Quantifying single-cell diacylglycerol signaling kinetics after uncaging.

Studying the role of molecularly distinct lipid species in cell signaling remains challenging due to a scarcity of methods for performing quantitative lipid biochemistry in living cells. We have recently used lipid uncaging to quantify lipid-protein affinities and rates of lipid trans-bilayer movement and turnover in the diacylglycerol signaling pathway. This approach is based on acquiring live-cell dose-response curves requiring light dose titrations and experimental determination of uncaging photoreaction efficiency. We here aimed to develop a methodological approach that allows us to retrieve quantitative kinetic data from uncaging experiments that 1) require only typically available datasets without the need for specialized additional constraints and 2) should in principle be applicable to other types of photoactivation experiments. Our new analysis framework allows us to identify model parameters such as diacylglycerol-protein affinities and trans-bilayer movement rates, together with initial uncaged diacylglycerol levels, using noisy single-cell data for a broad variety of structurally different diacylglycerol species. We find that lipid unsaturation degree and side-chain length generally correlate with faster lipid trans-bilayer movement and turnover and also affect lipid-protein affinities. In summary, our work demonstrates how rate parameters and lipid-protein affinities can be quantified from single-cell signaling trajectories with sufficient sensitivity to resolve the subtle kinetic differences caused by the chemical diversity of cellular signaling lipid pools.

Treatment with SGLT2 Inhibitors in Patients with Diabetes Mellitus and Extensive Coronary Artery Disease: Mortality and Cardiovascular Outcomes.

INTRODUCTION: Sodium-glucose type 2 cotransporter inhibitors (SGLT2-I) have shown solid benefits in reducing cardiovascular mortality and admissions for heart failure in patients with type 2 diabetes mellitus (T2DM) and cardiovascular disease. However, no specific studies exist in patients with high-risk coronary artery disease (CAD). METHODS: Single-center, retrospective, observational study including patients with T2DM and a new diagnosis of extensive CAD (defined as left main disease or three main coronary vessel disease). Patients were recruited from 2015 until 2020, with a follow-up of at least 12 months. The primary outcome was to compare all-cause mortality in patients treated with or without SGLT2-I at discharge and adjusted by inverse probability of treatment weighting (IPTW) propensity score. RESULTS: A total of 420 patients were included: 104 (24.7%) were treated with SGLT2-I and 316 (75.3%) were not (non-SGLT2-I group). The presentation was acute coronary syndrome in 44.3%. The mean age was 71.2 ± 10.5 years. The mean left ventricular ejection fraction was 51.5 ± 12.5%, and the mean estimated glomerular filtration rate was 73.9 ± 22 ml/min. After a mean follow-up of 3 ± 1.6 years, all-cause mortality was 16.4%, and cardiovascular mortality was 9.5%. After IPTW, the risk of all-cause death was lower in the SGLT2-I group with a hazard ratio of 0.32 (95% confidence interval 0.12-0.81), p = 0.016. With regard to secondary outcomes, patients in the SGLT2-I group were associated with less renal function deterioration but an increase in unplanned revascularizations. CONCLUSIONS: In patients with T2DM and extensive CAD, treatment with SGLT2-I after discharge was associated with a reduced risk of all-cause death.

Compartmentalized Cell-Free Expression Systems for Building Synthetic Cells.

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology. The open and simplified reaction environment of CFES has helped researchers discover fundamental concepts in the molecular biology of the cell. In recent decades, there has been a drive to encapsulate CFES reactions into cell-like compartments with the aim of building synthetic cells and multicellular systems. In this chapter, we discuss recent progress in compartmentalizing CFES to build simple and minimal models of biological processes that can help provide a better understanding of the process of self-assembly in molecularly complex systems.

Angiography-derived assessment of coronary microcirculatory resistance in patients with suspected myocardial ischaemia and non-obstructive coronary arteries.

