 John Wiley & Sons Ltd.)

Background: Pathophysiology of rumination syndrome (RS) is not well understood. Treatment with diaphragmatic breathing improves rumination syndrome. The aim of the study was to characterize vagal tone in patients with rumination syndrome during and after meals and during diaphragmatic breathing., Methods: We prospectively recruited 10 healthy volunteers (HV) and 10 patients with RS. Subjects underwent measurement of vagal tone using heart rate variability. Vagal tone was measured during baseline, test meal and intervention (diaphragmatic (DiaB), slow deep (SlowDB), and normal breathing). Vagal tone was assessed using mean values of root mean square of successive differences (RMSSD), and area under curves (AUC) were calculated for each period. We compared baseline RMSSD, the AUC and meal-induced discomfort scores between HV and RS. Furthermore, we assessed the effect of respiratory exercises on symptom scores, and number of rumination episodes., Key Results: There was no significant difference in baseline vagal tone between HV and RS. During the postprandial period, there was a trend to higher vagal tone in RS, but not significantly (P > .2 for all). RS had the higher total symptom scores than HV (P < .011). In RS, only DiaB decreased the number of rumination episodes during the intervention period (P = .028), while both DiaB and SlowDB increased vagal tone (P < .05 for both). The symptom scores with the 3 breathing exercises showed very similar trends., Conclusions and Inferences: Patients with RS do not have decreased vagal tone related to meals. DiaB reduced number of rumination events by a mechanism not related to changes in vagal tone., (© 2020 John Wiley & Sons Ltd.)

Background: Supragastric belching (SGB) may play a role in the pathophysiology of proton pump inhibitors (PPIs)-refractoriness in gastroesophageal reflux disease (GERD). SGB may be present in up to 40% of reflux symptoms in PPI-refractory GERD. Most reports on SGB have come from Western countries, and little is known about the prevalence and relevance of SGB in Asian refractory GERD patients. This study aimed at comparing the role of SGB in GERD patients in Japan and the UK., Methods: We re-analyzed impedance-pH monitoring tracings from patients who were referred to tertiary centers in Japan and the UK due to PPI-refractory reflux symptoms. The prevalence of excessive SGB and the impact of SGB on reflux symptoms were compared between the two countries., Results: Impedance-pH tracings from124 Japanese and 83 British patients were re-analyzed. Japanese patients were significantly younger and had smaller body mass index than the British (P < 0.001). Japanese patients had significantly lower prevalence of excessive SGB (18.5%) than the UK (36.1%) irrespective of reflux phenotype (P = 0.006). Logistic regression analysis showed that the geographical/cultural difference was the only factor associated with the different prevalence of SGB (odds ratio; 2.91, 95% CI 1.09-7.73, P = 0.032). SGB were related to typical reflux symptoms very rarely in Japan [0% (0-4.9)] compared to the UK [35% (0-54.1)] (P = 0.071)., Conclusions: The prevalence of SGB and their impact on reflux symptoms is significantly lower in Japan compared to the UK. The difference is not related to reflux parameters but might come from ethnic/cultural factors to be further characterized.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

Background/aims: Mean nocturnal baseline impedance (MNBI) during multichannel intraluminal impedance pH-monitoring (MII-pH) reflects the status of esophageal mucosal integrity. MNBI is suggested as an adjunctive method to distinguish patients with true gastroesophageal reflux disease (GERD) from functional heartburn (FH) and might predict outcomes for anti-reflux treatment. However, current methodology for calculation of MNBI is time-consuming and subject to operator-dependent selection bias. We aim to simplify and provide a more objective method to calculate MNBI., Methods: We retrospectively analyzed 100 MII-pH tracings from 20 patients with erosive reflux disease, 20 with non-erosive reflux disease (NERD), 20 with reflux hypersensitivity, 20 with functional heartburn (FH), and 20 healthy asymptomatic volunteers. We compared the current "conventional" MNBI analysis with our "simple" MNBI analysis measured by selecting the whole supine period using the impedance average calculation function in the MII-pH software., Results: Absolute values were very similar and there was a strong correlation between conventional and simple MNBI values in the most distal channel in all groups ( r ≥ 0.8, P < 0.001) including patients with increased supine acid reflux. Distal esophageal simple MNBI negatively correlated with acid exposure time ( r = -0.695, P < 0.001). Patients with erosive reflux disease and NERD had lower simple MNBI values in the most distal channel compared to other groups ( P < 0.001). With a cutoff value of 1785 ohms, simple MNBI can discriminate patients with GERD from those with reflux hypersensitivity and FH (sensitivity 80.0% and specificity 89.7%)., Conclusion: Simple MNBI analysis provides very similar values and has an excellent correlation with conventional MNBI analysis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis., Competing Interests: The authors declare no competing interest.

