Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography

Background: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. Methods: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. Results: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 μM–4 mM and 0.2 μM–0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 μM and 0.26 μM respectively whilst their limits of quantification were 1.14 μM and 0.88 μM respectively. Conclusion: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples

Uremic cardiomyopathy is characterised by loss of the cardioprotective effects of insulin

Chronic kidney disease is associated with a unique cardiomyopathy, characterised by a combination of structural and cellular remodelling, and an enhanced susceptibility to ischaemia-reperfusion injury. This may represent dysfunction of the reperfusion injury salvage kinase pathway, due to insulin resistance. Aims: The susceptibility of the uraemic heart to ischaemia-reperfusion injury and the cardioprotective effects of insulin and rosiglitazone were investigated. Methods and Results: Uraemia was induced in Sprague-Dawley rats by subtotal nephrectomy. Functional recovery from ischaemia was investigated in vitro in control and uraemic hearts ±insulin ±rosiglitazone. The response of myocardial oxidative metabolism to insulin was determined by 13C NMR spectroscopy. Activation of reperfusion injury salvage kinase pathway intermediates (Akt and GSK3β) were assessed by SDS-PAGE and immuno-precipitation. Insulin improved post-ischaemic rate pressure product in control but not uraemic hearts, (recovered rate pressure product (%), control 59.6±10.7 vs 88.9±8.5, p<0.05; uraemic 19.3±4.6 vs 28.5±10.4, p=ns). Rosiglitazone resensitised uraemic hearts to insulin-mediated cardio-protection (recovered rate pressure product (%) 12.7±7.0 vs. 61.8±15.9, p<0.05). Myocardial carbohydrate metabolism remained responsive to insulin in uraemic hearts. Uraemia was associated with increased phosphorylation of Akt (1.00±0.08 vs. 1.31±0.11, p<0.05) in normoxia, but no change in post-ischaemic phosphorylation of Akt or GSK3β. Akt2 isoform expression was decreased post-ischaemia in uraemic hearts (p<0.05). Conclusion: Uraemia is associated with enhanced susceptibility to ischaemia-reperfusion injury and a loss of insulin-mediated cardio-protection, which can be restored by administration of rosiglitazone. Altered Akt2 expression in uraemic hearts post ischaemia-reperfusion and impaired activation of reperfusion injury salvage kinase pathway may underlie these findings

Hepatic cysteine sulphinic acid decarboxylase depletion and defective taurine metabolism in a rat partial nephrectomy model of chronic kidney disease

© 2021, The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Background: Taurine depletion occurs in patients with end-stage chronic kidney disease (CKD). In contrast, in the absence of CKD, plasma taurine is reported to increase following dietary L-glutamine supplementation. This study tested the hypothesis that taurine biosynthesis decreases in a rat CKD model, but is rectified by L-glutamine supplementation. Methods: CKD was induced by partial nephrectomy in male Sprague-Dawley rats, followed 2 weeks later by 2 weeks of 12% w/w L-glutamine supplemented diet (designated NxT) or control diet (NxC). Sham-operated control rats (S) received control diet. Results: Taurine concentration in plasma, liver and skeletal muscle was not depleted, but steady-state urinary taurine excretion (a measure of whole-body taurine biosynthesis) was strongly suppressed (28.3 ± 8.7 in NxC rats versus 78.5 ± 7.6 μmol/24 h in S, P < 0.05), accompanied by reduced taurine clearance (NxC 0.14 ± 0.05 versus 0.70 ± 0.11 ml/min/Kg body weight in S, P < 0.05). Hepatic expression of mRNAs encoding key enzymes of taurine biosynthesis (cysteine sulphinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO)) showed no statistically significant response to CKD (mean relative expression of CSAD and CDO in NxC versus S was 0.91 ± 0.18 and 0.87 ± 0.14 respectively). Expression of CDO protein was also unaffected. However, CSAD protein decreased strongly in NxC livers (45.0 ± 16.8% of that in S livers, P < 0.005). L-glutamine supplementation failed to rectify taurine biosynthesis or CSAD protein expression, but worsened CKD (proteinuria in NxT 12.5 ± 1.2 versus 6.7 ± 1.5 mg/24 h in NxC, P < 0.05). Conclusion: In CKD, hepatic CSAD is depleted and taurine biosynthesis impaired. This is important in view of taurine’s reported protective effect against cardio-vascular disease - the leading cause of death in human CKD.Peer reviewe

Structurally optimised BODIPY derivatives for imaging of mitochondrial dysfunction in cancer and heart cells

The structural features required for mitochondrial uptake of BODIPY-based optical imaging agents have been explored. The first derivatives of this class of dyes shown to have mitochondrial membrane potential-dependent uptake in both cancer and heart cells have been developed

Improving diaper design to address incontinence associated dermatitis

<p>Abstract</p> <p>Background</p> <p>Incontinence associated dermatitis (IAD) is an inflammatory skin disease mainly triggered by prolonged skin contact with urine, feces but also liberal detergent use when cleansing the skin. To minimize the epidermal barrier challenge we optimized the design of adult incontinence briefs. In the fluid absorption area we interposed a special type of acidic, curled-type of cellulose between the top sheet in contact with the skin and the absorption core beneath containing the polyacrylate superabsorber. The intention was to minimize disturbance of the already weak acid mantle of aged skin. We also employed air-permeable side panels to minimize skin occlusion and swelling of the stratum corneum.</p> <p>Methods</p> <p>The surface pH of diapers was measured after repeated wetting with a urine substitute fluid at the level of the top sheet. Occlusive effects and hydration of the stratum corneum were measured after a 4 hour application of different side panel materials by corneometry on human volunteers. Finally, we evaluated skin symptoms in 12 patients with preexisting IAD for 21 days following the institutional switch to the optimized diaper design. Local skin care protocols remained in place unchanged.</p> <p>Results</p> <p>The improved design created a surface pH of 4.6 which was stable even after repeated wetting throughout a 5 hour period. The "standard design" briefs had values of 7.1, which is alkaline compared to the acidic surface of normal skin. Side panels made from non-woven material with an air-permeability of more than 1200 l/m²/s avoided excessive hydration of the stratum corneum when compared to the commonly employed air-impermeable plastic films. Resolution of pre-existing IAD skin lesions was noted in 8 out of 12 patients after the switch to the optimized brief design.</p> <p>Conclusions</p> <p>An improved design of adult-type briefs can create an acidic pH on the surface and breathable side panels avoid over-hydration of the stratum corneum and occlusion. This may support the epidermal barrier function and may help to reduce the occurrence of IAD.</p

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

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

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

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