ABSTRACT
BACKGROUND: Hyperglycemia is common in patients undergoing cardiovascular surgery with cardiopulmonary bypass. We hypothesize that intraoperative hyperglycemia may be, at least partially, attributable to insulin loss due to adhesion on artificial surfaces and/or degradation by hemolysis. Thus, our primary aim was to investigate the loss of insulin in 2 different isolated extracorporeal circulation circuits (ECCs), that is, a conventional ECC (cECC) with a roller pump, and a mini-ECC (MiECC) system with a centrifugal pump. The secondary aim was to assess and compare the relationship between changes in insulin concentration and the degree of hemolysis in our 2 ECC models. METHODS: Six cECC and 6 MiECC systems were primed with red packed blood cells and thawed fresh-frozen plasma (1:1). Four additional experiments were performed in cECC using only thawed fresh-frozen plasma. Human insulin (Actrapid) was added, targeting a plasma insulin concentration of 400 mU/L. Insulin concentration and hemolysis index were measured at baseline and hourly thereafter. The end points were the change in insulin level after 4 hours compared to baseline and hemolysis index after 4 hours. The insulin concentration and hemolysis index were analyzed by means of a saturated linear mixed-effect regression model with a random offset for each experiment to account for the repeated measure design of the study, resulting in mean estimates and 95% confidence intervals (CIs) of the primary end points as well as of pairwise contrasts with respect to ECC type. RESULTS: Insulin concentration decreased by 63% (95% CI, 48%-77%) in the MiECC and 92% (95% CI, 77%-106%) in the cECC system that contained red blood cells. Insulin loss was significantly higher in the cECC system compared to the MiECC (P = .022). In the cECC with only plasma, insulin did not significantly decrease (-4%; 95% CI, -21% to 14%). Hemolysis index in MiECC increased from 68 (95% CI, 46-91) to 76 (95% CI, 54-98) after 4 hours, in cECC from 81 (95% CI, 59-103) to 121 (95% CI, 99-143). Hemolysis index and percent change of insulin showed an excellent relationship (r = -0.99, P < .01). CONCLUSIONS: Our data showed that insulin levels substantially decreased during 4 hours of simulated cardiopulmonary bypass only in the ECC that contained hemoglobin. The decrease was more pronounced in the cECC, which also exhibited a greater degree of hemolysis. Our results suggest that insulin degradation by hemolysis products may be a stronger contributor to insulin loss than adhesion of insulin molecules to circuit surfaces.
ABSTRACT
Despite clinical benefits of tyrosine kinase inhibitors (TKIs) in cancer, most tumors can reactivate proliferation under TKI therapy. Here we present transcriptional profiling of HER2+ breast cancer cells transitioning from dormant drug tolerant cells to re-proliferating cells under continuous HER2 inhibitor (HER2i) therapy. Focusing on phosphatases, expression of dual-specificity phosphatase DUSP6 was found inhibited in dormant cells, but strongly induced upon regrowth. DUSP6 expression also selectively associated with poor patient survival in HER2+ breast cancers. DUSP6 overexpression conferred apoptosis resistance, whereas its pharmacological blockade prevented therapy tolerance development under HER2i therapy. DUSP6 targeting also synergized with clinically used HER2i combination therapies. Mechanistically DUSP6 is a positive regulator of HER3 expression, and its impact on HER2i tolerance was mediated by neuregulin-HER3 axis. In vivo, genetic targeting of DUSP6 reduced tumor growth in brain metastasis model, whereas its pharmacological targeting induced synthetic lethal therapeutic effect in combination with HER2i. Collectively this work demonstrates that DUSP6 drives escape from HER2i-induced dormancy, and that DUSP6 is a druggable target to overcome HER3-driven TKI resistance.
