The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice.

Simvastatin in Critically Ill Patients with Covid-19.

BACKGROUND: The efficacy of simvastatin in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear. METHODS: In an ongoing international, multifactorial, adaptive platform, randomized, controlled trial, we evaluated simvastatin (80 mg daily) as compared with no statin (control) in critically ill patients with Covid-19 who were not receiving statins at baseline. The primary outcome was respiratory and cardiovascular organ support-free days, assessed on an ordinal scale combining in-hospital death (assigned a value of -1) and days free of organ support through day 21 in survivors; the analyis used a Bayesian hierarchical ordinal model. The adaptive design included prespecified statistical stopping criteria for superiority (>99% posterior probability that the odds ratio was >1) and futility (>95% posterior probability that the odds ratio was <1.2). RESULTS: Enrollment began on October 28, 2020. On January 8, 2023, enrollment was closed on the basis of a low anticipated likelihood that prespecified stopping criteria would be met as Covid-19 cases decreased. The final analysis included 2684 critically ill patients. The median number of organ support-free days was 11 (interquartile range, -1 to 17) in the simvastatin group and 7 (interquartile range, -1 to 16) in the control group; the posterior median adjusted odds ratio was 1.15 (95% credible interval, 0.98 to 1.34) for simvastatin as compared with control, yielding a 95.9% posterior probability of superiority. At 90 days, the hazard ratio for survival was 1.12 (95% credible interval, 0.95 to 1.32), yielding a 91.9% posterior probability of superiority of simvastatin. The results of secondary analyses were consistent with those of the primary analysis. Serious adverse events, such as elevated levels of liver enzymes and creatine kinase, were reported more frequently with simvastatin than with control. CONCLUSIONS: Although recruitment was stopped because cases had decreased, among critically ill patients with Covid-19, simvastatin did not meet the prespecified criteria for superiority to control. (REMAP-CAP ClinicalTrials.gov number, NCT02735707.).

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

BACKGROUND: Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. METHODS: IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. RESULTS: We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. CONCLUSIONS: These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.

Factors associated with COVID-19-related death using OpenSAFELY.

Coronavirus disease 2019 (COVID-19) has rapidly affected mortality worldwide1. There is unprecedented urgency to understand who is most at risk of severe outcomes, and this requires new approaches for the timely analysis of large datasets. Working on behalf of NHS England, we created OpenSAFELY-a secure health analytics platform that covers 40% of all patients in England and holds patient data within the existing data centre of a major vendor of primary care electronic health records. Here we used OpenSAFELY to examine factors associated with COVID-19-related death. Primary care records of 17,278,392 adults were pseudonymously linked to 10,926 COVID-19-related deaths. COVID-19-related death was associated with: being male (hazard ratio (HR) 1.59 (95% confidence interval 1.53-1.65)); greater age and deprivation (both with a strong gradient); diabetes; severe asthma; and various other medical conditions. Compared with people of white ethnicity, Black and South Asian people were at higher risk, even after adjustment for other factors (HR 1.48 (1.29-1.69) and 1.45 (1.32-1.58), respectively). We have quantified a range of clinical factors associated with COVID-19-related death in one of the largest cohort studies on this topic so far. More patient records are rapidly being added to OpenSAFELY, we will update and extend our results regularly.

Conservative versus liberal oxygenation targets in critically ill children (Oxy-PICU): a UK multicentre, open, parallel-group, randomised clinical trial.