BACKGROUND: Myocardial ischaemia with non-obstructive coronary arteries (INOCA) represents a challenging and frequent, but largely underdiagnosed, condition. AIMS: We aimed to investigate the feasibility and diagnostic value of angiography-derived coronary microcirculatory resistance in patients with INOCA syndrome. METHODS: This is an investigator-driven, prospective and blinded study. The diagnostic yield of angiography-derived index of coronary microcirculatory resistance (angio-IMR) was investigated against thermodilution-derived IMR (thermo-IMR) in patients with clinically indicated coronary angiography due to suspected myocardial ischaemia and angiographically normal or non-obstructive coronary arteries. The angio-IMR was derived from resting angiograms (contrast-flow angio-IMR [cAngio-IMR]) by an expert analyst blinded to the thermo-IMR. An independent, blinded, physiology core laboratory analysed the raw intracoronary physiology data and provided the final thermo-IMR values. RESULTS: A total of 104 patients (108 coronary vessels) were analysed after fulfilling predefined inclusion criteria. Most patients were female (67%). Obstructive epicardial disease was angiographically (percent diameter stenosis <50%) and physiologically (fractional flow reserve>0.80) ruled out in all cases. Median thermo-IMR and cAngio-IMR were 16.6 (12.7, 23.0) and 16.8 (12.8, 23.1) units, respectively (median difference -0.31, 95% confidence interval: -1.53 to 1.00; p=0.654). cAngio-IMR showed good correlation (Pearson coefficient 0.76; p<0.001), agreement (mean bias 0.4), discriminatory power (area under the curve from the receiver operator characteristics 0.865; p<0.001) and accuracy (85%), compared to thermo-IMR (≥25 U). CONCLUSIONS: Evaluating coronary microcirculatory resistance in patients with INOCA syndrome using cAngio-IMR is feasible and accurate. By circumventing the need of coronary instrumentation and hyperaemic drugs, this method may facilitate the assessment of coronary microcirculatory resistance in patients with suspected INOCA. CLINICALTRIALS: gov: NCT04827498.

Effect of Vitamin C Supplementation for Pregnant Smokers on Offspring Airway Function and Wheeze at Age 5 Years: Follow-up of a Randomized Clinical Trial.

Importance: Vitamin C supplementation (500 mg/d) for pregnant smokers has been reported to increase offspring airway function as measured by forced expiratory flow (FEF) through age 12 months; however, its effects on airway function at age 5 years remain to be assessed. Objective: To assess whether vitamin C supplementation in pregnant smokers is associated with increased and/or improved airway function in their offspring at age 5 years and whether vitamin C decreases the occurrence of wheeze. Design, Setting, and Participants: This study followed up the Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function (VCSIP) double-blind, placebo-controlled randomized clinical trial conducted at 3 centers in the US (in Oregon, Washington, and Indiana) between 2012 and 2016. Investigators and participants remain unaware of the treatment assignments. Forced expiratory flow measurements at age 5 years were completed from 2018 to 2021. Interventions: Pregnant smokers were randomized to vitamin C (500 mg/d) or placebo treatment. Main Outcomes and Measures: The primary outcome was the prespecified measurement of FEF between 25% and 75% expired volume (FEF25-75) by spirometry at age 5 years. Secondary outcomes included FEF measurements at 50% and 75% of expiration (FEF50 and FEF75), forced expiratory volume in 1 second (FEV1), and occurrence of wheeze. Results: Of the 251 pregnant smokers included in this study, 125 (49.8%) were randomized to vitamin C and 126 (50.2%) were randomized to placebo. Of 213 children from the VCSIP trial who were reconsented into this follow-up study, 192 (90.1%) had successful FEF measurements at age 5 years; 212 (99.5%) were included in the analysis of wheeze. Analysis of covariance demonstrated that offspring of pregnant smokers allocated to vitamin C compared with placebo had 17.2% significantly higher mean (SE) measurements of FEF25-75 at age 5 years (1.45 [0.04] vs 1.24 [0.04] L/s; adjusted mean difference, 0.21 [95% CI, 0.13-0.30]; P < .001). Mean (SE) measurements were also significantly increased by 14.1% for FEF50 (1.59 [0.04] vs 1.39 [0.04] L/s; adjusted mean difference, 0.20 [95% CI, 0.11-0.30]; P < .001), 25.9% for FEF75 (0.79 [0.02] vs 0.63 [0.02] L/s; 0.16 [95% CI, 0.11-0.22]; P < .001), and 4.4% for FEV1 (1.13 [0.02] vs 1.09 [0.02] L; 0.05 [95% CI, 0.01-0.09]; P = .02). In addition, offspring of pregnant smokers randomized to vitamin C had significantly decreased wheeze (28.3% vs 47.2%; estimated odds ratio, 0.41 [95% CI, 0.23-0.74]; P = .003). Conclusions and Relevance: In this follow-up study of offspring of pregnant smokers randomized to vitamin C vs placebo, vitamin C supplementation during pregnancy resulted in significantly increased airway function of offspring at age 5 years and significantly decreased the occurrence of wheeze. These findings suggest that vitamin C supplementation for pregnant smokers may decrease the effects of smoking in pregnancy on childhood airway function and respiratory health. Trial Registration: ClinicalTrials.gov Identifier: NCT03203603.