Subject(s)
Breast Neoplasms , Dual Specificity Phosphatase 6 , Receptor, ErbB-2 , Receptor, ErbB-3 , Dual Specificity Phosphatase 6/metabolism , Dual Specificity Phosphatase 6/genetics , Humans , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Female , Receptor, ErbB-2/metabolism , Animals , Receptor, ErbB-3/metabolism , Receptor, ErbB-3/genetics , Receptor, ErbB-3/antagonists & inhibitors , Cell Line, Tumor , Mice , Drug Resistance, Neoplasm/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Protein Kinase Inhibitors/pharmacologyABSTRACT
BACKGROUND: Despite the fervent scientific effort, a state-of-the art assessment of the different causes of hypoxemia (shunt, ventilation-perfusion mismatch, and diffusion limitation) in COVID-19 acute respiratory distress syndrome (ARDS) is currently lacking. In this study, the authors hypothesized a multifactorial genesis of hypoxemia and aimed to measure the relative contribution of each of the different mechanism and their relationship with the distribution of tissue and blood within the lung. METHODS: In this cross-sectional study, the authors prospectively enrolled 10 patients with COVID-19 ARDS who had been intubated for less than 7 days. The multiple inert gas elimination technique (MIGET) and a dual-energy computed tomography (DECT) were performed and quantitatively analyzed for both tissue and blood volume. Variables related to the respiratory mechanics and invasive hemodynamics (PiCCO [Getinge, Sweden]) were also recorded. RESULTS: The sample (51 ± 15 yr; Pao2/Fio2, 172 ± 86 mmHg) had a mortality of 50%. The MIGET showed a shunt of 25 ± 16% and a dead space of 53 ± 11%. Ventilation and perfusion were mismatched (LogSD, Q, 0.86 ± 0.33). Unexpectedly, evidence of diffusion limitation or postpulmonary shunting was also found. In the well aerated regions, the blood volume was in excess compared to the tissue, while the opposite happened in the atelectasis. Shunt was proportional to the blood volume of the atelectasis (R2 = 0.70, P = 0.003). VËA/QËT mismatch was correlated with the blood volume of the poorly aerated tissue (R2 = 0.54, P = 0.016). The overperfusion coefficient was related to Pao2/Fio2 (R2 = 0.66, P = 0.002), excess tissue mass (R2 = 0.84, P < 0.001), and Etco2/Paco2 (R2 = 0.63, P = 0.004). CONCLUSIONS: These data support the hypothesis of a highly multifactorial genesis of hypoxemia. Moreover, recent evidence from post-mortem studies (i.e., opening of intrapulmonary bronchopulmonary anastomosis) may explain the findings regarding the postpulmonary shunting. The hyperperfusion might be related to the disease severity.
Subject(s)
COVID-19 , Pulmonary Atelectasis , Respiratory Distress Syndrome , Humans , Ventilation-Perfusion Ratio , Cross-Sectional Studies , COVID-19/complications , Respiratory Distress Syndrome/diagnostic imaging , Hypoxia/diagnostic imaging , Hypoxia/etiology , Tomography , Pulmonary Gas ExchangeABSTRACT
The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/metabolism , Membrane Transport Proteins/metabolism , Escherichia coli Proteins/metabolism , Ribosomes/metabolism , Arginine/metabolism , Cell Membrane/metabolismABSTRACT
Background: Obese patients frequently develop pulmonary atelectasis upon general anesthesia. The risk is increased during laparoscopic surgery. This prospective, observational single-center study evaluated atelectasis dynamics using Electric Impedance Tomography (EIT) in patients undergoing laparoscopic bariatric surgery. Methods: We included adult patients with ASA physical status I-IV and a BMI of ≥40. Exclusion criteria were known severe pulmonary hypertension, home oxygen therapy, heart failure, and recent pulmonary infections. The primary outcome was the proportion of poorly ventilated lung regions (low tidal variation areas) and the global inhomogeneity (GI) index assessed by EIT before discharge from the Post Anesthesia Care Unit compared to these same measures prior to initiation of anesthesia. Results: The median (IQR) proportion of low tidal variation areas at the different analysis points were T1 10.8% [3.6-15.1%] and T5 10.3% [2.6-18.9%], and the mean difference was -0.7% (95% CI: -5.8% -4.5%), i.e., lower than the predefined non-inferiority margin of 5% (p = 0.022). There were no changes at the four additional time points compared to T1 or postoperative pulmonary complications during the 14 days following the procedure. Conclusion: We found that obese patients undergoing laparoscopic bariatric surgery do not leave the Post Anesthesia Care Unit with increased low tidal variation areas compared to the preoperative period.
