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Highlights • A DEE/LP/MILP based deterministic global optimization algorithm and the MM-GBSA form of energy function were used to predict thermostable mutants of cephalosporin C acylase. • Computational protein design protocol has become an effective strategy for enzyme engineering with the aid of limited experimental validations. • Protein-protein interface was taken into account in the computational design to enhance the stability of enzymes. Abstract Computational protein design strategies can be used to increase enzyme stability without the need for high-throughput screening. In this report, computational methods were used to redesign cephalosporin C acylase from Pseudomonas strain SE83 to enhance its stability by repacking the hydrophobic core regions and reconstructing the protein-protein interactions in the segment interface regions. A nine-fold mutant with enhanced catalytic activity in the hydrolysis of cephalosporin C to 7-aminocephalosporanic acid, but with low stability, was used as a starting point. A computational enzyme design strategy was used to identify target regions to increase the protein melting temperature (T m). Single point mutations Asn2βThr, Asn2βVal, Cys470βSer, Leu154βPhe, and Leu180βPhe in hydrophobic core regions, and Ala100αSer and Ala37βSer in segment-segment interface regions, increased the T m by 4.7–19.7° C, while combining these confirmed single mutations increased the T m by up to 20.5° C. [ABSTRACT FROM AUTHOR]

Background Estimation of diffuse myocardial fibrosis, substrate for adverse events such as heart failure and arrhythmias in patients with various cardiac disorders, is presently done by histopathology or cardiac magnetic resonance. We sought to develop a non-contrast method to estimate the amount of diffuse myocardial fibrosis leveraging dual energy computed tomography (DECT) in phantoms and a suitable small animal model. Methods and Results Phantoms consisted of homogenized bovine myocardium with varying amounts of Type 1 collagen. Fifteen mice underwent sham surgery, no procedure, or transverse aortic constriction (TAC) for 5 or 8 weeks to produce moderate or severe fibrosis, respectively. Phantoms and ex vivo mouse hearts were imaged on a single source, DECT scanner equipped with kVp switching. Monochromatic images were reconstructed at 40–140 keV. Linear discriminant analysis (LDA) was performed on mean myocardial CT numbers derived from single energy (70 keV) images as well as images reconstructed across multiple energies. Classification of myocardial fibrosis severity as low, moderate or severe was more often correct using the multi-energy CT/LDA approach vs. single energy CT/LDA in both phantoms (80.0% vs. 70.0%) and mice (93.3% vs. 33.3%). Conclusions DECT myocardial imaging with multi-energy analysis better classifies myocardial fibrosis severity compared to a single energy-based approach. Non-contrast DECT can accurately and non-invasively estimate the extent of diffuse myocardial fibrosis in phantom and animal models. These data support further evaluation of this approach for in vivo myocardial fibrosis estimation. [ABSTRACT FROM AUTHOR]

Aims Prior work suggests that ischemic preconditioning increases the level of CD39 in the heart and contributes to cardiac protection. Therefore, we examined if targeted cardiac expression of CD39 protects against myocardial injury. Main methods Mice with cardiac-specific expression of human CD39 (αMHC/hCD39-Tg) were generated, characterized and subjected to left coronary artery ischemia-reperfusion injury and infarct size at 24 h following injury quantified. Key findings αMHC/hCD39-Tg mice have increased in cardiac ATPase and ADPase activity compared to WT littermates. The increased activity in αMHC/hCD39-mice was inhibited by the CD39 antagonist sodium polyoxotungstate (POM-1). Measurement of basal cardiac function by echocardiography revealed that αMHC/hCD39-Tg mice have a lower resting heart rate and increased stroke volume. In response to myocardial ischemia, systolic and diastolic function was better preserved in αMHC/hCD39-Tg compared to WT mice. Comparison of Tau also revealed preserved cardiac relaxation during ischemia in αMHC/hCD39-Tg hearts. Assessment of myocardial infarct size in response to 60 min of ischemia and 24 h of reperfusion demonstrated a significant reduction in infarct size in αMHC/hCD39-Tg hearts. Analysis of isolated cardiomyocytes revealed no basal difference in calcium transients between WT and αMHC/hCD39-Tg cardiomyocytes. However, in response to isoproterenol stimulation, there was a trend toward lower calcium transients in αMHC/hCD39 cardiomyocytes suggesting less calcium accumulation in response to metabolic stress. Significance Cardiac-specific expression of CD39 reduces myocardial dysfunction and infarct size following ischemia-reperfusion injury. Increasing nucleotidase expression in the heart may be a novel approach to protect the heart from ischemic injury. [ABSTRACT FROM AUTHOR]

