Changes in carotid arterial wall viscosity and carotid arterial stiffness in type 2 diabetes patients.

BACKGROUND AND AIMS: Changes in arterial wall viscosity (AWW) and stiffness during type 2 diabetes (T2D) have been little investigated. We explored changes in carotid AWV considering change in arterial stiffness and loading conditions, in patients with T2D. METHODS: This cross-sectional, monocentric study compared 19 middle-aged patients with T2D to 30 non-diabetic (ND) controls. The absolute viscosity (WV) was determined as the area of the pressure-lumen cross-sectional area (P-LCSA) loop obtained by carotid tonometry and contralateral echo-tracking. The relative viscosity was determined as the ratio between WV and the elastic energy stored within the arterial wall (WV/WE). Carotid geometry, midwall stress, distensibility and elastic modulus were also compared between groups. RESULTS: T2D patients were older and more frequently had hypertension. Internal diameter, mean central and pulse blood pressure were higher in T2D patients but midwall stress was similar compared to ND controls. WV and WV/WE were higher in T2D patients when compared with ND controls (23 [16-41] vs. 11 [7-18] mm Hg.mm2, p=0.007 and 21% [17-25] vs. 12% [8-17], p < 0.001 respectively) even after adjustment on confounding factors. Carotid arterial stiffness was higher in T2D patients, but after adjustment this difference was only observed for the highest levels of midwall stress. CONCLUSIONS: Carotid AWV and stiffness are increased in T2D patients but only AWV is significantly increased after considering loading conditions. Whether this increase in energy dissipation within the arterial wall contributes to alter cardiovascular coupling in T2D remains to be established.

Multiple sclerosis and bowel symptoms: Frequency and barriers to their management.

BACKGROUND: The frequency of bowel symptoms (BSs) is still a matter for debate in multiple sclerosis (MS) patients. However, BSs have been shown to cause significant distress. Our study aimed to (i) investigate the frequency of BSs, particularly those that are not managed, (ii) identify potential predictors for help-seeking care for patients with BSs, and (iii) evaluate the ability of the Neurogenic Bowel Dysfunction (NBD) score to screen for BSs. METHOD: Three hundred sixty-nine MS patients completed a cross-sectional demographic and clinical survey of MS and BSs and their management. RESULTS: BSs were reported by 47.7% of MS patients. Eighty-eight percent of MS patients had a very minor-minor Neurogenic Bowel Disorder (NBD) score and 12% had a moderate-severe NBD score. Forty-one percent of patients did not report their BS to a healthcare provider, mainly because they preferred not to talk about the problem. BS duration was the only significant predictor of help-seeking for BS management. Female sex, visual impairment, a digestive history, and longer MS duration were good predictors of BSs. Patients with BSs (86%) were correctly identified with an NBD score >2. CONCLUSION: BSs are under-detected in MS populations. This is partially related to non-declaration by patients. Targeting BSs using the NBD score is a good way to increase reporting.

Inhibition of Soluble Epoxide Hydrolase Does Not Promote or Aggravate Pulmonary Hypertension in Rats.

Inhibitors of soluble epoxide hydrolase (sEH), which catalyzes the hydrolysis of various natural epoxides to their corresponding diols, present an opportunity for developing oral drugs for a range of human cardiovascular and inflammatory diseases, including, among others, diabetes and neuropathic pain. However, some evidence suggests that their administration may precipitate the development of pulmonary hypertension (PH). We thus evaluated the impact of chronic oral administration of the sEH inhibitor TPPU (N-[1-(1-Oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]-urea) on hemodynamics, pulmonary vascular reactivity, and remodeling, as well as on right ventricular (RV) dimension and function at baseline and in the Sugen (SU5416) + hypoxia (SuHx) rat model of severe PH. Treatment with TPPU started 5 weeks after SU5416 injection for 3 weeks. No differences regarding the increase in pulmonary vascular resistance, remodeling, and inflammation, nor the abolishment of phenylephrine-induced pulmonary artery constriction, were noted in SuHx rats. In addition, TPPU did not modify the development of RV dysfunction, hypertrophy, and fibrosis in SuHx rats. Similarly, none of these parameters were affected by TPPU in normoxic rats. Complementary in vitro data demonstrated that TPPU reduced the proliferation of cultured human pulmonary artery-smooth muscle cells (PA-SMCs). This study demonstrates that inhibition of sEH does not induce nor aggravate the development of PH and RV dysfunction in SuHx rats. In contrast, a potential beneficial effect against pulmonary artery remodeling in humans is suggested.

