The YjgF/YER057c/UK114 family of proteins is conserved in all domains of life, suggesting that the role of these proteins arose early and was maintained throughout evolution. Metabolic consequences ...of lacking this protein in Salmonella enterica and other organisms have been described, but the biochemical function of YjgF remained unknown. This work provides the first description of a conserved biochemical activity for the YjgF protein family. Our data support the conclusion that YjgF proteins have enamine/imine deaminase activity and accelerate the release of ammonia from reactive enamine/imine intermediates of the pyridoxal 5′-phosphate-dependent threonine dehydratase (IlvA). Results from structure-guided mutagenesis experiments suggest that YjgF lacks a catalytic residue and that it facilitates ammonia release by positioning a critical water molecule in the active site. YjgF is renamed RidA (reactive intermediate/imine deaminase A) to reflect the conserved activity of the protein family described here. This study, combined with previous physiological studies on yjgF mutants, suggests that intermediates of pyridoxal 5′-phosphate-mediated reactions may have metabolic consequences in vivo that were previously unappreciated. The conservation of the RidA/YjgF family suggests that reactive enamine/imine metabolites are of concern to all organisms.
Background: YjgF proteins are conserved across the three domains of life.
Results: YjgF proteins deaminate unstable products of PLP-dependent enzyme threonine dehydratase by positioning the enamine/imine close to water in the active site.
Conclusion: YjgF is an enamine/imine deaminase and is renamed RidA.
Significance: RidA removes reactive metabolites released by PLP-dependent enzymes before they can damage cellular components.
Members of the RidA (YjgF/YER057c/UK114) protein family are broadly conserved across the domains of life. In vitro, these proteins deaminate 3- or 4-carbon enamines that are generated as mechanistic ...intermediates of pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases. The three-carbon enamine 2-aminoacrylate can inactivate some enzymes by forming a covalent adduct via a mechanism that has been well characterized in vitro. The biochemical activity of RidA suggested that the phenotypes of ridA mutant strains were caused by the accumulation of reactive enamine metabolites. The data herein show that in ridA mutant strains of Salmonella enterica, a stable 2-aminoacrylate (2-AA)/PLP adduct forms on the biosynthetic alanine racemase, Alr, indicating the presence of 2-aminoacrylate in vivo. This study confirms the deleterious effect of 2-aminoacrylate generated by metabolic enzymes and emphasizes the need for RidA to quench this reactive metabolite.
Paediatric in-patients are at high risk of malnutrition but validated paediatric screening tools suitable for use by nursing staff are scarce. The present study aimed to assess the diagnostic ...accuracy of the new Paediatric Yorkhill Malnutrition Score (PYMS). During a pilot introduction in a tertiary referral hospital and a district general hospital, two research dietitians assessed the validity of the PYMS by comparing the nursing screening outcome with a full dietetic assessment, anthropometry and body composition measurements. An additional PYMS form was completed by the research dietitians to assess its inter-rater reliability with the nursing staff and for comparison with the Screening Tool for the Assessment of Malnutrition in Paediatrics (STAMP) and the Paediatric Subjective Global Nutritional Assessment (SGNA). Of the 247 children studied, the nurse-rated PYMS identified 59 % of those rated at high risk by full dietetic assessment. Of those rated at high risk by the nursing PYMS, 47 % were confirmed as high risk on full assessment. The PYMS showed moderate agreement with the full assessment (κ = 0·46) and inter-rater reliability (κ = 0·53) with the research dietitians. Children who screened as high risk for malnutrition had significantly lower lean mass index than those at moderate or low risk, but no difference in fat. When completed by the research dietitians, the PYMS showed similar sensitivity to the STAMP, but a higher positive predictive value. The SGNA had higher specificity than the PYMS but much lower sensitivity. The PYMS screening tool is an acceptable screening tool for identifying children at risk of malnutrition without producing unmanageable numbers of false-positive cases.
Summary
Background
Faecal calprotectin decreases during exclusive enteral nutrition in children with active Crohn's disease. It is unknown how faecal calprotectin changes during food re‐introduction ...and the influence of maintenance enteral nutrition.
Aims
To study changes to faecal calprotectin during exclusive enteral nutrition and at food reintroduction, and explore associations with maintenance enteral nutrition.
Methods
Children with Crohn's disease were followed during exclusive enteral nutrition and during food‐reintroduction. Faecal calprotectin was measured before, at 33 and 54 days of exclusive enteral nutrition, and at 17, 52 and 72 days after food‐reintroduction. Maintenance enteral nutrition use was recorded with estimated weight food diaries. Data are presented with medians and Q1:Q3.
