Regional differences of energetics, mechanics, and kinetics of myosin cross-bridge in human ureter smooth muscle

This study provides information about baseline mechanical properties of the entire muscle and the molecular contractile mechanism in human ureter smooth muscle and proposed to investigate if changes in mechanical motor performance in different regions of isolated human ureter are attributable to differences in myosin crossbridge interactions. Classic mechanical, contraction and energetic parameters derived from the tension-velocity relationship were studied in ureteral smooth muscle strips oriented longitudinally and circularly from abdominal and pelvic human ureter parts. By applying of Huxley’s mathematical model we calculated the total working crossbridge number per mm2 (Ψ), elementary force per single crossbridge (Π0), duration of maximum rate constant of crossbridge attachment 1/f1 and detachment 1/g2 and peak mechanical efficiency (Eff.max). Abdominal longitudinal smooth muscle strips exhibited significantly higher maximum isometric tension and faster maximum unloaded shortening velocity compared to pelvic ones. Contractile differences were associated with significantly higher crossbridge number per mm2. Abdominal longitudinal muscle strips showed a lower duration of maximum rate constant of crossbridge attachment and detachment and higher peak mechanical efficiency than pelvic ones. Such data suggest that the abdominal human ureter showed better mechanical motor performance mainly related to a higher crossbridge number and crossbridge kinetics differences. Such results were more evident in the longitudinal rather than in the circular layer

STZ-diabetic rat heart maintains developed tension amplitude by increasing sarcomere length and crossbridge density

New Findings: What is the central question of this study? In the papillary muscle from type I diabetic rats, does diabetes-associated altered ventricular function result from changes of acto-myosin interactions and are these modifications attributable to a possible sarcomere rearrangement? What is the main finding and its importance? For the first time, we showed that type-I diabetes altered sarcomeric ultrastructure, as seen by transmission electron microscopy, consistent with physiological parameters. The diabetic condition induced slower timing parameters, which is compatible with a diastolic dysfunction. At the sarcomeric level, augmented β-myosin heavy chain content and increased sarcomere length and crossbridges' number preserve myocardial stroke and could concur to maintain the ejection fraction. Abstract: We investigated whether diabetes-associated altered ventricular function, in a type I diabetes animal model, results from a modification of acto-myosin interactions, through the in vitro recording of left papillary muscle mechanical parameters and examination of sarcomere morphology by transmission electron microscopy (TEM). Experiments were performed on streptozotocin-induced diabetic and age-matched control female Wistar rats. Mechanical isometric and isotonic indexes and timing parameters were determined. Using Huxley's equations, we calculated mechanics, kinetics and energetics of myosin crossbridges. Sarcomere length and A-band length were measured on TEM images. Type I and III collagen and β-myosin heavy chain (MHC) expression were determined by immunoblotting. No variation in resting and developed tension or maximum extent of shortening was evident between groups, but diabetic rats showed lower maximum shortening velocity and prolonged timing parameters. Compared to controls, diabetics also displayed a higher number of crossbridges with lower unitary force. Moreover, no change in type I and III collagen was associated to diabetes, but pathological rats showed a two-fold enhancement of β-MHC content and longer sarcomeres and A-band, detected by ultrastructural morphometry. Overall, these data address whether a preserved systolic function accompanied by an altered diastolic phase results from a recruitment of super-relaxed myosin heads or the phosphorylation of the regulatory light chain site in myosin. Although the early signs of diabetic cardiomyopathy were well expressed, the striking finding of our study was that, in diabetics, sarcomere modification may be a possible compensatory mechanism that preserves systolic function

Developing an ML pipeline for asthma and COPD: The case of a Dutch primary care service

A complex combination of clinical, demographic and lifestyle parameters determines the correct diagnosis and the most effective treatment for asthma and Chronic Obstructive Pulmonary Disease patients. Artificial Intelligence techniques help clinicians in devising the correct diagnosis and designing the most suitable clinical pathway accordingly, tailored to the specific patient conditions. In the case of machine learning (ML) approaches, availability of real-world patient clinical data to train and evaluate the ML pipeline deputed to assist clinicians in their daily practice is crucial. However, it is common practice to exploit either synthetic data sets or heavily preprocessed collections cleaning and merging different data sources. In this paper, we describe an automated ML pipeline designed for a real-world data set including patients from a Dutch primary care service, and provide a performance comparison of different prediction models for (i) assessing various clinical parameters, (ii) designing interventions, and (iii) defining the diagnosis