BACKGROUND: The optimal target for systemic oxygenation in critically ill children is unknown. Liberal oxygenation is widely practiced, but has been associated with harm in paediatric patients. We aimed to evaluate whether conservative oxygenation would reduce duration of organ support or incidence of death compared to standard care. METHODS: Oxy-PICU was a pragmatic, multicentre, open-label, randomised controlled trial in 15 UK paediatric intensive care units (PICUs). Children admitted as an emergency, who were older than 38 weeks corrected gestational age and younger than 16 years receiving invasive ventilation and supplemental oxygen were randomly allocated in a 1:1 ratio via a concealed, central, web-based randomisation system to conservative peripheral oxygen saturations ([SpO2] 88-92%) or liberal (SpO2 >94%) targets. The primary outcome was the duration of organ support at 30 days following random allocation, a rank-based endpoint with death either on or before day 30 as the worst outcome (a score equating to 31 days of organ support), with survivors assigned a score between 1 and 30 depending on the number of calendar days of organ support received. The primary effect estimate was the probabilistic index, a value greater than 0·5 indicating more than 50% probability that conservative oxygenation is superior to liberal oxygenation for a randomly selected patient. All participants in whom consent was available were included in the intention-to-treat analysis. The completed study was registered with the ISRCTN registry (ISRCTN92103439). FINDINGS: Between Sept 1, 2020, and May 15, 2022, 2040 children were randomly allocated to conservative or liberal oxygenation groups. Consent was available for 1872 (92%) of 2040 children. The conservative oxygenation group comprised 939 children (528 [57%] of 927 were female and 399 [43%] of 927 were male) and the liberal oxygenation group included 933 children (511 [56%] of 920 were female and 409 [45%] of 920 were male). Duration of organ support or death in the first 30 days was significantly lower in the conservative oxygenation group (probabilistic index 0·53, 95% CI 0·50-0·55; p=0·04 Wilcoxon rank-sum test, adjusted odds ratio 0·84 [95% CI 0·72-0·99]). Prespecified adverse events were reported in 24 (3%) of 939 patients in the conservative oxygenation group and 36 (4%) of 933 patients in the liberal oxygenation group. INTERPRETATION: Among invasively ventilated children who were admitted as an emergency to a PICU receiving supplemental oxygen, a conservative oxygenation target resulted in a small, but significant, greater probability of a better outcome in terms of duration of organ support at 30 days or death when compared with a liberal oxygenation target. Widespread adoption of a conservative oxygenation saturation target (SpO2 88-92%) could help improve outcomes and reduce costs for the sickest children admitted to PICUs. FUNDING: UK National Institute for Health and Care Research Health Technology Assessment Programme.

Temporal Trends in Mortality of Critically Ill Patients with Sepsis in the United Kingdom, 1988-2019.

Rationale: Sepsis is a frequent cause of ICU admission and mortality. Objectives: To evaluate temporal trends in the presentation and outcomes of patients admitted to the ICU with sepsis and to assess the contribution of changing case mix to outcomes. Methods: We conducted a retrospective cohort study of patients admitted to 261 ICUs in the United Kingdom during 1988-1990 and 1996-2019 with nonsurgical sepsis. Measurements and Main Results: A total of 426,812 patients met study inclusion criteria. The patients had a median (interquartile range) age of 66 (53-75) years, and 55.6% were male. The most common sites of infection were respiratory (60.9%), genitourinary (11.5%), and gastrointestinal (10.3%). Compared with patients in 1988-1990, patients in 2017-2019 were older (median age, 66 vs. 63 yr), were less acutely ill (median Acute Physiology and Chronic Health Evaluation II acute physiology score, 14 vs. 20), and more often had genitourinary sepsis (13.4% vs. 2.0%). Hospital mortality decreased from 54.6% (95% confidence interval [CI], 51.0-58.1%) in 1988-1990 to 32.4% (95% CI, 32.1-32.7%) in 2017-2019, with an adjusted odds ratio of 0.64 (95% CI, 0.54-0.75). The adjusted absolute hospital mortality reduction from 1988-1990 to 2017-2019 was 8.8% (95% CI, 5.6-12.1). Thus, of the observed 22.2-percentage point reduction in hospital mortality, 13.4 percentage points (60% of total reduction) were explained by case mix changes, whereas 8.8 percentage points (40% of total reduction) were not explained by measured factors and may be a result of improvements in ICU management. Conclusions: Over a 30-year period, mortality for ICU admissions with sepsis decreased substantially. Although changes in case mix accounted for the majority of observed mortality reduction, there was an 8.8-percentage point reduction in mortality not explained by case mix.

Reversal of High Fat Diet-Induced Obesity, Systemic Inflammation, and Astrogliosis by the NLRP3 Inflammasome Inhibitors NT-0249 and NT-0796.