Population Pharmacokinetics of Levosimendan and its Metabolites in Critically Ill Neonates and Children Supported or Not by Extracorporeal Membrane Oxygenation.

BACKGROUND: Levosimendan (LVSMD) is a calcium-sensitizer inotropic and vasodilator agent whose use might have a beneficial effect on the weaning of venoarterial extracorporeal membrane oxygenation (VA-ECMO). In light of LVSMD pharmacological characteristics, we hypothesized that ECMO may induce major pharmacokinetic (PK) modifications for LVSMD and its metabolites. OBJECTIVE: The aim of this study was to investigate the PK of LVSMD and its metabolites, and to assess the effects of ECMO on PK parameters. METHODS: We conducted a multicentric, prospective study (NCT03681379). Twenty-seven infusions of LVSMD were performed, allowing for the collection of 255 blood samples. Non-linear mixed-effects modeling software (MONOLIX®) was used to develop a parent-metabolite PK model of LVSMD and its metabolites. RESULTS: Most patients received a 0.2 µg/kg/min infusion of LVSMD over 24 h. After elimination of non-reliable samples or concentrations below the limit of quantification, 166, 101 and 85 samples were considered for LVSMD, OR-1855 and OR-1896, respectively, of which 81, 53 and 41, respectively, were drawn under ECMO conditions. Parent-metabolite PK modeling revealed that a two-compartment model with first-order elimination best described LVSMD PK. Use of a transit compartment allowed for an explanation of the delayed appearance of circulating OR-1855 and OR-1896, with the latter following a first-order elimination. Patient weight influenced the central volume of distribution and elimination of LVSMD. ECMO support increased the elimination rate of LVSMD by 78%, and ECMO also slowed down the metabolite formation rate by 85% for OR-1855, which in turn is converted to the active metabolite OR-1896, 14% slower than without ECMO. Simulated data revealed that standard dosing may not be appropriate for patients under ECMO, with a decrease in the steady-state concentration of LVSMD and lower exposure to the active metabolite OR-1896. CONCLUSIONS: ECMO altered PK parameters for LVSMD and its metabolites. An infusion of LVSMD over 48 h, instead of 24 h, with a slightly higher dose may promote synthesis of the active metabolite OR-1896, which is responsible for the long-term efficacy of LVSMD. Further trials evaluating ECMO effects using a PK/pharmacodynamic approach may be of interest. REGISTRATION: ClinicalTrials.gov identifier number NCT03681379.

CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury.

INTRODUCTION: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown. OBJECTIVES: This study aimed to assess in vivo the physiological role of sEH-P. METHODS: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity. RESULTS: The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury. CONCLUSION: Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.

Determination of Lipoxygenase, CYP450, and Non-Enzymatic Metabolites of Arachidonic Acid in Essential Hypertension and Type 2 Diabetes.

Type 2 diabetes (T2D) and hypertension (HTN) are common risk factors of cardiovascular diseases (CVD) characterized by chronic low-grade systemic inflammation and impaired endothelial function. This study aimed to assess whether levels of non-enzymatic, lipoxygenase (LOX)- and cytochrome P450 (CYP)-derived arachidonic acid (ARA) metabolites, which are known regulators of vascular homeostasis, are affected by HTN and T2D. For this objective, 17 plasma level derivatives of ARA were quantitated by chromatography coupled with mass spectrometry in 44 patients (12 healthy, 8 HTN, 7 T2D, and 17 HTN + T2D). Effects of hyperglycemic and hyperinsulinemic clamps on ARA metabolite levels were assessed in seven healthy subjects. No significant differences in the plasma levels of ARA metabolites were observed for T2D patients compared with healthy volunteers. HTN was associated with an alteration of ARA metabolite correlation patterns with increased 20-, 19-, 15-, and 8-hydroxyeicosatrienoic acid (HETE). A decrease of 20-HETE was also observed during both hyperglycemic and hyperinsulinemic clamps. Additional experiments are needed to assess whether the modulation of HETE metabolites in HTN may be of interest. Furthermore, although not affected by T2D, it remains to investigate whether the decrease of 20-HETE observed during clamps may be related to the regulation of glucose tolerance and insulin signaling.

Relationship between anal functional lumen imaging probe (EndoFLIP®) results and the clinical presentation of faecal incontinence.