Transvascular Balloon Occlusion: A Novel Bailout Strategy in Large Left Main Perforations.

Several complications have been reported in rotational atherectomy, and these complications are closely associated with cardiac tamponade, emergent surgery, and death. Here we describe a case of left main coronary artery, bullet-like perforation treated with a novel approach-transvascular balloon occlusion. (Level of Difficulty: Advanced.).

Changes in light absorption and composition of chromophoric marine-dissolved organic matter across a microbial bloom.

Marine chromophoric dissolved organic matter (m-CDOM) mediates many vital photochemical processes at the ocean's surface. Isolating m-CDOM within the chemical complexity of marine dissolved organic matter has remained an analytical challenge. The SeaSCAPE campaign, a large-scale mesocosm experiment, provided a unique opportunity to probe the in situ production of m-CDOM across phytoplankton and microbial blooms. Results from mass spectrometry coupled with UV-VIS spectroscopy reveal production of a chemodiverse set of compounds well-correlated with increases in absorbance after a bacterial bloom, indicative of autochthonous m-CDOM production. Notably, many of the absorbing compounds were found to be enriched in nitrogen, which may be essential to chromophore function. From these results, quinoids, porphyrins, flavones, and amide-like compounds were identified via structural analysis and may serve as important photosensitizers in the marine boundary layer. Overall, this study demonstrates a step forward in identifying and characterizing m-CDOM using temporal mesocosm data and integrated UV-VIS spectroscopy and mass spectrometry analyses.

A Large Segmental Mid-Diaphyseal Femoral Defect Sheep Model: Surgical Technique.

Background:There are numerous animal models available to study bone healing as well as test strategies to accelerate bone formation. Sheep are commonly used for evaluation of long bone fractures due to similar dimensions and weight bearing environments compared to patients. Large critical-size defects can be created in sheep to facilitate the study of implantable materials, osteogenic proteins, and stem cell treatments. Studies have been published using plates to stabilize large critical size defects in femoral, tibial, and metatarsal defects. External fixators have also been used to stabilize tibial defects in sheep.Methods: The purpose of the current paper is to detail the surgical technique for creation of a 42 mm mid-diaphyseal femoral defect stabilization with an intramedullary device in sheep. Additional surgical details are provided for dynamization, reverse dynamization, and device removal.Conclusion: The article provides multiple technical tips applicable to this and other ovine osteotomy models and concludes with a discussion comparing the use of each stabilization technique in clinically significant large critical-size bone defects.

Cell-Free Gene Expression Dynamics in Synthetic Cell Populations.

The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

Score2: A New Updated Algorithm to Predict Cardiovascular Disease Risk in Europe.

The recently published guidelines of the European Society of Cardiology for the prevention of cardiovascular diseases presented in August 2021 at the virtual European Society of Cardiology congress recommend the use of the SCORE2 risk index instead of the classic SCORE risk index to calculate the cardiovascular risk (specifically, ten-year fatal and nonfatal risk) in a healthy population under the age of 70 years, with a level of evidence IB. This new risk index was developed with the collaboration of about 200 investigators, including 45 cohorts in 13 countries with 700,000 participants, and covers the known risk factors for heart and circulatory diseases such as age, sex, lipid levels, blood pressure, and smoking. In addition, it divides the countries into four groups of risk and uses a competing risk model, adjusting the risk for the probability of having another event, which enables better estimation of the risk of fatal and nonfatal events in a younger population (40-69 years).