Background: Recent studies reported that impaired proximal duodenal mucosa, assessed by duodenal biopsy, could play an important role in the development of dyspeptic symptoms. The aims of this study were (a) to develop a method to measure "in vivo" duodenal and jejunal baseline impedance (BI) and (b) to assess small bowel mucosal integrity in patients with functional dyspepsia (FD) and healthy controls (HC)., Methods: We recruited 16 patients with FD and 15 HC. All subjects underwent ambulatory duodeno-jejunal manometry combined with impedance (HRM/Z), BI were determined by measuring impedance immediately after the passage of nocturnal migrating motor complex (MMC) phase IIIs., Results: The number of MMC phase IIIs in FD was significantly lower than that in HC (2.6 ± 1.4 vs 4.8 ± 1.7, p < 0.001). The BI in patients was significantly lower than that in HC in D1(164.2 ± 59.8 Ω in FD and 243.1 ± 40.5 Ω in HC, p = 0.0061), D2 (191.2 ± 34.1 and 256.5 ± 91.4 Ω, p = 0.01), D3 (214.0 ± 76.9 and 278.1 ± 45.3 Ω, p = 0.009), D4 (270.8 ± 54.2 and 351.8 ± 50.2 Ω, p < 0.001), and J1 (312.2 ± 55.4 and 379.3 ± 38.3 Ω, p = 0.001)., Conclusions: This is the first study reporting the duodenal and jejunal BI in vivo. The results have shown significantly lowered BI in the proximal small intestine in patients with FD compared to HC. Furthermore it suggests that measurements of small bowel BI could be used as a biomarker for diagnosis and follow up of patients with FD.

Objectives: Up to 40% of children presenting with reflux symptoms do not respond to standard medical interventions. In adults, 20% of patients presenting with Proton Pump Inhibitors refractory Gastro-Esophageal Reflux Disease (GERD) have rumination syndrome. The management of GERD and rumination differ significantly. Our study aimed to identify rumination syndrome amongst children presenting with persistent GERD symptoms based on a distinct pattern on impedance-pH monitoring., Methods: The parameters of impedance-pH monitoring were compared between children with rumination syndrome (n = 12), diagnosed on high-resolution manometry impedance (HRM/Z), children with GERD (n = 18), children with an alternative diagnosis (non-GERD, n = 12) and children negative for rumination based on HRM/Z (n = 14). The parameters that distinguish the rumination group were identified and incorporated into a scoring system, which was blindly applied on a separate group of children with refractory GERD (n = 18) to define its sensitivity and specificity., Results: Rumination syndrome presents in 44% of children with refractory GERD. Children with rumination syndrome present with a large number of proximal reflux episodes (>57.5 episodes/24 hours); a high frequency of nonacid reflux events in the postprandial period (>2/hour); and a highly positive symptom-reflux association analysis (SAP ≥ 95%). A score of ≥2 out of the 3 points distinguishes children with rumination syndrome with 75% sensitivity and 80% specificity., Conclusions: Children with rumination syndrome have a distinct pattern of impedance-pH monitoring and can be distinguished amongst children presenting with refractory GERD. Applying a simple scoring system during impedance-pH analysis could lead to early diagnosis of children with rumination syndrome.