AIM: Faecal incontinence (FI) subtypes (urge, passive, mixed) are linked to the physiopathological mechanism of FI. Previous studies have failed to demonstrate a consistent relationship between FI subtype and anal sphincter dysfunction. Our aim was to evaluate the relationship between anal sphincter function, assessed using the new EndoFLIP® technology, and FI subtype. METHOD: Patients referred for FI were prospectively enrolled between October 2015 and May 2021 in a registry, and data were retrospectively examined. Each patient underwent a clinical assessment as well as three-dimensional high-resolution or water-perfused anorectal manometry, anal EndoFLIP®, and anorectal electrophysiological and endoanal ultrasound tests. The results of the investigations were compared across FI subtypes. RESULTS: The cohort included 133 patients, 54 (41%) of whom met the criteria for urge FI, 40 (30%) for passive FI and 39 (29%) for mixed FI. The resting anal distensibility index (DI) at 50 ml of distension was significantly lower in patients with urge FI than in patients with passive FI (p = 0.04). At rest, a DI at 50 ml of distension ≥7.3 mm2  mmHg-1 and a DI at 40 ml of distension <1.3 mm2  mmHg-1 were associated with the passive and urge FI subtypes, respectively, with poor discriminatory power (an accuracy of 0.49 compared with 0.33 for random assignment). There were no differences in anorectal manometry, endoanal ultrasound or electrophysiological test results among the urge, passive and mixed FI subgroups (all p > 0.05). CONCLUSION: The anal sphincter DI using the EndoFLIP® system displayed poor predictive performance in distinguishing among FI subtypes.

Adaptation of Arterial Wall Viscosity to the Short-Term Reduction of Heart Rate: Impact of Aging.

Background Changes in arterial wall viscosity, which dissipates the energy stored within the arterial wall, may contribute to the beneficial effect of heart rate (HR) reduction on arterial stiffness and cardiovascular coupling. However, it has never been assessed in humans and could be altered by aging. We evaluated the effect of a selective HR-lowering agent on carotid arterial wall viscosity and the impact of aging on this effect. Methods and Results This randomized, placebo-controlled, double-blind, crossover study performed in 19 healthy volunteers evaluated the effects of ivabradine (5 mg BID, 1-week) on carotid arterial wall viscosity, mechanics, hemodynamics, and cardiovascular coupling. Arterial wall viscosity was evaluated by the area of the hysteresis loop of the pressure-lumen cross-sectional area relationship, representing the energy dissipated (WV), and by the relative viscosity (WV/WE), with WE representing the elastic energy stored. HR reduction by ivabradine increased WV and WE whereas WV/WE remained stable. In middle-aged subjects (n=11), baseline arterial stiffness and cardiovascular coupling were less favorable, and WE was similar but WV and therefore WV/WE were lower than in youth (n=8). HR reduction increased WV/WE in middle-aged but not in young subjects, owing to a larger increase in WV than WE. These results were supported by the age-related linear increase in WV/WE after HR reduction (P=0.009), explained by a linear increase in WV. Conclusion HR reduction increases arterial wall energy dissipation proportionally to the increase in WE, suggesting an adaptive process to bradycardia. This mechanism is altered during aging resulting in a larger than expected energy dissipation, the impact of which should be assessed. Registration URL: https://www.clinicaltrials.gov; Unique identifier: 2015/077/HP. URL: https://www. eudract.ema.europa.eu; Unique identifier: 2015-002060-17.

Ten-year Evaluation of a Large Retrospective Cohort Treated by Sacral Nerve Modulation for Fecal Incontinence: Results of a French Multicenter Study.

OBJECTIVE: The aim of this study was to assess the effectiveness of sacral nerve modulation (SNM) in a large cohort of patients implanted for at least 10 years, quantify adverse event rates, and identify predictive factors of long-term success. SUMMARY BACKGROUND DATA: Few studies have evaluated the long-term success of SNM. METHODS: Data collected prospectively from patients implanted for fecal incontinence (FI) in 7 French centers between January 1998 and December 2008 were retrospectively analyzed. Patient FI severity scores were assessed before and 10 years after implantation. The main evaluation criterion was the success of SNM defined by the continuation of the treatment without additional therapies. The secondary evaluation criteria were the rate of device revisions and explantations. Preoperative predictors of success at 10 years were sought. RESULTS: Of the 360 patients (27 males, mean age: 59 ± 12 years) implanted for FI, 162 (45%) had a favorable outcome 10 years post-implantation, 115 (31.9%) failed, and 83 (23.1%) were lost to follow-up. The favorable outcome derived from the time-to-event Kaplan-Meier curve at 10 years was 0.64 (95% CI 0.58-0.69). FI severity scores were significantly better 10 years post-implantation compared to preimplantation (7.4 ± 4.3 vs 14.0 ± 3.2; P < 0.0001). During the 10-year follow-up, 233 patients (64.7%) had a surgical revision and 94 (26.1%) were explanted. A history of surgery for FI and sex (male) were associated with an increased risk of an unfavorable outcome. CONCLUSIONS: Long-term efficacy was maintained in approximately half of the FI patients treated by SNM at least 10 years post-implantation.