ABSTRACT
AIMS: To compare the time required for perioperative glucose management using fully automated closed-loop versus standard insulin therapy. METHODS: We performed a time-motion study to quantify the time requirements for perioperative glucose management with fully closed-loop (FCL) and standard insulin therapy applied to theoretical scenarios. Following an analysis of workflows in different periods of perioperative care in elective surgery patients receiving FCL or standard insulin therapy upon hospital admission (pre- and intra-operatively, at the intermediate care unit and general wards), the time of process-specific tasks was measured by shadowing hospital staff. Each task was measured 20 times and its average duration in combination with its frequency according to guidelines was used to calculate the cumulative staff time required for blood glucose management. Cumulative time was calculated for theoretical scenarios consisting of elective minor and major abdominal surgeries (pancreatic surgery and sleeve gastrectomy, respectively) to account for the different care settings and lengths of stay. RESULTS: The FCL insulin therapy reduced the time required for perioperative glucose management compared to standard insulin therapy, across all assessed care periods and for both perioperative pathways (range 2.1-4.5). For a major abdominal surgery, total time required was 248.5 min using FCL versus 753.9 min using standard insulin therapy. For a minor abdominal surgery, total time required was 68.6 min and 133.2 min for FCL and standard insulin therapy, respectively. CONCLUSIONS: The use of fully automated closed-loop insulin delivery for inpatient glucose management has the potential to alleviate the workload of diabetes management in an environment with adequately trained staff.
Subject(s)
Diabetes Mellitus, Type 1 , Insulin , Humans , Insulin/therapeutic use , Hypoglycemic Agents/therapeutic use , Blood Glucose/metabolism , Glucose , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/surgery , Insulin Infusion SystemsABSTRACT
Aims: To investigate the effect of empagliflozin on glucose dynamics in individuals suffering from postbariatric hypoglycemia (PBH) after Roux-en-Y gastric bypass (RYGB). Methods: Twenty-two adults with PBH after RYGB were randomized to empagliflozin 25 mg or placebo once daily over 20 days in a randomized, double-blind, placebo-controlled, crossover trial. The primary efficacy outcome was the amplitude of plasma glucose excursion (peak to nadir) during a mixed-meal tolerance test (MMTT). Outcomes of the outpatient period were assessed using continuous glucose monitoring (CGM) and an event-tracking app. Results: The amplitude of glucose excursion during the MMTT was 8.1 ± 2.4 mmol/L with empagliflozin versus 8.1 ± 2.6 mmol/L with placebo (mean ± standard deviation, P = 0.807). CGM-based mean amplitude of glucose excursion during the 20-day period was lower with empagliflozin than placebo (4.8 ± 1.3 vs. 5.2 ± 1.6. P = 0.028). Empagliflozin reduced the time spent with CGM values >10.0 mmol/L (3.8 ± 3.5% vs. 4.7 ± 3.8%, P = 0.009), but not the time spent with CGM values <3.0 mmol/L (1.7 ± 1.6% vs. 1.5 ± 1.5%, P = 0.457). No significant difference was observed in the quantity and quality of recorded symptoms. Eleven adverse events occurred with empagliflozin (three drug-related) and six with placebo. Conclusions: Empagliflozin 25 mg reduces glucose excursions but not hypoglycemia in individuals with PBH. Clinical Trial Registration: Clinicaltrials.gov: NCT05057819.