Background: Discontinuation of long-term proton pump inhibitors (PPIs) on patients with reflux symptoms can be challenging, as symptoms often exacerbate after stopping. The mechanism remains unknown. Our aim was to evaluate the impact of stopping long-term PPIs on patients with heartburn, and its association with esophageal acid exposure., Methods: Patients with heartburn on long-term PPIs underwent symptom questionnaire, high-resolution manometry, and 24h ambulatory impedance-pH studies, following a 7-day PPIs discontinuation. We investigated the association between exacerbation of symptoms and findings on ambulatory reflux studies., Key Results: We studied 37 patients. After stopping PPIs, 27 patients (73%) had exacerbation of heartburn. Esophageal acid exposure time% (AET) in patients with exacerbation of heartburn was not significantly higher than in patients without (3.5% [1.3-9.7] vs 2.5% [1.3-8.7], NS). Fourteen of 27 patients with exacerbation had physiological AET (<4%) as compared with 6 of 10 patients with physiological AET (NS). All questioned symptoms (heartburn, regurgitation, epigastric discomfort/pain, bloating/belch) worsened after stopping PPIs (NS)., Conclusions & Inferences: Exacerbation of heartburn after discontinuation of PPIs does not appear to be due to increased esophageal acid exposure., (© 2019 John Wiley & Sons Ltd.)

Objectives: Up to 20% of patients with refractory gastroesophageal reflux disease (GERD) might have postprandial rumination. The aim of this study was to distinguish persistent GERD-related postprandial regurgitation from rumination in patients with refractory GERD undergoing ambulatory multichannel intraluminal impedance-pH (MII-pH) monitoring., Methods: We first characterized 24-hour and postprandial MII-pH patterns in 28 consecutive patients with confirmed rumination syndrome (positive clinical and high-resolution manometry/impedance). We compared such MII-pH patterns with those from 30 patients with typical GERD symptoms (10 nonerosive reflux disease, 10 hyperactive esophagus, and 10 functional heartburn) and 27 healthy controls. Using ROC curves, we selected the best MII-pH parameters to prepare an MII-pH rumination score. We prospectively tested the performance of the new MII-pH rumination score in 26 consecutive patients with refractory GERD (predominant regurgitation)., Results: Compared with GERD controls, patients with rumination were more often females, younger, and had significantly more postprandial early nonacid reflux episodes with high proximal extent. Postprandial reflux in ruminators had a distinct nadir pH profile over time (from nonacid to acid). Despite increased reflux events, baseline impedance in ruminators was similar to that in healthy subjects. Ruminators marked postprandial symptoms earlier and much more often than patients with GERD. An MII-pH-based rumination score (using postprandial nonacid reflux/hour and Symptom Index) diagnosed rumination in 46% of patients with refractory GERD and persistent regurgitation (sensitivity 91.7% and specificity 78.6%)., Discussion: Postprandial rumination is very common in refractory GERD with persistent regurgitation. A simple MII-pH score detects rumination in these patients with high sensitivity and specificity.

Background/aims: Single swallow integrated relaxation pressure (IRP) on high-resolution manometry (HRM) does not always accurately predict esophagogastric outflow obstruction on timed barium esophagogram (TBE). Furthermore, neither single swallow IRP or TBE is reliable in predicting symptoms, particularly after treatment with dilatation or myotomy. A 200 mL rapid drink challenge (RDC) has been proposed as an adjunctive test during HRM. This serves as a "stress-test" to the esophagogastric junction, and may yield clinically useful parameters. We aim to assess HRM parameters during RDC, and their ability to predict outflow obstruction on TBE in patients with dysphagia, and to correlate with symptoms in patients' achalasia., Methods: Thirty patients with dysphagia were recruited. All underwent standard single swallow HRM analysis, 200 mL RDC, then TBE. RDC parameters, including esophagogastric pressure gradient, IRP, and RDC duration were evaluated. Multiple regression analysis was performed to assess the best predictive parameter for obstruction on TBE. A further 21 patients with achalasia were evaluated with Eckhardt score, single swallow HRM, RDC, and TBE. Parameter correlation with Eckhardt score was evaluated., Results: Mean IRP during RDC was the best HRM parameter at predicting outflow obstruction on TBE. This performed much better in untreated patients (sensitivity 100% and specificity 85.5%) than in previously treated patients (sensitivity 50% and specificity 66%). In patients with achalasia, mean IRP during RDC was the only parameter that correlated with symptom score., Conclusion: Mean IRP during RDC appears to be a clinically useful "stress test" to the esophagogastric junction during HRM.