Systemic and cerebral inflammatory responses are implicated in the pathogenesis of obesity and associated metabolic impairment. While the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been linked to obesity-associated inflammation, whether it contributes to the development or maintenance of obesity is unknown. We provide support for a direct role of saturated fatty acids, such as palmitic acid, as NLRP3 activating stimuli in obese states. To investigate whether NLRP3 activation contributes to the pathogenesis of diet-induced obesity (DIO) in mice, we tested two different clinical-stage NLRP3 inflammasome inhibitors. We demonstrate a contributory role of this key inflammasome to established obesity and associated systemic and cerebral inflammation. By comparing their effects to calorie restriction, we aimed to identify specific NLRP3-sensitive mechanisms contributing to obesity-induced inflammation (as opposed to be those regulated by weight loss per se). In addition, a direct comparison of an NLRP3 inhibitor to a glucagon like peptide-1 receptor agonist, semaglutide (Wegovy), in the DIO model allowed an appreciation of the relative efficacy of these two therapeutic strategies on obesity, its associated systemic inflammatory response, and cerebral gliosis. We show that two structurally distinct, NLRP3 inhibitors, NT-0249 and NT-0796, reverse obesity in the DIO mouse model and that brain exposure appears necessary for efficacy. In support of this, we show that DIO-driven hypothalamic glial fibrillary acidic protein expression is blocked by dosing with NT-0249/NT-0796. While matching weight loss driven by semaglutide or calorie restriction, remarkably, NLRP3 inhibition provided enhanced improvements in disease-relevant biomarkers of acute phase response, cardiovascular inflammation, and lipid metabolism. SIGNIFICANCE STATEMENT: Obesity is a global health concern that predisposes individuals to chronic disease such as diabetes and cardiovascular disease at least in part by promoting systemic inflammation. We report that in mice fed a high-fat, obesogenic diet, obesity is reversed by either of two inhibitors of the intracellular inflammatory mediator NLRP3. Furthermore, NLRP3 inhibition reduces both hypothalamic gliosis and circulating biomarkers of cardiovascular disease risk beyond what can be achieved by either the glucagon like peptide-1 agonist semaglutide or calorie restriction alone.

Target Cell Activation of a Structurally Novel NOD-Like Receptor Pyrin Domain-Containing Protein 3 Inhibitor NT-0796 Enhances Potency.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1ß and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: The Catalytic Roles of Tyr24 and Asp80.

The human enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (HsDNPH1) catalyses the hydrolysis of 5-hydroxymethyl-2'-deoxyuridine 5'-phosphate to generate 5-hydroxymethyluracil and 2-deoxyribose-5-phosphate via a covalent 5-phospho-2-deoxyribosylated enzyme intermediate. HsDNPH1 is a promising target for inhibitor development towards anticancer drugs. Here, site-directed mutagenesis of conserved active-site residues, followed by HPLC analysis of the reaction and steady-state kinetics are employed to reveal the importance of each of these residues in catalysis, and the reaction pH-dependence is perturbed by each mutation. Solvent deuterium isotope effects indicate no rate-limiting proton transfers. Crystal structures of D80N-HsDNPH1 in unliganded and substrate-bound states, and of unliganded D80A- and Y24F-HsDNPH1 offer atomic level insights into substrate binding and catalysis. The results reveal a network of hydrogen bonds involving the substrate and the E104-Y24-D80 catalytic triad and are consistent with a proposed mechanism whereby D80 is important for substrate positioning, for helping modulate E104 nucleophilicity, and as the general acid in the first half-reaction. Y24 positions E104 for catalysis and prevents a catalytically disruptive close contact between E104 and D80.

DESI-MSI-guided exploration of metabolic-phenotypic relationships reveals a correlation between PI 38:3 and proliferating cells in clear cell renal cell carcinoma via single-section co-registration of multimodal imaging.

A workflow has been evaluated that utilizes a single tissue section to obtain spatially co-registered, molecular, and phenotypical information suitable for AI-enabled image analysis. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to obtain molecular information followed by conventional histological staining and immunolabelling. The impact of varying DESI-MSI conditions (e.g., heated transfer line (HTL) temperature, scan rate, acquisition time) on the detection of small molecules and lipids as well as on tissue integrity crucial for integration into typical clinical pathology workflows was assessed in human kidney. Increasing the heated transfer line temperature from 150 to 450 °C resulted in a 1.8-fold enhancement in lipid signal at a scan rate of 10 scans/s, while preserving histological features. Moreover, increasing the acquisition speed to 30 scans/s yielded superior lipid signal when compared to 10 scans/s at 150 °C. Tissue morphology and protein epitopes remained intact allowing full histological assessment and further multiplex phenotyping by immunofluorescence (mIF) and immunohistochemistry (mIHC) of the same section. The successful integration of the workflow incorporating DESI-MSI, H&E, and immunolabelling on a single tissue section revealed an accumulation of ascorbic acid in regions of focal chronic inflammatory cell infiltrate within non-cancerous kidney tissue. Additionally, a strong positive correlation between PI 38:3 and proliferating cells was observed in clear cell renal cell carcinoma (ccRCC) showing the utility of this approach in uncovering molecular associations in disease pathology.