Subject(s)
Gastric Bypass , Hypoglycemia , Adult , Humans , Gastric Bypass/adverse effects , Blood Glucose , Blood Glucose Self-Monitoring , Cross-Over Studies , Hypoglycemia/drug therapy , Hypoglycemia/etiology , Hypoglycemia/prevention & control , Glucose , Double-Blind MethodABSTRACT
OBJECTIVE: Continuous glucose monitoring (CGM) may be challenged by extreme conditions during cardiac surgery using hypothermic extracorporeal circulation (ECC). RESEARCH DESIGN AND METHODS: We evaluated the Dexcom G6 sensor in 16 subjects undergoing cardiac surgery with hypothermic ECC, of whom 11 received deep hypothermic circulatory arrest (DHCA). Arterial blood glucose, quantified by the Accu-Chek Inform II meter, served as reference. RESULTS: Intrasurgery mean absolute relative difference (MARD) of 256 paired CGM/reference values was 23.8%. MARD was 29.1% during ECC (154 pairs) and 41.6% immediately after DHCA (10 pairs), with a negative bias (signed relative difference: -13.7%, -26.6%, and -41.6%). During surgery, 86.3% pairs were in Clarke error grid zones A or B and 41.0% of sensor readings fulfilled the International Organization for Standardization (ISO) 15197:2013 norm. Postsurgery, MARD was 15.0%. CONCLUSIONS: Cardiac surgery using hypothermic ECC challenges the accuracy of the Dexcom G6 CGM although recovery appears to occur thereafter.
Subject(s)
Cardiac Surgical Procedures , Diabetes Mellitus, Type 1 , Humans , Blood Glucose , Blood Glucose Self-Monitoring , Reproducibility of ResultsABSTRACT
The central role of pancreas in glucose regulation imposes high demands on perioperative glucose management in patients undergoing pancreatic surgery. In a post hoc subgroup analysis of a randomized controlled trial, we evaluated the perioperative use of subcutaneous (SC) fully closed-loop (FCL; CamAPS HX) versus usual care (UC) insulin therapy in patients undergoing partial or total pancreatic resection. Glucose control was compared using continuous glucose monitoring (CGM) metrics (% time with CGM values between 5.6 and 10.0 mmol/L and more). Over the time of hospitalization, FCL resulted in better glucose control than UC with more time spent in the target range 5.6-10.0 mmol/L (mean [standard deviation] % time in target 77.7% ± 4.6% and 41.1% ± 19.5% in FCL vs. UC subjects, respectively; mean difference 36.6% [95% confidence interval 18.5-54.8]), without increasing the risk of hypoglycemia. Findings suggest that an adaptive SC FCL approach effectively accommodated the highly variable insulin needs in patients undergoing pancreatic surgery. Clinical trials registration: ClinicalTrials.gov, NCT04361799.
Subject(s)
Diabetes Mellitus, Type 1 , Insulin , Humans , Insulin/therapeutic use , Blood Glucose , Hypoglycemic Agents/therapeutic use , Blood Glucose Self-Monitoring , Diabetes Mellitus, Type 1/drug therapy , Insulin Infusion Systems/adverse effects , Insulin, Regular, Human/therapeutic useABSTRACT
INTRODUCTION: Postprandial hypoglycaemia after gastric bypass surgery (also known as postbariatric hypoglycaemia or PBH) is an increasingly encountered clinical problem. PBH is characterised by meal-induced rapid spikes and consequent falls in glycaemia, resulting in both hypoglycaemia burden and high glycaemic variability. Despite its frequency, there is currently no approved pharmacotherapy. The purpose of this investigation is to evaluate efficacy and safety of empagliflozin 25 mg, a sodium-glucose cotransporter 2-inhibitor, to reduce glucose excursions and hypoglycaemia burden in patients with PBH after gastric bypass surgery. METHODS AND ANALYSIS: In a prospective, single-centre, randomised, double-blind, placebo-controlled, crossover trial, we plan to enrol 22 adults (≥18 years) with PBH after Roux-en-Y gastric bypass