Search

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.