Pharmacokinetic modeling of morphine and its glucuronides: Comparison of nebulization versus intravenous route in healthy volunteers.

Intravenous (i.v.) morphine is a safe, robust, and recommended treatment for severe pain using the titration principle. Despite its high efficacy, it is impacted by organizational constraints related to venous access. Nebulized (NEB) morphine may represent an alternative for titration but pharmacokinetic (PK) properties of short nebulization using routine devices need evaluation. Twenty-seven healthy volunteers were included to receive NEB or i.v. morphine administration using increasing amounts according to Dixon's reference method. Plasma morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) were quantified. PK modeling and simulations were performed using Monolix. Dixon's method exhibited a significantly higher morphine dose regimen in the NEB group versus the i.v. group (6.2 [5.3-7.1] vs. 3.0 [2.0-4.0] mg, p < 0.001). Morphine, M3G, and M6G dose-normalized exposure were significantly lower in the NEB group versus the i.v. group: morphine (19 [13-23] vs. 1044 [702-1266] µg min/L, p < 0.001), M3G (245 [162-287] vs. 3752 [2487-5165] µg min/L, p < 0.001) and M6G (28 [21-43] vs. 466 [370-723] µg min/L, p < 0.001). The model that best fitted the data consisted in a transit compartment for morphine absorption, three compartments for morphine distribution followed by multiple transit compartments (8.2 and 57.5-min transit time for M3G and M6G, respectively) and a first order elimination for M3G and M6G. Morphine bioavailability in the NEB group was 3.5% using the i.v. group as reference. Administration route and sex significantly influenced morphine and metabolite PKs. This work aimed to evaluate the PKs of NEB morphine compared with the i.v. route. Despite a bioavailability to improve, NEB morphine administration using a routine device is suitable to plan morphine titration.

Preventing the Increase in Lysophosphatidic Acids: A New Therapeutic Target in Pulmonary Hypertension?

Cardiovascular diseases (CVD) are the leading cause of premature death and disability in humans that are closely related to lipid metabolism and signaling. This study aimed to assess whether circulating lysophospholipids (LPL), lysophosphatidic acids (LPA) and monoacylglycerols (MAG) may be considered as potential therapeutic targets in CVD. For this objective, plasma levels of 22 compounds (13 LPL, 6 LPA and 3 MAG) were monitored by liquid chromatography coupled with tandem mass spectrometry (HPLC/MS2) in different rat models of CVD, i.e., angiotensin-II-induced hypertension (HTN), ischemic chronic heart failure (CHF) and sugen/hypoxia(SuHx)-induced pulmonary hypertension (PH). On one hand, there were modest changes on the monitored compounds in HTN (LPA 16:0, 18:1 and 20:4, LPC 16:1) and CHF (LPA 16:0, LPC 18:1 and LPE 16:0 and 18:0) models compared to control rats but these changes were no longer significant after multiple testing corrections. On the other hand, PH was associated with important changes in plasma LPA with a significant increase in LPA 16:0, 18:1, 18:2, 20:4 and 22:6 species. A deleterious impact of LPA was confirmed on cultured human pulmonary smooth muscle cells (PA-SMCs) with an increase in their proliferation. Finally, plasma level of LPA(16:0) was positively associated with the increase in pulmonary artery systolic pressure in patients with cardiac dysfunction. This study demonstrates that circulating LPA may contribute to the pathophysiology of PH. Additional experiments are needed to assess whether the modulation of LPA signaling in PH may be of interest.

Investigation of Coagulation Biomarkers to Assess Clinical Deterioration in SARS-CoV-2 Infection.