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome-one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte-derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid-type glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte-derived tertiary plastids: "gapC1h" acquired from the haptophyte endosymbiont and "gapC1p" inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid-type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT-derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Objectives: Excessive supragastric belching (SGB) manifests as troublesome belching, and can be associated with reflux and significant impact on quality of life (QOL). In some GERD patients, SGB-associated reflux contributes to up to 1/3 of the total esophageal acid exposure. We hypothesized that a cognitive-behavioral intervention (CBT) might reduce SGB, improve QOL, and reduce acid gastroesophageal reflux (GOR). We aimed to assess the effectiveness of CBT in patients with pathological SGB., Methods: Patients with SGB were recruited at the Royal London Hospital. Patients attended CBT sessions focused on recognition of warning signals and preventative exercises. Objective outcomes were the number of SGBs, esophageal acid exposure time (AET), and proportion of AET related to SGBs. Subjective evaluation was by patient-reported questionnaires., Results: Of 51 patients who started treatment, 39 completed the protocol, of whom 31 had a follow-up MII-pH study. The mean number of SGBs decreased significantly after CBT (before: 116 (47-323) vs. after 45 (22-139), P<0.0003). Sixteen of 31 patients were shown to have a reduction in SGB by >50%. In patients with increased AET at baseline, AET after CBT was decreased: 9.0-6.1% (P=0.005). Mean visual analog scale severity scores decreased after CBT (before: 260 (210-320) mm vs. after: 140 (80-210) mm, P<0.0001)., Conclusions: Cognitive behavioral therapy reduced the number of SGB and improved social and daily activities. Careful analysis of MII-pH allows identification of a subgroup of GERD patients with acid reflux predominantly driven by SGB. In these patients, CBT can reduce esophageal acid exposure.

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering.

All members of the order Trypanosomatida known to date are parasites that are most likely descendants of a free-living ancestor. Trypanosomatids are an excellent model to assess the transition from a free-living to a parasitic lifestyle, because a large amount of experimental data has been accumulated for well-studied members that are harmful to humans and livestock (Trypanosoma spp. and Leishmania spp.). However, recent advances in our understanding of the diversity of trypanosomatids and their close relatives (i.e., members of the class Kinetoplastea) have suggested that the change in lifestyle took place multiple times independently from that which gave rise to the extant trypanosomatid parasites. In the current study, transcriptomic data of two parasitic kinetoplastids belonging to orders other than Trypanosomatida, namely Azumiobodo hoyamushi (Neobodonida) and Trypanoplasma borreli (Parabodonida), were generated. We re-examined the transition from a free-living to a parasitic lifestyle in the evolution of kinetoplastids by combining (i) the relationship among the five orders in Kinetoplastea and (ii) that among free-living and parasitic species within the individual orders. The former relationship was inferred from a large-scale multigene alignment including the newly generated data from Azumiobodo and Trypanoplasma, as well as the data from another parasitic kinetoplastid, Perkinsela sp., deposited in GenBank; and the latter was inferred from a taxon-rich small subunit ribosomal DNA alignment. Finally, we discuss the potential value of parasitic kinetoplastids identified in Parabodonida and Neobodonida for studying the evolutionary process that turned a free-living species into a parasite.

