Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD

The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 angstroms resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 angstroms, beta = 106.41 degrees. The final R-factor and R-free were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.YesThe manuscript was peer-reviewed by two anonymous reviewers

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Identification of active retinaldehyde dehydrogenase isoforms in the postnatal human eye.

Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-trans-retinoic acid (atRA) synthesis in response to visual signals in animal models of myopia. To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye.In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA

Western blot analysis and quantification of RALDH2 in postnatal human ocular tissues.

<p><b>(A)</b> Cytosol fractions from ocular tissues of donors aged 16–46 years were immunblotted with anti-RALDH2 or anti-RPE65. <i>Top Panel</i>: RALDH2 immunopositive bands (∼ 55–59 kDa) were present in the choroid (C), sclera (S), retina (R), and RPE (E) of postnatal human eyes. <i>Bottom Panel</i>: anti-RPE65 was used to identify the presence of RPE contamination in the retina, choroid, and sclera samples. 3 μg total protein/lane was loaded for each blot. <b>(B)</b> Relative abundance of RALDH2 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH2-immunopositive bands. <b>(C)</b> Average RALDH2 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 5). *<i>p</i> < 0.05, **<i>p</i> < 0.01 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).</p

Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues.

<p><b>(A)</b> Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. <i>Top panel</i>: RALDH1 (∼ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. <i>Bottom Panel</i>: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 μg total protein/lane was loaded on the blot. 1.25 μg recombinant human RALDH3 (R3) was loaded as a positive control. <b>(B)</b> Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH1-immunopositive bands. <b>(C)</b> Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).</p

SDS-PAGE minigel of cytosol fractions from human ocular tissues.

<p>3 μg total protein from choroid (C), sclera (S), retina (R), and RPE (E) cytosol fractions (100,000g supernatants) from human donors aged 17 and 30 years were separated on a 1.0 mm 10% Bis/Tris gel.</p

Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody.

<p><b>(A)</b> Low power magnification of ocular tissues demonstrating RALDH1 labeling <i>(red)</i> in the retina and choroid. <b>(B)</b> Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. <b>(C, D)</b> Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (<i>blue)</i>. BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C, D.</p

Confocal images of RALDH2 <i>(red)</i> and αSMA <i>(green)</i> expressing cells after immunolabeling with anti-RALDH2 and anti-αSMA.

<p><b>(A)</b> Low power magnification of ocular tissues demonstrating RALDH2 labeling in the retina, RPE, and choroid. <b>(B)</b> Negative control section (primary antibody pre-absorbed with recombinant RALDH2) demonstrating absence of labeling in the retina and choroid and slight auto-fluorescence in the RPE. <b>(C, D)</b> Choroid demonstrating RALDH2 labeling in extravascular cells closely associated with blood vessels and in slender cells throughout the stroma (arrowhead). <b>(E, F)</b> Double labeling of the choroid with anti-RALDH2 and anti-αSMA demonstrates RALDH2 positive cells are distinct from vascular and extravascular smooth muscle cells. Asterisk (*) indicates an αSMA-positive extravascular smooth muscle cell in (E). Upward arrow in (C-F) indicates orientation for the scleral side of the choroid tissue. Nuclei were counterstained with DAPI (<i>blue)</i>. BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C-F.</p