Results
Sixty‐six patients started exclusive enteral nutrition and 41 (62%) achieved clinical remission (weighted paediatric Crohn's disease activity index <12.5). Baseline faecal calprotectin (mg/kg) decreased after 4 and 8 weeks of exclusive enteral nutrition (Start: 1433 Q1: 946, Q3: 1820 vs 33 days: 844 314, 1438 vs 54 days: 453 165, 1100; P < .001). Within 17 days of food reintroduction, faecal calprotectin increased to 953 Q1: 519, Q3: 1611 and by 52 days to 1094 660, 1625 (both P < .02). Fifteen of 41 (37%) children in remission used maintenance enteral nutrition (333 kcal or 18% of energy intake). At 17 days of food reintroduction, faecal calprotectin was lower in maintenance enteral nutrition users than non‐users (651 Q1: 271, Q3: 1781 vs 1238 749, 2102, P = .049) and correlated inversely with maintenance enteral nutrition volume (rho: −0.573, P = .041), kcals (rho: −0.584, P = .036) and % energy intake (rho: −0.649, P = .016). Maintenance enteral nutrition use was not associated with longer periods of remission (P = .7). Faecal calprotectin at the end of exclusive enteral nutrition did not predict length of remission.
Conclusions
The effect of exclusive enteral nutrition on faecal calprotectin is diminished early during food reintroduction. Maintenance enteral nutrition at ~18% of energy intake is associated with a lower faecal calprotectin at the early phase of food reintroduction but is ineffective in maintaining longer term remission.
Summary
The RidA/Yer057/UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have ...enamine deaminase activity, and in their absence the reactive 2‐aminoacrylate (2‐AA) accumulates and inactivates at least some pyridoxal 5′‐phosphate (PLP)‐containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2‐AA was a ubiquitous cellular stressor that was generated in central metabolism. Phenotypically, strains of S. enterica that lack RidA accumulated significantly more pyruvate in the growth medium than wild‐type strains. Here we dissected this ridA mutant phenotype and showed it was an indirect consequence of damage to serine hydroxymethyltransferase (GlyA; E.C. 2.1.2.1). The results here identified a fourth PLP enzyme as a target of enamine stress in Salmonella.
The gastrointestinal microbiota is considered important in inflammatory bowel disease (IBD) pathogenesis. Discoveries from established disease cohorts report reduced bacterial diversity, changes in ...bacterial composition, and a protective role for Faecalibacterium prausnitzii in Crohn's disease (CD). The majority of studies to date are however potentially confounded by the effect of treatment and a reliance on established rather than de-novo disease.
Microbial changes at diagnosis were examined by biopsying the colonic mucosa of 37 children: 25 with newly presenting, untreated IBD with active colitis (13 CD and 12 ulcerative colitis (UC)), and 12 pediatric controls with a macroscopically and microscopically normal colon. We utilized a dual-methodology approach with pyrosequencing (threshold >10,000 reads) and confirmatory real-time PCR (RT-PCR).
Threshold pyrosequencing output was obtained on 34 subjects (11 CD, 11 UC, 12 controls). No significant changes were noted at phylum level among the Bacteroidetes, Firmicutes, or Proteobacteria. A significant reduction in bacterial α-diversity was noted in CD vs. controls by three methods (Shannon, Simpson, and phylogenetic diversity) but not in UC vs. controls. An increase in Faecalibacterium was observed in CD compared with controls by pyrosequencing (mean 16.7% vs. 9.1% of reads, P=0.02) and replicated by specific F. prausnitzii RT-PCR (36.0% vs. 19.0% of total bacteria, P=0.02). No disease-specific clustering was evident on principal components analysis.
Our results offer a comprehensive examination of the IBD mucosal microbiota at diagnosis, unaffected by therapeutic confounders or changes over time. Our results challenge the current model of a protective role for F. prausnitzii in CD, suggesting a more dynamic role for this organism than previously described.
Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can ...exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD.
100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis.
129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312).
This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon.
This study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633).
The anti-inflammatory effect of exclusive enteral nutrition on the gut of children with Crohn's disease is rapidly lost after food reintroduction. This study assessed disease dietary triggers ...following successful treatment with exclusive enteral nutrition.
Nutrient intake, dietary patterns and dietary biomarkers in faeces (gluten immunogenic peptides, undigestible starch, short chain fatty acids) were assessed in 14 children with Crohn's disease during early food reintroduction, following exclusive enteral nutrition. Groups above (Group A) and below (Group B) the median levels of faecal calprotectin after food reintroduction were assigned for comparative analysis.
Intakes of fibre, gluten-containing cereals and red and processed meat were significantly higher in Group A than Group B; (median Q1, Q3, g/day; Fibre: 12.1 11.2, 19.9 vs. 9.9 7.6, 12.1, p = 0.03; Red and processed meat: 151 66.7, 190 vs. 63.3 21.7, 67, p = 0.02; gluten-containing cereals: 289 207, 402 vs. 203 61, 232, p = 0.035). A diet consisting of cereals and meat products was predictive (92% accuracy) of higher faecal calprotectin levels after food reintroduction. In faeces, butyrate levels, expressed as absolute concentration and relative abundance, were higher in Group A than Group B by 28.4 µmol/g (p = 0.015) and 6.4% (p = 0.008), respectively. Levels of gluten immunogenic peptide and starch in faeces did not differ between the two groups.