Functionally and morphologically degenerate mitochondria, so-called mitochondrion-related organelles (MROs), are frequently found in eukaryotes inhabiting hypoxic or anoxic environments. In the last decade, MROs have been discovered from a phylogenetically broad range of eukaryotic lineages and these organelles have been revealed to possess diverse metabolic capacities. In this study, the biochemical characteristics of an MRO in the free-living anaerobic protist Cantina marsupialis, which represents an independent lineage in stramenopiles, were inferred based on RNA-seq data. We found transcripts for proteins known to function in one form of MROs, the hydrogenosome, such as pyruvate:ferredoxin oxidoreductase, iron-hydrogenase, acetate:succinate CoA-transferase, and succinyl-CoA synthase, along with transcripts for acetyl-CoA synthetase (ADP-forming). These proteins possess putative mitochondrial targeting signals at their N-termini, suggesting dual ATP generation systems through anaerobic pyruvate metabolism in Cantina MROs. In addition, MROs in Cantina were also shown to share several features with canonical mitochondria, including amino acid metabolism and an "incomplete" tricarboxylic acid cycle. Transcripts for all four subunits of complex II (CII) of the electron transport chain were detected, while there was no evidence for the presence of complexes I, III, IV, or F1Fo ATPase. Cantina MRO biochemistry challenges the categories of mitochondrial organelles recently proposed., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background & Aims: Management of patients with dysphagia, regurgitation, and related symptoms after antireflux surgery is challenging. This prospective, case-control study tested the hypothesis that compared with standard high-resolution manometry (HRM) with single water swallows (SWS), adding multiple water swallows (MWS) and a solid test meal increases diagnostic yield and clinical impact of physiological investigations., Methods: Fifty-seven symptomatic and 12 asymptomatic patients underwent HRM with SWS, MWS, and a solid test meal. Dysphagia and reflux were assessed by validated questionnaires. Diagnostic yield of standard and full HRM studies with 24-hour pH-impedance monitoring was compared. Pneumatic dilatation was performed for outlet obstruction on HRM studies. Clinical outcome was assessed by questionnaires and an analogue scale with "satisfactory" defined as at least 40% symptom improvement requiring no further treatment., Results: Postoperative esophagogastric junction pressure was similar in all groups. Abnormal esophagogastric junction morphology (double high pressure band) was more common in symptomatic than in control patients (13 of 57 vs 0 of 12, P = .004). Diagnostic yield of HRM was 11 (19%), 11 (19%), and 33 of 57 (58%), with SWS, MWS, and solids, respectively (P < .001); it was greatest for solids in patients with dysphagia (19 of 27, 70%). Outlet obstruction was present in 4 (7%), 11 (19%), and 15 of 57 patients (26%) with SWS, MWS, and solids, respectively (P < .009). No asymptomatic control had clinically relevant dysfunction on solid swallows. Dilatation was performed in 12 of 15 patients with outlet obstruction during the test meal. Symptom response was satisfactory, good, or excellent in 7 of 12 (58%) with no serious complications., Conclusions: The addition of MWS and a solid test meal increases the diagnostic yield of HRM studies in patients with symptoms after fundoplication and identifies additional patients with outlet obstruction who benefit from endoscopic dilatation., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background/aims: Supragastric belching (SGB) is a phenomenon during which air is sucked into the esophagus and then rapidly expelled through the mouth. Patients often complain of severely impaired quality of life. Our objective was to establish the prevalence of ex-cessive SGB within a high-volume gastrointestinal physiology unit, and evaluate its association with symptoms, esophageal mo-tility and gastresophageal reflux disease., Methods: We established normal values for SGB by analyzing 24-hour pH-impedance in 40 healthy asymptomatic volunteers. We searched 2950 consecutive patient reports from our upper GI Physiology Unit (from 2010-2013) for SGB. Symptoms were re-corded by a standardized questionnaire evaluating for reflux, dysphagia, and dyspepsia symptoms. We reviewed the predom-inant symptoms, 24-hour pH-impedance and high-resolution esophageal manometry results., Results: Excessive SGB was defined as > 13 per 24 hours. We identified 100 patients with excessive SGB. Ninety-five percent of these patients suffered from typical reflux symptoms, 86% reported excessive belching, and 65% reported dysphagia. Forty-one per-cent of patients with excessive SGB had pathological acid reflux. Compared to the patients with normal acid exposure these patients trended towards a higher number of SGB episodes. Forty-four percent of patients had esophageal hypomotility. Patients with hypomotility had a significantly higher frequency of SGB compared to those with normal motility (118.3 ± 106.1 vs 80.6 ± 75.7, P = 0.020)., Conclusions: Increased belching is rarely a symptom in isolation. Pathological acid exposure and hypomotility are associated with more SGB frequency. Whether SGB is a disordered response to other esophageal symptoms or their cause is unclear.