surgery (plasma or sensor glucose <3.0 mmol/L). Eligible patients will be randomised to receive empagliflozin 25 mg and placebo once daily, each for 20 days, in random order. Study periods will be separated by a 2-6 weeks wash-out period. The primary efficacy outcome will be the amplitude of plasma glucose excursion (peak to nadir) during a mixed meal tolerance test. Results will be presented as paired-differences±SD plus 95% CIs with p values and hypothesis testing for primary and secondary outcomes according to intention-to-treat. Secondary outcomes include continuous glucose monitoring-based outcomes, further metabolic measures and safety. ETHICS AND DISSEMINATION: The DEEP-EMPA trial (original protocol title: Randomized, double-blind, placebo-controlled crossover trialassessing the impact of the SGLT2 inhibitor empagliflozin onpostprandial hypoglycaemia after gastric bypass) was approved by the Bern Ethics Committee (ID 2021-01187) and Swissmedic (Ref. Number: 102663190) in October and November 2021, respectively. First results are expected in the first quarter of 2023 and will be disseminated via peer-reviewed publications and presented at national and international conferences. The acronym DEEP was derived from an overarching project title (DEciphering the Enigma of Postprandial Hyperinsulinaemic Hypoglycaemia after Bariatric Surgery), the term EMPA stands for the drug empagliflozin. TRIAL REGISTRATION NUMBER: NCT05057819.
Subject(s)
Gastric Bypass , Hypoglycemia , Sodium-Glucose Transporter 2 Inhibitors , Adult , Benzhydryl Compounds/therapeutic use , Blood Glucose/metabolism , Blood Glucose Self-Monitoring , Cross-Over Studies , Double-Blind Method , Gastric Bypass/adverse effects , Glucosides/therapeutic use , Humans , Hypoglycemia/etiology , Hypoglycemia/prevention & control , Prospective Studies , Randomized Controlled Trials as Topic , Sodium-Glucose Transporter 2 Inhibitors/therapeutic useABSTRACT
OBJECTIVE: Perioperative management of glucose levels remains challenging. We aimed to assess whether fully closed-loop subcutaneous insulin delivery would improve glycemic control compared with standard insulin therapy in insulin-requiring patients undergoing elective surgery. RESEARCH DESIGN AND METHODS: We performed a single-center, open-label, randomized controlled trial. Patients with diabetes (other than type 1) undergoing elective surgery were recruited from various surgical units and randomly assigned using a minimization schedule (stratified by HbA1c and daily insulin dose) to fully closed-loop insulin delivery with fast-acting insulin aspart (closed-loop group) or standard insulin therapy according to local clinical practice (control group). Study treatment was administered from hospital admission to discharge (for a maximum of 20 days). The primary end point was the proportion of time with sensor glucose in the target range (5.6-10.0 mmol/L). RESULTS: Forty-five patients were enrolled and assigned to the closed-loop (n = 23) or the control (n = 22) group. One patient (closed-loop group) withdrew from the study before surgery and was not analyzed. Participants underwent abdominal (57%), vascular (23%), orthopedic (9%), neuro (9%), or thoracic (2%) surgery. The mean proportion of time that sensor glucose was in the target range was 76.7 ± 10.1% in the closed-loop and 54.7 ± 20.8% in the control group (mean difference 22.0 percentage points [95% CI 11.9; 32.0%]; P < 0.001). No episodes of severe hypoglycemia (<3.0 mmol/L) or hyperglycemia with ketonemia or any study-related adverse events occurred in either group. CONCLUSIONS: In the context of mixed elective surgery, the use of fully closed-loop subcutaneous insulin delivery improves glucose control without a higher risk of hypoglycemia.