Osseosurface electronics-thin, wireless, battery-free and multimodal musculoskeletal biointerfaces.

Bioelectronic interfaces have been extensively investigated in recent years and advances in technology derived from these tools, such as soft and ultrathin sensors, now offer the opportunity to interface with parts of the body that were largely unexplored due to the lack of suitable tools. The musculoskeletal system is an understudied area where these new technologies can result in advanced capabilities. Bones as a sensor and stimulation location offer tremendous advantages for chronic biointerfaces because devices can be permanently bonded and provide stable optical, electromagnetic, and mechanical impedance over the course of years. Here we introduce a new class of wireless battery-free devices, named osseosurface electronics, which feature soft mechanics, ultra-thin form factor and miniaturized multimodal biointerfaces comprised of sensors and optoelectronics directly adhered to the surface of the bone. Potential of this fully implanted device class is demonstrated via real-time recording of bone strain, millikelvin resolution thermography and delivery of optical stimulation in freely-moving small animal models. Battery-free device architecture, direct growth to the bone via surface engineered calcium phosphate ceramic particles, demonstration of operation in deep tissue in large animal models and readout with a smartphone highlight suitable characteristics for exploratory research and utility as a diagnostic and therapeutic platform.

Engineered acetoacetate-inducible whole-cell biosensors based on the AtoSC two-component system.

Whole-cell biosensors hold potential in a variety of industrial, medical, and environmental applications. These biosensors can be constructed through the repurposing of bacterial sensing mechanisms, including the common two-component system (TCS). Here we report on the construction of a range of novel biosensors that are sensitive to acetoacetate, a molecule that plays a number of roles in human health and biology. These biosensors are based on the AtoSC TCS. An ordinary differential equation model to describe the action of the AtoSC TCS was developed and sensitivity analysis of this model used to help inform biosensor design. The final collection of biosensors constructed displayed a range of switching behaviours at physiologically relevant acetoacetate concentrations and can operate in several Escherichia coli host strains. It is envisaged that these biosensor strains will offer an alternative to currently available commercial strip tests and, in future, may be adopted for more complex in vivo or industrial monitoring applications.

Low-Density Lipoprotein Cholesterol Targets in Patients With Coronary Heart Disease in Extremadura (Spain): LYNX Registry.

BACKGROUND: Low-density lipoprotein cholesterol (LDL-C) contributes decisively to the development of cardiovascular disease (CVD). In the LYNX registry we determined the rate of achievement of the target value of LDL-C, the use of lipid-lowering therapy (LLT) and the predictive factors of not reaching the target in patients with stable coronary heart disease (CHD). METHODS: LYNX included consecutive patients with stable CHD treated at the University Hospital of Caceres, Extremadura (Spain) from September 2016 to September 2018, and those who must have an LDL-C target below 70 mg/dL according to the European Society of Cardiology (ESC) 2016 guidelines. The variables independently associated with the breach of the LDL-C objective were evaluated by multivariable logistic regression. RESULTS: A total of 674 patients with stable CHD were included. The average LDL-C levels were 68.3 ± 24.5 mg/dL, with 56.7% showing a level below 70 mg/dL. LLT was used by 96.7% of patients, 71.7% were treated with high-powered statins and 30.1% with ezetimibe. The risk of not reaching the target value of LDL-C was higher in women, in active smokers, and in those who had multivessel CHD or had atrial fibrillation. Patients with diabetes mellitus, those who took potent statins or co-administration treatment with ezetimibe were more likely to reach the target level of LDL-C. CONCLUSIONS: The treatment of dyslipidemia in patients with chronic CHD remains suboptimal; however, an increasing number of very high-risk patients achieve the LDL-C objective, although there is still enormous potential to improve cardiovascular outcome through the use of more intensive LLT.