Ethnicity and Observed Oxygen Saturations, Fraction of Inspired Oxygen, and Clinical Outcomes: A Post Hoc Analysis of the Oxy-PICU Trial of Conservative Oxygenation.

OBJECTIVES: A conservative oxygenation strategy, targeting peripheral oxygen saturations (Spo2) between 88% and 92% in mechanically ventilated children in PICU, was associated with a shorter duration of organ support and greater survival compared with Spo2 greater than 94% in our recent Oxy-PICU trial. Spo2 monitors may overestimate arterial oxygen saturation (Sao2) in patients with higher levels of skin pigmentation compared with those with less skin pigmentation. We investigated if ethnicity was associated with changes in distributions of Spo2 and Fio2 and outcome. DESIGN: Post hoc analysis of a pragmatic, open-label, multicenter randomized controlled trial. SETTING: Fifteen PICUs across the United Kingdom and Scotland. PATIENTS: Children aged 38 weeks corrected gestational age to 15 years accepted to a participating PICU as an unplanned admission and receiving invasive mechanical ventilation with supplemental oxygen for abnormal gas exchange. METHODS: Hierarchical regression models for Spo2 and Fio2, and ordinal models for the primary trial outcome of a composite of the duration of organ support at 30 days and death, were used to examine the effects of ethnicity, accounting for baseline Spo2, Fio2, and mean airway pressure and trial allocation. MEASUREMENTS AND MAIN RESULTS: Ethnicity data were available for 1577 of 1986 eligible children, 1408 (89.3%) of which were White, Asian, or Black. Spo2 and Fio2 distributions did not vary according to Black or Asian ethnicity compared with White children. The trial primary outcome measure also did not vary significantly with ethnicity. The point estimate for the treatment effect of conservative oxygenation in Black children was 0.64 (95% CI, 0.33-1.25) compared with 0.84 (0.68-1.04) in the overall trial population. CONCLUSIONS: These data do not suggest that the association between improved outcomes and conservative oxygenation strategy in mechanically ventilated children in PICU is modified by ethnicity.

Protocol for a Randomized Controlled Trial to Evaluate a Permissive Blood Pressure Target Versus Usual Care in Critically Ill Children with Hypotension (PRESSURE).

OBJECTIVES: Management of hypotension is a fundamental part of pediatric critical care, with cardiovascular support in the form of fluids or vasoactive drugs offered to every hypotensive child. However, optimal blood pressure (BP) targets are unknown. The PRotocolised Evaluation of PermiSSive BP Targets Versus Usual CaRE (PRESSURE) trial aims to evaluate the clinical and cost-effectiveness of a permissive mean arterial pressure (MAP) target of greater than a fifth centile for age compared with usual care. DESIGN: Pragmatic, open, multicenter, parallel-group randomized control trial (RCT) with integrated economic evaluation. SETTING: Eighteen PICUs across the United Kingdom. PATIENTS: Infants and children older than 37 weeks corrected gestational age to 16 years accepted to a participating PICU, on mechanical ventilation and receiving vasoactive drugs for hypotension. INTERVENTIONS: Adjustment of hemodynamic support to achieve a permissive MAP target greater than fifth centile for age during invasive mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Randomization is 1:1 to a permissive MAP target or usual care, stratified by site and age group. Due to the emergency nature of the treatment, approaching patients for written informed consent will be deferred until after randomization. The primary clinical outcome is a composite of death and days of ventilatory support at 30 days. Baseline demographics and clinical status will be recorded as well as daily measures of BP and organ support, and discharge outcomes. This RCT received Health Research Authority approval (reference 289545), and a favorable ethical opinion from the East of England-Cambridge South Research Ethics Committee on May 10, 2021 (reference number 21/EE/0084). The trial is registered and has an International Standard RCT Number (reference 20609635). CONCLUSIONS: Trial findings will be disseminated in U.K. national and international conferences and in peer-reviewed journals.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.

The enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

Placental endocrine insufficiency programs anxiety, deficits in cognition and atypical social behaviour in offspring.

Abnormally elevated expression of the imprinted PHLDA2 gene has been reported in the placenta of human babies that are growth restricted in utero in several studies. We previously modelled this gene alteration in mice and found that just 2-fold increased expression of Phlda2 resulted in placental endocrine insufficiency. In addition, elevated Phlda2 was found to drive fetal growth restriction (FGR) of transgenic offspring and impaired maternal care by their wildtype mothers. Being born small and being exposed to suboptimal maternal care have both been associated with the increased risk of mental health disorders in human populations. In the current study we probed behavioural consequences of elevated Phlda2 for the offspring. We discovered increased anxiety-like behaviours, deficits in cognition and atypical social behaviours, with the greatest impact on male offspring. Subsequent analysis revealed alterations in the transcriptome of the adult offspring hippocampus, hypothalamus and amygdala, regions consistent with these behavioural observations. The inclusion of a group of fully wildtype controls raised in a normal maternal environment allowed us to attribute behavioural and molecular alterations to the adverse maternal environment induced by placental endocrine insufficiency rather than the specific gene change of elevated Phlda2. Our work demonstrates that a highly common alteration reported in human FGR is associated with negative behavioural outcomes later in life. Importantly, we also establish the experimental paradigm that placental endocrine insufficiency can program atypical behaviour in offspring highlighting the under-appreciated role of placental endocrine insufficiency in driving disorders of later life behaviour.

Pathophysiology of Hypertension: The Mosaic Theory and Beyond.

Dr Irvine Page proposed the Mosaic Theory of Hypertension in the 1940s advocating that hypertension is the result of many factors that interact to raise blood pressure and cause end-organ damage. Over the years, Dr Page modified his paradigm, and new concepts regarding oxidative stress, inflammation, genetics, sodium homeostasis, and the microbiome have arisen that allow further refinements of the Mosaic Theory. A constant feature of this approach to understanding hypertension is that the various nodes are interdependent and that these almost certainly vary between experimental models and between individuals with hypertension. This review discusses these new concepts and provides an introduction to other reviews in this compendium of Circulation Research.

Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension.

[Figure: see text].

The effect of blood pressure on mortality following out-of-hospital cardiac arrest: a retrospective cohort study of the United Kingdom Intensive Care National Audit and Research Centre database.

BACKGROUND: Hypotension following out-of-hospital cardiac arrest (OHCA) may cause secondary brain injury and increase mortality rates. Current guidelines recommend avoiding hypotension. However, the optimal blood pressure following OHCA is unknown. We hypothesised that exposure to hypotension and hypertension in the first 24 h in ICU would be associated with mortality following OHCA. METHODS: We conducted a retrospective analysis of OHCA patients included in the Intensive Care National Audit and Research Centre Case Mix Programme from 1 January 2010 to 31 December 2019. Restricted cubic splines were created following adjustment for important prognostic variables. We report the adjusted odds ratio for associations between lowest and highest mean arterial pressure (MAP) and systolic blood pressure (SBP) in the first 24 h of ICU care and hospital mortality. RESULTS: A total of 32,349 patients were included in the analysis. Hospital mortality was 56.2%. The median lowest and highest MAP and SBP were similar in survivors and non-survivors. Both hypotension and hypertension were associated with increased mortality. Patients who had a lowest recorded MAP in the range 60-63 mmHg had the lowest associated mortality. Patients who had a highest recorded MAP in the range 95-104 mmHg had the lowest associated mortality. The association between SBP and mortality followed a similar pattern to MAP. CONCLUSIONS: We found an association between hypotension and hypertension in the first 24 h in ICU and mortality following OHCA. The inability to distinguish between the median blood pressure of survivors and non-survivors indicates the need for research into individualised blood pressure targets for survivors following OHCA.

High tissue-sodium associates with systemic inflammation and insulin resistance in obese individuals.