Subject(s)
Diabetes Mellitus, Type 1 , Hypoglycemia , Blood Glucose , Cross-Over Studies , Diabetes Mellitus, Type 1/drug therapy , Glucose/therapeutic use , Humans , Hypoglycemia/chemically induced , Hypoglycemic Agents/therapeutic use , Insulin , Insulin Infusion Systems , Insulin, Regular, Human/therapeutic use , Treatment OutcomeABSTRACT
The role and outcome of the muscarinic M2 acetylcholine receptor (M2R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M2R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the Gi-mediated Rac1 activation into a Gi-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (- 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart.
Subject(s)
Heart Failure , Myocytes, Cardiac , Animals , Cholinergic Agents , Heart Ventricles , Rats , Receptors, MuscarinicABSTRACT
BACKGROUND: Pulmonary hypertension (PH) is a known complication of hereditary hemorrhagic telangiectasia (HHT) in patients with hepatic arteriovenous malformations (HAVM). Orthotopic liver transplantation (OLT) is a recognized treatment of HAVM in HHT, but its effect on PH has not been reported in detail before. METHODS: Systematic review on HHT patients with pre- or postcapillary PH who underwent OLT and report of a case. RESULTS: Twenty-one patients were included from 7 articles, all case reports or case series. All had high-output cardiac failure prior to OLT. Two patients had precapillary PH, both related to ALK1 mutations. All patients but 1 showed significant improvement or complete resolution of PH after transplantation. One patient died of acute cardiac failure postoperatively. We also report the case of a 72-year-old woman with type 3 HHT and severe mixed pre- and postcapillary PH. The patient presented with multiple HAVM, left-to-right shunting, and severe but partially reversible combined pre- and postcapillary PH, without ALK1 mutation. After recurrent cholangitis episodes, liver abscesses, and severe obstruction of the right-sided biliary tree, an interdisciplinary decision was taken to proceed with OLT despite PH. Intraoperatively, PH resolved almost instantly after hepatic artery ligation and hepatectomy. CONCLUSIONS: In our patient, OLT completely abrogated mixed pre- and postcapillary PH. Based on this systematic review, we suggest that OLT should be considered a viable treatment option in patients with HHT, HAVM, and mixed pre- and postcapillary PH, featuring cardiac failure and drug responsive PH, rather than being seen as a major risk factor for cardiopulmonary complications.
Subject(s)
Hypertension, Pulmonary , Liver Transplantation , Telangiectasia, Hereditary Hemorrhagic , Aged , Female , Hepatic Artery , Humans , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/surgery , Liver , Telangiectasia, Hereditary Hemorrhagic/complications , Telangiectasia, Hereditary Hemorrhagic/surgeryABSTRACT
Mcm2-7 is the catalytic core of the eukaryotic replicative helicase, which together with CDC45 and the GINS complex unwind parental DNA to generate templates for DNA polymerase. Being a highly regulated and complex enzyme that operates via an incompletely understood multi-step mechanism, molecular probes of Mcm2-7 that interrogate specific mechanistic steps would be useful tools for research and potential future chemotherapy. Based upon a synthetic lethal approach, we previously developed a budding yeast multivariate cell-based high throughput screening (HTS) assay to identify putative Mcm inhibitors by their ability to specifically cause a growth defect in an mcm mutant relative to a wild-type strain[1]. Here, as proof of concept, we used this assay to screen a 1280-member compound library (LOPAC) for potential Mcm2-7 inhibitors. Primary screening and dose-dependent retesting identified twelve compounds from this library that specifically inhibited the growth of the Mcm mutant relative to the corresponding wild-type strain (0.9 % hit rate). Secondary assays were employed to rule out non-specific DNA damaging agents, establish direct protein-ligand interaction via biophysical methods, and verify in vivo DNA replication inhibition via fluorescence activated cell sorter analysis (FACS). We identified one agent (ß-carboline-3-carboxylic acid N-methylamide, CMA) that physically bound to the purified Mcm2-7 complex (Kdapp119 µM), and at slightly higher concentrations specifically blocked S-phase cell cycle progression of the wild-type strain. In total, identification of Mcm2-7 as a CMA target validates our synthetic lethal HTS assay paradigm as a tool to identify chemical probes for the Mcm2-7 replicative helicase.