This pilot study identified potential dietary triggers of gut inflammation in children with Crohn's disease after food reintroduction following treatment with exclusive enteral nutrition.
Clinical trials.gov registration number: NCT02341248; Clinical trials.gov URL: https://clinicaltrials.gov/ct2/show/NCT02341248 (retrospectively registered).
To evaluate acute-onset postoperative endophthalmitis following cataract surgery and to compare rates between resident and attending physician-performed cohorts.
Bascom Palmer Eye Institute, Miami, ...Florida.
Retrospective chart review.
Records of patients diagnosed with endophthalmitis within 6 weeks of surgery performed by attending and resident physicians between January 1, 2015, and December 31, 2020, were reviewed. Total cataract volume was obtained from institutional records, and resident case totals were obtained from case logs. Endophthalmitis cases were obtained from billing records and confirmed with chart review.
There were 22 cases of endophthalmitis among 32 505 cases (0.068%). Endophthalmitis occurred in 6 of 6447 (0.093%) resident cases and 16 of 26 058 (0.061%) attending cases ( P = .55). The most common bacterial isolates were coagulase-negative Staphylococcus (8/22, 36.3%) and Streptococcus species (3/22, 13.6%), with negative cultures in 10 (10/22, 45.5%). Initial treatment with vitreous tap and injection of intravitreal antibiotics was performed in 21 eyes (21/22, 95.4%) and vitrectomy with intravitreal antibiotic injection in one (1/22, 4.5%). Vitrectomy was performed secondarily in 9 patients (9/22, 40.9%). Corrected distance visual acuity (CDVA) at last follow-up was ≥20/40 in 13 eyes (13/22, 59%) and ≤hand motions in 3 eyes (3/22, 13.6%). CDVA (logMAR mean ± SD) was 1.22 ± 1.16 in resident and 0.49 ± 0.79 in attending cases ( P = .11).
In the current study, acute-onset postoperative endophthalmitis developed infrequently following cataract surgery. The rates and visual outcomes of endophthalmitis were similar in resident and attending cases.
Weekends off antiretroviral therapy (ART) may help engage HIV-1-infected young people facing lifelong treatment. BREATHER showed short cycle therapy (SCT; 5 days on, 2 days off ART) was non-inferior ...to continuous therapy (CT) over 48 weeks. Planned follow-up was extended to 144 weeks, maintaining original randomisation.
BREATHER was an open-label, non-inferiority trial. Participants aged 8-24yrs with virological suppression on efavirenz-based first-line ART were randomised 1:1, stratified by age and African/non-African sites, to remain on CT or change to SCT. The Kaplan-Meier method was used to estimate the proportion of participants with viral rebound (confirmed VL≥50 copies/mL) under intent-to-treat at 48 weeks (primary outcome), and in extended follow-up at 96, 144, and 192 weeks. SCT participants returned to CT following viral rebound, 3 VL blips or discontinuation of efavirenz.
Of 199 participants (99 SCT, 100 CT), 97 per arm consented to extended follow-up. Median follow-up was 185.3 weeks (IQR 160.9-216.1). 69 (70%) SCT participants remained on SCT at last follow-up. 105 (53%) were male, baseline median age 14 years (IQR 12-18), median CD4 count 735 cells/μL (IQR 576-968). 16 SCT and 16 CT participants had confirmed VL≥50 copies/mL by the end of extended follow-up (HR 1.00, 95% CI 0.50-2.00). Estimated difference in percentage with viral rebound (SCT minus CT) by week 144 was 1.9% (90% CI -6.6-10.4; p = 0.72) and was similar in a per-protocol analysis. There were no significant differences between arms in proportions of participants with grade 3/4 adverse events (18 SCT vs 16 CT participants; p = 0.71) or ART-related adverse events (10 vs 12; p = 0.82). 20 versus 8 serious adverse events (SAEs) were reported in 16 SCT versus 4 CT participants, respectively (p = 0.005 comparing proportions between groups; incidence rate ratio 2.49, 95%CI 0.71-8.66, p = 0.15). 75% of SAEs (15 SCT, 6 CT) were hospitalisations for a wide range of conditions. 3 SCT and 6 CT participants switched to second-line ART following viral failure (p = 0.50).
Sustainable non-inferiority of virological suppression in young people was shown for SCT versus CT over median 3.6 years. Standard-dose efavirenz-based SCT is a viable option for virologically suppressed HIV-1 infected young people on first-line ART with 3-monthly VL monitoring.
EudraCT 2009-012947-40 ISRCTN 97755073 ClinicalTrials.gov NCT01641016.
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