Since December 2019, a pandemic caused by a new coronavirus has spread to more than 170 countries around the world. Worsening infected patients requiring intensive care unit (ICU) admission associated with 30% of mortality. A part of worsening is induced by hemostasis deregulation. The aim of this study was to investigate the association of coagulation activation in COVID-19 progression. Thirty-five of the 99 patients got clinically worse. The final model of the logistic regression analysis revealed that O2 requirement (RR = 7.27 [1.50-19.31]), monocytes below 0.2G/L (RR = 2.88 [1.67-3.19]), fibrinogen levels (RR = 1.45 [1.17-1.82] per g/L increase), prothrombin fragments 1+2 higher than 290 pM (RR = 2.39 [1.20-3.30]), and thrombin peak (RR = 1.28 [1.03-1.59] per 50 nM increase) were associated with an increased risk of clinical worsening. A fibrinogen level threshold of 5.5 g/L, a thrombin peak measurement threshold of 99 pM, and O2 requirement associated with clinical outcome in more than 80% of our cohort. In conclusion, we identified fibrinogen and thrombin peak at admission as coagulation biomarkers associated with an increased risk of ICU admission or death. This finding allows initiating steroids and triage for worsening patients. Our results should therefore be considered as exploratory and deserve confirmation.

Preservation of epoxyeicosatrienoic acid bioavailability prevents renal allograft dysfunction and cardiovascular alterations in kidney transplant recipients.

This study addressed the hypothesis that epoxyeicosatrienoic acids (EETs) synthesized by CYP450 and catabolized by soluble epoxide hydrolase (sEH) are involved in the maintenance of renal allograft function, either directly or through modulation of cardiovascular function. The impact of single nucleotide polymorphisms (SNPs) in the sEH gene EPHX2 and CYP450 on renal and vascular function, plasma levels of EETs and peripheral blood monuclear cell sEH activity was assessed in 79 kidney transplant recipients explored at least one year after transplantation. Additional experiments in a mouse model mimicking the ischemia-reperfusion (I/R) injury suffered by the transplanted kidney evaluated the cardiovascular and renal effects of the sEH inhibitor t-AUCB administered in drinking water (10 mg/l) during 28 days after surgery. There was a long-term protective effect of the sEH SNP rs6558004, which increased EET plasma levels, on renal allograft function and a deleterious effect of K55R, which increased sEH activity. Surprisingly, the loss-of-function CYP2C9*3 was associated with a better renal function without affecting EET levels. R287Q SNP, which decreased sEH activity, was protective against vascular dysfunction while CYP2C8*3 and 2C9*2 loss-of-function SNP, altered endothelial function by reducing flow-induced EET release. In I/R mice, sEH inhibition reduced kidney lesions, prevented cardiac fibrosis and dysfunction as well as preserved endothelial function. The preservation of EET bioavailability may prevent allograft dysfunction and improve cardiovascular disease in kidney transplant recipients. Inhibition of sEH appears thus as a novel therapeutic option but its impact on other epoxyfatty acids should be carefully evaluated.

Pharmacogenetics of Direct Oral Anticoagulants: A Systematic Review.

Dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban are direct oral anticoagulants (DOACs). Their inter-individual variability in pharmacodynamics and pharmacokinetics (transport and metabolism) is high, and could result from genetic polymorphisms. As recommended by the French Network of Pharmacogenetics (RNPGx), the management of some treatments in cardiovascular diseases (as antiplatelet agents, oral vitamin K antagonists, and statins) can rely on genetic testing in order to improve healthcare by reducing therapeutic resistance or toxicity. This paper is a review of association studies between single nucleotide polymorphisms (SNPs) and systemic exposure variation of DOACs. Most of the results presented here have a lot to do with some SNPs of CES1 (rs2244613, rs8192935, and rs71647871) and ABCB1 (rs1128503, rs2032582, rs1045642, and rs4148738) genes, and dabigatran, rivaroxaban, and apixaban. Regarding edoxaban and betrixaban, as well as SNPs in the CYP3A4 and CYP3A5 genes, literature is scarce, and further studies are needed.

The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.

Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 µg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes.

Fostering global data sharing: highlighting the recommendations of the Research Data Alliance COVID-19 working group.