BACKGROUND AND AIMS: High sodium intake is associated with obesity and insulin resistance, and high extracellular sodium content may induce systemic inflammation, leading to cardiovascular disease. In this study, we aim to investigate whether high tissue sodium accumulation relates with obesity-related insulin resistance and whether the pro-inflammatory effects of excess tissue sodium accumulation may contribute to such association. METHODS AND RESULTS: In a cross-sectional study of 30 obese and 53 non-obese subjects, we measured insulin sensitivity determined as glucose disposal rate (GDR) using hyperinsulinemic euglycemic clamp, and tissue sodium content using 23Na magnetic resonance imaging. Median age was 48 years, 68% were female and 41% were African American. Median (interquartile range) BMI was 33 (31.5, 36.3) and 25 (23.5, 27.2) kg/m2 in the obese and non-obese individuals, respectively. In obese individuals, insulin sensitivity negatively correlated with muscle (r = -0.45, p = 0.01) and skin sodium (r = -0.46, p = 0.01). In interaction analysis among obese individuals, tissue sodium had a greater effect on insulin sensitivity at higher levels of high-sensitivity C-reactive protein (p-interaction = 0.03 and 0.01 for muscle and skin Na+, respectively) and interleukin-6 (p-interaction = 0.024 and 0.003 for muscle and skin Na+, respectively). In interaction analysis of the entire cohort, the association between muscle sodium and insulin sensitivity was stronger with increasing levels of serum leptin (p-interaction = 0.01). CONCLUSIONS: Higher muscle and skin sodium are associated with insulin resistance in obese patients. Whether high tissue sodium accumulation has a mechanistic role in the development of obesity-related insulin resistance through systemic inflammation and leptin dysregulation remains to be examined in future studies. CLINICALTRIALS: gov registration: NCT02236520.

Randomization to a Liberal Versus Conservative Oxygenation Target: Redox Responses in Critically Ill Children.

RATIONALE: Optimal systemic oxygenation targets in pediatric critical illness are unknown. A U-shaped relationship exists between blood oxygen levels and PICU mortality. Redox stress or iatrogenic injury from intensive treatments are potential mechanisms of harm from hyperoxia. OBJECTIVES: To measure biomarkers of oxidative status in children admitted to PICU and randomized to conservative (oxygen-hemoglobin saturation [Sp o2 ] 88-92%) versus liberal (Sp o2 > 94%) peripheral oxygenation targets. DESIGN: Mechanistic substudy nested within the Oxygen in PICU (Oxy-PICU) pilot randomized feasibility clinical trial ( ClinicalTrials.gov : NCT03040570). SETTING: Three U.K. mixed medical and surgical PICUs in university hospitals. PATIENTS: Seventy-five eligible patients randomized to the Oxy-PICU randomized feasibility clinical trial. INTERVENTIONS: Randomization to a conservative (Sp o2 88-92%) versus liberal (Sp o2 > 94%) peripheral oxygenation target. MEASUREMENTS AND MAIN RESULTS: Blood and urine samples were collected at two timepoints: less than 24 hours and up to 72 hours from randomization in trial participants (March 2017 to July 2017). Plasma was analyzed for markers of ischemic/oxidative response, namely thiobarbituric acid-reactive substances (TBARS; lipid peroxidation marker) and ischemia-modified albumin (protein oxidation marker). Total urinary nitrate/nitrite was measured as a marker of reactive oxygen and nitrogen species (RONS). Blood hypoxia-inducible factor (HIF)-1a messenger RNA (mRNA) expression (hypoxia response gene) was measured by reverse transcription- polymerase chain reaction. Total urinary nitrate/nitrite levels were greater in the liberal compared with conservative oxygenation group at 72 hours (median difference 32.6 µmol/mmol of creatinine [95% CI 13.7-93.6]; p < 0.002, Mann-Whitney test). HIF-1a mRNA expression was increased in the conservative group compared with liberal in less than 24-hour samples (6.0-fold [95% CI 1.3-24.0]; p = 0.032). There were no significant differences in TBARS or ischemia-modified albumin. CONCLUSIONS: On comparing liberal with conservative oxygenation targets, we show, first, significant redox response (increase in urinary markers of RONS), but no changes in markers of lipid or protein oxidation. We also show what appears to be an early hypoxic response (increase in HIF-1a gene expression) in subjects exposed to conservative rather than liberal oxygenation targets.