Subject(s)
Eukaryota , High-Throughput Screening Assays , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Replication , Eukaryota/metabolism , Minichromosome Maintenance Proteins/genetics , Minichromosome Maintenance Proteins/metabolismABSTRACT
Background: Coronary autoregulation is a feedback system, which maintains near-constant myocardial blood flow over a range of mean arterial pressure (MAP). Yet in emergency or peri-operative situations, hypotensive or hypertensive episodes may quickly arise. It is not yet established how rapid blood pressure changes outside of the autoregulation zone (ARZ) impact left (LV) and right ventricular (RV) function. Using cardiovascular magnetic resonance (CMR) imaging, measurements of myocardial tissue oxygenation and ventricular systolic and diastolic function can comprehensively assess the heart throughout a range of changing blood pressures. Design and methods: In 10 anesthetized swine, MAP was varied in steps of 10-15 mmHg from 29 to 196 mmHg using phenylephrine and urapidil inside a 3-Tesla MRI scanner. At each MAP level, oxygenation-sensitive (OS) cine images along with arterial and coronary sinus blood gas samples were obtained and blood flow was measured from a surgically implanted flow probe on the left anterior descending coronary artery. Using CMR feature tracking-software, LV and RV circumferential systolic and diastolic strain parameters were measured from the myocardial oxygenation cines. Results: LV and RV peak strain are compromised both below the lower limit (LV: Δ1.2 ± 0.4%, RV: Δ4.4 ± 1.2%, p < 0.001) and above the upper limit (LV: Δ2.1 ± 0.4, RV: Δ5.4 ± 1.4, p < 0.001) of the ARZ in comparison to a baseline of 70 mmHg. LV strain demonstrates a non-linear relationship with invasive and non-invasive measures of oxygenation. Specifically for the LV at hypotensive levels below the ARZ, systolic dysfunction is related to myocardial deoxygenation (ß = -0.216, p = 0.036) in OS-CMR and both systolic and diastolic dysfunction are linked to reduced coronary blood flow (peak strain: ß = -0.028, p = 0.047, early diastolic strain rate: ß = 0.026, p = 0.002). These relationships were not observed at hypertensive levels. Conclusion: In an animal model, biventricular function is compromised outside the coronary autoregulatory zone. Dysfunction at pressures below the lower limit is likely caused by insufficient blood flow and tissue deoxygenation. Conversely, hypertension-induced systolic and diastolic dysfunction points to high afterload as a cause. These findings from an experimental model are translatable to the clinical peri-operative environment in which myocardial deformation may have the potential to guide blood pressure management, in particular at varying individual autoregulation thresholds.
ABSTRACT
Understanding the mechanism of SARS-CoV-2 infection and identifying potential therapeutics are global imperatives. Using a quantitative systems pharmacology approach, we identified a set of repurposable and investigational drugs as potential therapeutics against COVID-19. These were deduced from the gene expression signature of SARS-CoV-2-infected A549 cells screened against Connectivity Map and prioritized by network proximity analysis with respect to disease modules in the viral-host interactome. We also identified immuno-modulating compounds aiming at suppressing hyperinflammatory responses in severe COVID-19 patients, based on the transcriptome of ACE2-overexpressing A549 cells. Experiments with Vero-E6 cells infected by SARS-CoV-2, as well as independent syncytia formation assays for probing ACE2/SARS-CoV-2 spike protein-mediated cell fusion using HEK293T and Calu-3 cells, showed that several predicted compounds had inhibitory activities. Among them, salmeterol, rottlerin, and mTOR inhibitors exhibited antiviral activities in Vero-E6 cells; imipramine, linsitinib, hexylresorcinol, ezetimibe, and brompheniramine impaired viral entry. These novel findings provide new paths for broadening the repertoire of compounds pursued as therapeutics against COVID-19.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Drug Evaluation, Preclinical/methods , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Drug Repositioning , HEK293 Cells , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/physiology , Humans , Imidazoles/pharmacology , Pyrazines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Salmeterol Xinafoate/pharmacology , Vero CellsABSTRACT
Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.