Objective: Current diagnostic methods for gastro-oesophageal reflux disease (GORD) have moderate sensitivity/specificity and can be invasive and expensive. Pepsin detection in saliva has been proposed as an 'office-based' method for GORD diagnosis. The aims of this study were to establish normal values of salivary pepsin in healthy asymptomatic subjects and to determine its value to discriminate patients with reflux-related symptoms (GORD, hypersensitive oesophagus (HO)) from functional heartburn (FH)., Design: 100 asymptomatic controls and 111 patients with heartburn underwent MII-pH monitoring and simultaneous salivary pepsin determination on waking, after lunch and dinner. Cut-off value for pepsin positivity was 16 ng/mL. Patients were divided into GORD (increased acid exposure time (AET), n=58); HO (normal AET and + Symptom Association Probability (SAP), n=26) and FH (normal AET and-SAP, n=27)., Results: 1/3 of asymptomatic subjects had pepsin in saliva at low concentration (0(0-59)ng/mL). Patients with GORD and HO had higher prevalence and pepsin concentration than controls (HO, 237(52-311)ng/mL and GORD, 121(29-252)ng/mL)(p<0.05). Patients with FH had low prevalence and concentration of pepsin in saliva (0(0-40) ng/mL). A positive test had 78.6% sensitivity and 64.9% specificity for diagnosis of GORD+HO (likelihood ratio: 2.23). However, one positive sample with >210 ng/mL pepsin suggested presence of GORD+HO with 98.2% specificity (likelihood ratio: 25.1). Only 18/84 (21.4%) of GORD+HO patients had 3 negative samples., Conclusion: In patients with symptoms suggestive of GORD, salivary pepsin testing may complement questionnaires to assist office-based diagnosis. This may lessen the use of unnecessary antireflux therapy and the need for further invasive and expensive diagnostic methods., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Esophageal function testing should be used for differential diagnosis of dysphagia. Dysphagia can be the consequence of hypermotility or hypomotility of the muscles of the esophagus. Decreased esophageal or esophagogastric junction distensibility can provoke dysphagia. The most well established esophageal dysmotility is achalasia. Other motility disorders can also cause dysphagia. High-resolution manometry (HRM) is the gold standard investigation for esophageal motility disorders. Simultaneous measurement of HRM and intraluminal impedance can be useful to assess motility and bolus transit. Impedance planimetry measures distensibility of the esophageal body and gastroesophageal junction in patients with achalasia and eosinophilic esophagitis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Goals: We aimed to quantify pharyngeal exposure to gastric contents in patients diagnosed with reflux-related hoarseness and healthy controls using new diagnostic techniques., Background: Hoarseness with typical signs on laryngoscopy is commonly thought to be caused by esophagopharyngeal reflux. New methods are proposed to assess pharyngeal exposure to gastric contents. They are suggested to measure: (1) liquid or mixed gas-liquid acid and nonacid reflux with impedance pH, (2) aerosolized acid reflux (Dx-pH measuring system), and (3) pepsin in the saliva., Study: Twenty-one patients with hoarseness and positive laryngoscopy and 10 controls underwent simultaneous impedance pH, Dx-pH monitoring, and saliva pepsin sampling (5 samples in 24 h)., Results: Of the 21 patients, 10 had impedance pH-detected reflux plus at least 1 other test positive. These patients were more likely to have symptomatic relief after proton pump inhibitor therapy. Three of the 21 patients had all 3 tests positive and 4 had all tests negative. None of the controls had impedance pH-detected reflux. Two controls had a positive Dx-pH "RYAN score" and 1 control had >1 saliva sample positive for pepsin. Only 11% of Dx-pH drops to pH<4, 15% pH drops to pH<5, and 10% of pH drops to pH<5.5 coincided with impedance pH-detected reflux in the esophageal body. Positive pepsin saliva samples were preceded by more reflux events [3 (range, 0 to 10)] in the previous 60 minutes than negative samples [0 (range, 0 to 7)] (P<0.0001)., Conclusion: A subgroup of patients with hoarseness (10/21) had objective detection of the esophagopharyngeal reflux. We propose that these patients are more likely to benefit from further intense antireflux therapy. Detection of pepsin in the saliva may be a useful screening tool in these patients.