The systemic challenges of the COVID-19 pandemic require cross-disciplinary collaboration in a global and timely fashion. Such collaboration needs open research practices and the sharing of research outputs, such as data and code, thereby facilitating research and research reproducibility and timely collaboration beyond borders. The Research Data Alliance COVID-19 Working Group recently published a set of recommendations and guidelines on data sharing and related best practices for COVID-19 research. These guidelines include recommendations for clinicians, researchers, policy- and decision-makers, funders, publishers, public health experts, disaster preparedness and response experts, infrastructure providers from the perspective of different domains (Clinical Medicine, Omics, Epidemiology, Social Sciences, Community Participation, Indigenous Peoples, Research Software, Legal and Ethical Considerations), and other potential users. These guidelines include recommendations for researchers, policymakers, funders, publishers and infrastructure providers from the perspective of different domains (Clinical Medicine, Omics, Epidemiology, Social Sciences, Community Participation, Indigenous Peoples, Research Software, Legal and Ethical Considerations). Several overarching themes have emerged from this document such as the need to balance the creation of data adherent to FAIR principles (findable, accessible, interoperable and reusable), with the need for quick data release; the use of trustworthy research data repositories; the use of well-annotated data with meaningful metadata; and practices of documenting methods and software. The resulting document marks an unprecedented cross-disciplinary, cross-sectoral, and cross-jurisdictional effort authored by over 160 experts from around the globe. This letter summarises key points of the Recommendations and Guidelines, highlights the relevant findings, shines a spotlight on the process, and suggests how these developments can be leveraged by the wider scientific community.

Soluble Epoxide Hydrolase Inhibition Prevents Experimental Type 4 Cardiorenal Syndrome.

Objectives: Cardiovascular diseases (CVD) remain the leading cause of morbimortality in patients with chronic kidney disease (CKD). The aim of this study was to assess the cardiovascular impact of the pharmacological inhibition of soluble epoxide hydrolase (sEH), which metabolizes the endothelium-derived vasodilatory and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acid (DHETs), in the 5/6 nephrectomy (Nx) mouse model. Methods and Results: Compared to sham-operated mice, there was decrease in EET-to-DHET ratio 3 months after surgery in vehicle-treated Nx mice but not in mice treated with the sEH inhibitor t-AUCB. Nx induced an increase in plasma creatinine and in urine albumin-to-creatinine ratio as well as the development of kidney histological lesions, all of which were not modified by t-AUCB. In addition, t-AUCB did not oppose Nx-induced blood pressure increase. However, t-AUCB prevented the development of cardiac hypertrophy and fibrosis induced by Nx, as well as normalized the echocardiographic indices of diastolic and systolic function. Moreover, the reduction in endothelium-dependent flow-mediated dilatation of isolated mesenteric arteries induced by Nx was blunted by t-AUCB without change in endothelium-independent dilatation to sodium nitroprusside. Conclusion: Inhibition of sEH reduces the cardiac remodelling, and the diastolic and systolic dysfunctions associated with CKD. These beneficial effects may be mediated by the prevention of endothelial dysfunction, independent from kidney preservation and antihypertensor effect. Thus, inhibition of sEH holds a therapeutic potential in preventing type 4 cardiorenal syndrome.

Exploring the microvascular impact of red blood cell transfusion in intensive care unit patients.

BACKGROUND: Red blood cell (RBC) transfusion is a common treatment for hospitalized patients. However, the effects of RBC transfusion on microvascular function remain controversial. METHODS: In a medical ICU in a tertiary teaching hospital, we prospectively included anemic patients requiring RBC transfusion. Skin microvascular reactivity was measured before and 30 min after RBC transfusion. Plasma was collected to analyze intravascular hemolysis and draw the lipidomic and cytokine profiles. RESULTS: In a cohort of 59 patients, the median age was 66 [55-81] years and SAPS II was 38 [24-48]. After RBC transfusion, endothelium-dependent microvascular reactivity improved in 35 (59%) patients, but worsened in 24 others (41%). Comparing clinical and biological markers revealed that baseline blood leucokyte counts distinguished improving from worsening patients (10.3 [5.7; 19.7] vs. 4.6 [2.1; 7.3] × 109/L; p = 0.001) and correlated with variations of microvascular reactivity (r = 0.36, p = 0.005). Blood platelet count was also higher in improving patients (200 [97; 280] vs 160 [40; 199] × 103/mL, p = 0.03) but did not correlate with variations of microvascular reactivity. We observed no intravascular hemolysis (HbCO, heme, bilirubin, LDH), but recorded a significant increase in RBC microparticle levels specific to improving patients after transfusion (292 [108; 531] vs. 53 [34; 99] MP/µL; p = 0.03). The improvement in microvascular dilation was positively correlated with RBC microparticle levels (R = 0.83, p < 0.001) and conversion of arachidonic acid into vasodilating eicosanoids. CONCLUSIONS: Patients displaying an improved microvascular reactivity after RBC transfusion had high blood leukocyte counts, increased RBC microparticle formation, and enhanced metabolism of arachidonic acid into vasodilating lipids. Our data suggested a contribution of recipient leukocytes to the vascular impact of RBC transfusion.