Molecular interactions of carbapenem antibiotics with the multidrug efflux transporter acrb of escherichia coli

The drug/proton antiporter AcrB, engine of the major efflux pump AcrAB(Z)-TolC of Escherichia coli and other bacteria, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multi-drug resistance (MDR) phenotype. Although hundreds of small molecules are known to be AcrB substrates, only a few co-crystal structures are available to date. Computational methods have been therefore intensively employed to provide structural and dynamical fingerprints related to transport and inhibition of AcrB. In this work, we performed a systematic computational investigation to study the interaction between representative carbapenem antibiotics and AcrB. We focused on the interaction of carbapenems with the so-called distal pocket, a region known for its importance in binding inhibitors and substrates of AcrB. Our findings reveal how the different physico-chemical nature of these antibiotics is reflected on their binding preference for AcrB. The molecular-level information provided here could help design new antibiotics less susceptible to the efflux mechanism

Holo-like and Druggable Protein Conformations from Enhanced Sampling of Binding Pocket Volume and Shape

Understanding molecular recognition of small molecules by proteins in atomistic detail is key for drug design. Molecular docking is a widely used computational method to mimic ligand-protein association in silico. However, predicting conformational changes occurring in proteins upon ligand binding is still a major challenge. Ensemble docking approaches address this issue by considering a set of different conformations of the protein obtained either experimentally or from computer simulations, e.g., molecular dynamics. However, holo structures prone to host (the correct) ligands are generally poorly sampled by standard molecular dynamics simulations of the apo protein. In order to address this limitation, we introduce a computational approach based on metadynamics simulations called ensemble docking with enhanced sampling of pocket shape (EDES) that allows holo-like conformations of proteins to be generated by exploiting only their apo structures. This is achieved by defining a set of collective variables that effectively sample different shapes of the binding site, ultimately mimicking the steric effect due to the ligand. We assessed the method on three challenging proteins undergoing different extents of conformational changes upon ligand binding. In all cases our protocol generates a significant fraction of structures featuring a low RMSD from the experimental holo geometry. Moreover, ensemble docking calculations using those conformations yielded in all cases native-like poses among the top-ranked ones

Chronic Red Bull Consumption during Adolescence: Effect on Mesocortical and Mesolimbic Dopamine Transmission and Cardiovascular System in Adult Rats

Energy drinks are very popular nonalcoholic beverages among adolescents and young adults for their stimulant effects. Our study aimed to investigate the effect of repeated intraoral Red Bull (RB) infusion on dopamine transmission in the nucleus accumbens shell and core and in the medial prefrontal cortex and on cardiac contractility in adult rats exposed to chronic RB consumption. Rats were subjected to 4 weeks of RB voluntary consumption from adolescence to adulthood. Monitoring of in vivo dopamine was carried out by brain microdialysis. In vitro cardiac contractility was studied on biomechanical properties of isolated left-ventricular papillary muscle. The main finding of the study was that, in treated animals, RB increased shell dopamine via a nonadaptive mechanism, a pattern similar to that of drugs of abuse. No changes in isometric and isotonic mechanical parameters were associated with chronic RB consumption. However, a prolonged time to peak tension and half-time of relaxation and a slower peak rate of tension fall were observed in RB-treated rats. It is likely that RB treatment affects left-ventricular papillary muscle contraction. The neurochemical results here obtained can explain the addictive properties of RB, while the cardiovascular investigation findings suggest a hidden papillary contractility impairment

A framework for dissecting affinities of multidrug efflux transporter AcrB to fluoroquinolones

Sufficient concentration of antibiotics close to their target is key for antimicrobial action. Among the tools exploited by bacteria to reduce the internal concentration of antibiotics, multidrug efflux pumps stand out for their ability to capture and expel many unrelated compounds out of the cell. Determining the specificities and efflux efficiency of these pumps towards their substrates would provide quantitative insights into the development of antibacterial strategies. In this light, we developed a competition efflux assay on whole cells, that allows measuring the efficacy of extrusion of clinically used quinolones in populations and individual bacteria. Experiments reveal the efficient competitive action of some quinolones that restore an active concentration of other fluoroquinolones. Computational methods show how quinolones interact with the multidrug efflux transporter AcrB. Combining experiments and computations unveils a key molecular mechanism acting in vivo to detoxify bacterial cells. The developed assay can be generalized to the study of other efflux pumps