Objective: Patients with non-erosive reflux disease (NERD) have impaired oesophageal mucosal integrity (dilated intercellular spaces). Oesophageal mucosal integrity reflects the balance between repeated reflux damage and mucosal recovery. The relationship between mucosal integrity and acid sensitivity is unclear. Oesophageal impedance may be used for in vivo mucosal integrity measurement. We studied acid-induced changes in oesophageal mucosal integrity and acid perception in patients with heartburn., Design: 50 patients with heartburn whithout oesophagitis underwent impedance monitoring before, during and after 10 min oesophageal perfusion with neutral (pH 6.5) and acid solutions (pH 1). Symptoms and impedance were recorded during perfusion. Impedance recovery was assessed for 2 h post-perfusion in ambulatory conditions followed by 24-h impedance-pH study., Results: Reflux monitoring discriminated 20 NERD and 30 functional heartburn (FH) patients. Neutral perfusion caused impedance fall that recovered within 10 min. Acid perfusion caused impedance fall with slow recovery: 6.5 Ω/min (IQR 3.3-12.0 Ω/min). Patients with slow recovery (< 25th percentile) had lower baseline impedance (1273 Ω ± 208 Ω vs. 3220 Ω ± 275 Ω ±, p < 0.01) and more frequent acid sensitivity (10/12 vs. 4/12, p = 0.04) than those with fast (> 75th percentile) recovery. Patients with NERD had lower baseline impedance (1669 ± 182 Ω vs. 2384 ± 211 Ω, p = 0.02) and slower impedance recovery (6.0 ± 0.9 Ω/min vs. 10.7 ± 1.6 Ω/min, p = 0.03) than patients with FH., Conclusion: Impaired mucosal integrity might be the consequence of repeated reflux episodes with slow recovery. Mucosal integrity, recovery capacity and symptom perception are linked. Low basal impedance and slow recovery after acid challenge are associated with increased acid sensitivity.

The development of the high-resolution esophageal manometry (HRM) and the Chicago classification have improved the diagnosis and management of esophageal motility disorders. However, some conditions have yet to be addressed by this classification. This review describes findings in HRM which are not included in the current Chicago classification based on the experience in our center. This includes the analysis of the upper esophageal sphincter, proximal esophagus, longitudinal muscle contraction, disorders related to gastroesophageal reflux disease and respiratory symptoms. The utility of provocative tests and the use of HRM in the evaluation of rumination syndrome and post-surgical patients will also be discussed. We believe that characterization of the manometric findings in these areas will eventually lead to incorporation of new criteria into the existing classification.

The primary role of the esophagus is to propel swallowed food or fluid into the stomach and to prevent or clear gastroesophageal reflux. This function is achieved by an organized pattern that involves a sensory pathway, neural reflexes, and a motor response that includes esophageal tone, peristalsis, and shortening. The motor function of the esophagus is controlled by highly complex voluntary and involuntary mechanisms. There are three different functional areas in the esophagus: the upper esophageal sphincter, the esophageal body, and the LES. This article focused on anatomy and physiology of the esophageal body., (© 2011 Copyright the Authors. Journal compilation © 2011, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

Background: Acid infusion in humans induces primary and secondary oesophageal hypersensitivity. The effects of pregabalin, a centrally-acting modulator of voltage-sensitive calcium channels, on development of acid-induced oesophageal hypersensitivity remain unknown., Aim: To study the effects of pregabalin on development of secondary oesophageal hypersensitivity in healthy humans., Methods: Placebo-controlled, double-blind, randomised, cross-over study of 15 healthy volunteers (six women, age 21-56 years). After oesophageal manometry, baseline pain thresholds (PTs) to proximal oesophageal electrical stimulation were determined using bipolar ring electrodes. A 30-min infusion of HCl was performed in the distal oesophagus followed by PT measurements at 30 and 90 min. This protocol was repeated after administration of pregabalin (dosing schedule: 75 mg twice daily for 3 days then 150 mg twice daily for 1 day and then 150 mg on the morning of study) or placebo., Results: T0 PTs were similar in patients after receiving placebo or pregabalin [mean (s.d.) 32.9 mA (20.5) vs. 34.1 (15.7), P = 0.42]. Pregabalin reduced development of acid-induced hypersensitivity in the proximal oesophagus at 30 min [mean change in PT (C.I.) placebo -6.2 mA (-11.3 to +1.3) vs. pregabalin +0.20 mA (-2.7 to +3.3)] and 90 min [placebo -3.7 mA (-10.0 to +2.0) vs. pregabalin +0.7 mA (-4.7 to 7.3)] overall P = 0.001. Pregabalin reduced median visual analogue scale score for acid-induced pain (1/10 vs. placebo 3/10, P = 0.027)., Conclusions: Pregabalin attenuates development of secondary hypersensitivity in the proximal oesophagus after distal oesophageal acidification; it may thus have a role in treatment of patients with proven oesophageal pain hypersensitivity., (© 2011 Blackwell Publishing Ltd.)