Abstract A resonant fiber optic gyro (RFOG) is a high-accuracy inertial rotation sensor based on the Sagnac effect. Knowledge of the laser frequency noise, especially the study of its transfer characteristics is critical to optimize the resonant frequency servo loop parameters in the RFOG. In this paper, a Mach–Zehnder interferometer (MZI) is used to measure the changes of the power spectral density (PSD) of the laser frequency noise. Three different observation points are set to investigate the transfer characteristics of the laser beam along the transmission loop of the RFOG. Through real-time observation and analysis of the PSDs of the laser frequency noise, we found that the effects of the laser frequency noise on the RFOG can be clearly determined, and the resonant frequency servo loop parameters can be optimized according to the required gyro bandwidth. We also demonstrate the significance of reducing the residual error at the lock-in frequency while ensuring a sufficient gyro bandwidth. [ABSTRACT FROM AUTHOR]

[Display omitted] • TRPV1 overexpressed in neurons were activated by thermal effect of ultrasound. • Activation of TRPV1 overexpressed neurons by ultrasound was effective and harmful. • Protein TRPV1 was an effectively sensitive-protein for sonothermogenetics. Sonogenetics is preferred for neuroregulation and the treatment of brain diseases due to its noninvasive properties. Ultrasonic stimulation produces thermal and mechanical effects, among others. Since transient receptor potential vanilloid 1 (TRPV1) could be activated at 42 °C, it is overexpressed in the M1 region of the mouse motor cortex to sense the change of temperature upon being stimulated by focused ultrasound. Whether the heat generated by ultrasonic stimulation could activate TRPV1 in the M1 region and induce changes in electromyography (EMG) signals collected from the mice's triceps was carefully verified. The position of the focused ultrasound and the temperature of the tissue at the location of the focused position were simulated using COMSOL software and verified via experiments. For Neuro-2a cells with TRPV1 overexpression, 42 °C could activate the TRPV1 and induce calcium influx. For mice with TRPV1 overexpression in the M1 region, tissue temperature of >42 °C in the M1 region induces an increased number of cfos, suggesting that neurons with overexpressed TRPV1 in the M1 region can be activated using focused ultrasound. Furthermore, when the temperature is >42 °C, the peak-to-peak value of the EMG signal for mice with TRPV1 overexpression in the M1 region was higher than that for mice without TRPV1 overexpression. The immunohistochemical results showed that ultrasound was not harmful to the stimulation site. The noninvasive ultrasound stimulation combined with thermosensitive protein TRPV1 overexpressed in neurocytes as sonothermogenetics technology has great potential to be used for the treatment of neurological diseases. [ABSTRACT FROM AUTHOR]

Abstract: Aims: High-fat diet (HFD) is associated with adipose inflammation, which contributes to key components of metabolic abnormalities. The expanded adipose tissue mass associated with obesity is the result of hyperplasia and hypertrophy of adipocytes. In this study, we investigated the effects of long-term HFD on adipocyte progenitor cell (APC) population and adipose-specific gene profiles in both white and brown adipose, and the role of perivascular adipose in the alteration of vascular function in response to HFD. Main methods: Male C57BL/6 mice were fed a standard normal diet (ND) or HFD for about 8months. Glucose metabolism was assessed by an intraperitoneal glucose tolerance test. APC population and adipose-related gene profile were evaluated, and vascular function was measured in the presence or absence of perivascular adipose. Adiponectin and AMPK activity were also investigated. Key findings: HFD induced insulin resistance and glucose intolerance, and resulted in a decrease in APC population in brown, but not in white adipose tissue, when compared with animals fed a ND, with differential alterations of white and brown adipocyte-specific gene expression in brown and white adipose. Additionally, HFD led to altered vascular function in arteries in the presence of perivascular adipose tissue, which is associated with increased superoxide production. Adiponectin and AMPK activity were significantly decreased in response to long-term HFD. Significance: These findings suggest that long-term high-fat intake differentially alters adipocyte progenitor population and adipose-related gene expression in adipose tissue, and adiponectin-AMPK signaling might be involved. In addition, HFD induces changes in perivascular adipose-mediated vascular function. [Copyright &y& Elsevier]