Subject(s)
Drug Discovery/methods , Enzyme Stability/drug effects , High-Throughput Screening Assays/methods , Small Molecule Libraries , Triose-Phosphate Isomerase/chemistry , Drug Evaluation, Preclinical/methods , Genes, Reporter , HEK293 Cells , Humans , Mutation , Triose-Phosphate Isomerase/antagonists & inhibitors , Triose-Phosphate Isomerase/deficiency , Triose-Phosphate Isomerase/geneticsABSTRACT
Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) therapy is a rescue strategy for severe cardiopulmonary failure. The estimation of cardiac output during VA-ECMO is challenging. A lung circuit ([Formula: see text]Lung) and an ECMO circuit ([Formula: see text]ECMO) with oxygenators for CO2 removal ([Formula: see text]CO2) and O2 uptake ([Formula: see text]O2) simulated the setting of VA-ECMO with varying ventilation/perfusion ([Formula: see text]/[Formula: see text]) ratios and shunt. A metabolic chamber with a CO2/N2 blend simulated [Formula: see text]CO2 and [Formula: see text]O2. [Formula: see text] Lung was estimated with a modified Fick principle: [Formula: see text]Lung = [Formula: see text]ECMO × ([Formula: see text] CO2 or [Formula: see text]O2Lung)/([Formula: see text]CO2 or [Formula: see text]O2ECMO). A normalization procedure corrected [Formula: see text]CO2 values for a [Formula: see text]/[Formula: see text] of 1. Method agreement was evaluated by Bland-Altman analysis. Calculated [Formula: see text]Lung using gaseous [Formula: see text]CO2 and [Formula: see text]O2 correlated well with measured [Formula: see text]Lung with a bias of 103 ml/min [- 268 to 185] ml/min; Limits of Agreement: - 306 ml/min [- 241 to - 877 ml/min] to 512 ml/min [447 to 610 ml/min], r2 0.85 [0.79-0.88]). Blood measurements of [Formula: see text]CO2 showed an increased bias (- 260 ml/min [- 1503 to 982] ml/min), clinically not applicable. Shunt and [Formula: see text]/[Formula: see text] mismatch decreased the agreement of methods significantly. This in-vitro simulation shows that [Formula: see text]CO2 and [Formula: see text]O2 in steady-state conditions allow for clinically applicable calculations of [Formula: see text]Lung during VA-ECMO therapy.
Subject(s)
Carbon Dioxide/blood , Extracorporeal Membrane Oxygenation , Models, Cardiovascular , Oxygen Consumption , Oxygen/blood , Child , HumansABSTRACT
The aim of this study was to characterize the variability of exogenous insulin requirements during fully closed-loop insulin delivery in hospitalized patients with type 2 diabetes or new-onset hyperglycaemia, and to determine patient-related characteristics associated with higher variability of insulin requirements. We retrospectively analysed data from two fully closed-loop inpatient studies involving adults with type 2 diabetes or new-onset hyperglycaemia requiring insulin therapy. The coefficient of variation quantified day-to-day variability of exogenous insulin requirements during up to 15 days using fully automated closed-loop insulin delivery. Data from 535 days in 67 participants were analysed. The coefficient of variation of day-to-day exogenous insulin requirements was 30% ± 16%, and was higher between nights than between any daytime period (56% ± 29% overnight [11:00 pm to 4:59 am] compared with 41% ± 21% in the morning [5:00 am to 10:59 am], 39% ± 15% in the afternoon [11:00 am to 4:59 pm] and 45% ± 19% during the evening [5:00 pm to 10:59 pm]; all P < 0.01). There is high day-to-day variability of exogenous insulin requirements in inpatients, particularly overnight, and diabetes management approaches should account for this variability.