Stereotypical changes in pH occur along the gastrointestinal (GI) tract. Classically, there is an abrupt increase in pH on exit from the stomach, followed later by a sharp fall in pH, attributed to passage through the ileocecal region. However, the precise location of this latter pH change has never been conclusively substantiated. We aimed to determine the site of fall in pH using a dual-scintigraphic technique. On day 1, 13 healthy subjects underwent nasal intubation with a 3-m-long catheter, which was allowed to progress to the distal ileum. On day 2, subjects ingested a pH-sensitive wireless motility capsule labeled with 4 MBq (51)Chromium [EDTA]. The course of this, as it travelled through the GI tract, was assessed with a single-headed γ-camera using static and dynamic scans. Capsule progression was plotted relative to a background of 4 MBq ¹¹¹Indium [diethylenetriamine penta-acetic acid] administered through the catheter. Intraluminal pH, as recorded by the capsule, was monitored continuously, and position of the capsule relative to pH was established. A sharp fall in pH was recorded in all subjects; position of the capsule relative to this was accurately determined anatomically in 9/13 subjects. In these nine subjects, a pH drop of 1.5 ± 0.2 U, from 7.6 ± 0.05 to 6.1 ± 0.1 occurred a median of 7.5 min (1-16) after passage through the ileocecal valve; location was either in the cecum (n = 5), ascending colon (n = 2), or coincident with a move from the cecum to ascending colon (n = 2). This study provides conclusive evidence that the fall in pH seen within the ileocolonic region actually occurs in the proximal colon. This phenomenon can be used as a biomarker of transition between the small and large bowel and validates assessment of regional GI motility using capsule technology that incorporates pH measurement.

Background: Slow transit constipation (STC) is associated with upper gastrointestinal tract motor abnormalities in a subset of patients. This could influence the clinical approach, particularly in those rare cases where surgical management is considered., Aims: To identify factors that predict proximal gut dysmotility in patients with STC., Methods: Esophageal and small bowel motor function were evaluated in 77 patients with STC. Severity and pattern of colonic transit delay, prevalence of a co-existent rectal evacuatory disorder, and type (if present), and duration, and onset of constipation symptoms were compared., Results: Of the 77 patients studied, 43% exhibited altered motor function in the esophagus and/or small bowel. Frequency of defecation was lower in these patients than in those without upper gastrointestinal dysmotility (0.5 +/- 0.1 vs. 1.3 +/- 0.3 bowel movements/per week, respectively; P = 0.04). Severity and patterns of colonic transit delay and the mechanism associated with the onset of constipation symptoms or with their duration were similar in subjects with or without upper gastrointestinal tract dysmotility. Small bowel but not esophageal motor dysfunction was more frequently associated with a co-existent rectal evacuatory disorder (P = 0.01)., Conclusion: Upper gastrointestinal tract dysmotility in patients with STC is frequent, but prediction on the basis of clinical history and characteristics of colonic transit is problematic.

Interdigestive human small bowel motility is characterized by the migrating motor complex (MMC). The aims of this study were to: (i) establish the normal range of variables of the nocturnal jejunal MMC and (ii) incorporate these data in a subsequent meta-analysis. Eighty-one recordings were performed by prolonged (24 h) ambulatory manometry in 51 subjects in two centres. Quantitative analysis was undertaken of 419 Phase III and 332 Phase II episodes. Adjusted mean values of seven variables were calculated using a mixed-effects model. Meta-analysis of pooled published data to generate a reliable 95% reference range was also performed. Adjusted mean values and confidence intervals are presented for all seven variables. Intrasubject variances were large in comparison with intersubject. Meta-analysis of 19 studies (356 pooled patients) meeting inclusion criteria produced wide reference ranges. At least five such ranges are useful for the detection of abnormality in the individual. This is the largest study of normal volunteers presented to date, with ranges for many variables produced using appropriate statistical methodology. A model for definition of abnormality has been proposed. We recommend that these data may be used by investigators in this field as a complement to other existing indicators of small bowel dysmotility.