Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP3) assay measures 3H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M1 acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers

Lung Adenocarcinoma Identification via Cellular Phenotyping and Neighborhood Analysis with Imaging Mass Cytometry

https://openworks.mdanderson.org/sumexp21/1210/thumbnail.jp

High-Pressure-Sintering-Induced Microstructural Engineering for an Ultimate Phonon Scattering of Thermoelectric Half-Heusler Compounds

Thermal management is of vital importance in various modern technologies such as portable electronics, photovoltaics, and thermoelectric devices. Impeding phonon transport remains one of the most challenging tasks for improving the thermoelectric performance of certain materials such as half-Heusler compounds. Herein, a significant reduction of lattice thermal conductivity (κL) is achieved by applying a pressure of ≈1 GPa to sinter a broad range of half-Heusler compounds. Contrasting with the common sintering pressure of less than 100 MPa, the gigapascal-level pressure enables densification at a lower temperature, thus greatly modifying the structural characteristics for an intensified phonon scattering. A maximum κL reduction of ≈83% is realized for HfCoSb from 14 to 2.5 W m−1 K−1 at 300 K with more than 95% relative density. The realized low κL originates from a remarkable grain-size refinement to below 100 nm together with the abundant in-grain defects, as determined by microscopy investigations. This work uncovers the phonon transport properties of half-Heusler compounds under unconventional microstructures, thus showing the potential of high-pressure compaction in advancing the performance of thermoelectric materials

Unveiling the phonon scattering mechanisms in half-Heusler thermoelectric compounds

Half-Heusler (HH) compounds are among the most promising thermoelectric (TE) materials for large-scale applications due to their superior properties such as high power factor, excellent mechanical and thermal reliability, and non-toxicity. Their only drawback is the remaining-high lattice thermal conductivity. Various mechanisms were reported with claimed effectiveness to enhance the phonon scattering of HH compounds including grain-boundary scattering, phase separation, and electron–phonon interaction. In this work, however, we show that point-defect scattering has been the dominant mechanism for phonon scattering other than the intrinsic phonon–phonon interaction for ZrCoSb and possibly many other HH compounds. Induced by the charge-compensation effect, the formation of Co/4d Frenkel point defects is responsible for the drastic reduction of lattice thermal conductivity in ZrCoSb1−xSnx. Our work systematically depicts the phonon scattering profile of HH compounds and illuminates subsequent material optimizations

Analysis of Load-Sharing and Contact Characteristics of the Concentric Face Gear Split-Torque Transmission System with Elastic Supports

The concentric face gear split-torque transmission system (CFGSTTS) is a new form of drive that is primarily used in helicopter transmission systems. Its load-sharing performance among different branches and tooth contact characteristics have a great impact on the service life of helicopter transmissions. It contains ten meshing pairs, the load distribution is complicated, and the tooth contact areas are difficult to determine. Therefore, based on the multi-point constraint method and nonconforming grid, a quasi-static analysis model of the CFGSTTS coupled with flexible supports was established and the load-sharing performance and contact characteristics were studied. The model considered the support stiffness, backlash, installation error, and web structure of the upper face gear, which could comprehensively reflect the meshing state of the system. The load-sharing coefficient curves, tooth contact area diagram, and meshing force were obtained. The results indicated that (1) a larger idler support stiffness and a smaller input gear support stiffness could achieve better load equalization performance; (2) better load equalization between idler gears could be acquired with a lower face gear support stiffness factor of approximately 0.9; (3) increasing the axial mounting error caused the contact area to shift to the top and inner end of the face gear tooth, which was detrimental to the transmission; and (4) adjusting the backlash of the idler gears, input gears, and tail gear had little influence on the load balance and contact

Analysis of Load-Sharing and Contact Characteristics of the Concentric Face Gear Split-Torque Transmission System with Elastic Supports

The concentric face gear split-torque transmission system (CFGSTTS) is a new form of drive that is primarily used in helicopter transmission systems. Its load-sharing performance among different branches and tooth contact characteristics have a great impact on the service life of helicopter transmissions. It contains ten meshing pairs, the load distribution is complicated, and the tooth contact areas are difficult to determine. Therefore, based on the multi-point constraint method and nonconforming grid, a quasi-static analysis model of the CFGSTTS coupled with flexible supports was established and the load-sharing performance and contact characteristics were studied. The model considered the support stiffness, backlash, installation error, and web structure of the upper face gear, which could comprehensively reflect the meshing state of the system. The load-sharing coefficient curves, tooth contact area diagram, and meshing force were obtained. The results indicated that (1) a larger idler support stiffness and a smaller input gear support stiffness could achieve better load equalization performance; (2) better load equalization between idler gears could be acquired with a lower face gear support stiffness factor of approximately 0.9; (3) increasing the axial mounting error caused the contact area to shift to the top and inner end of the face gear tooth, which was detrimental to the transmission; and (4) adjusting the backlash of the idler gears, input gears, and tail gear had little influence on the load balance and contact

Meshing Stiffness Parametric Vibration of Coaxial Contrarotating Encased Differential Gear Train

Planetary gears are widely used in mechanical transmission systems, but the vibration and noise affect their reliability and life. In this paper, the torsional dynamic model of an encased differential planetary gear with coaxial contrarotating outputs considering the time-varying meshing stiffness, damping, and phase difference of all gear pairs is established. By solving the equations of the derived system, three types of natural frequencies with different multiplicities of the system are obtained. The multiscale method is used to study the parametric vibration stability caused by the time-varying meshing stiffness, and the results are verified by numerical simulation. The dynamic characteristics of elastic meshing force are analyzed from time domain and frequency domain. The variation of the dynamic load factor of each gear pair with input speed and the relationship between its peak position and the natural frequency of the derived system are discussed. The results show that there is an unequal coupling phenomenon of meshing frequency between the meshing forces of different planetary sets. In the absence of external excitation, the meshing stiffness parameters not only excite the main resonance response of the system but also cause superharmonic resonance, subharmonic resonance, and combined resonance

Study on Friction and Wear Properties of Zr–Cu–Ni–Al Crystalline Powder Cladding and Amorphous Composite Powder Cladding by Laser

In order to improve the friction and wear performance and surface hardness of AISI 1045 steel and expand its application range, this paper carried out the research on friction and wear performance and surface hardness of Zr65Al7.5Ni10Cu17.5 crystalline powder (CP) and amorphous powder (AP) after laser cladding on AISI 1045 steel surface. The results show that both CP and amorphous powder (AP) formed a cladding layer on the surface of AISI 1045 steel under laser irradiation. The thickness of the cladding layer is about 400 μm, and the thickness of the AP cladding layer is slightly larger than that of the CP cladding layer. The results show that there are many holes in the AP cladding layer, and holes can be observed at the junction with the matrix; while the CP cladding layer is well combined with the matrix and no holes are observed. The friction performance of CP cladding layer is better than that of AP cladding layer. In the wear marks of the AP cladding layer, there are bonding areas, while the wear marks of the CP cladding layer have a furrow-like morphology, and part of the matrix is exposed. The surface microhardness and average microhardness of AP cladding layer are 49% and 94% higher than that of CP cladding layer, respectively. Hardness modification has obvious advantages. The reasons for porosity, large friction coefficient and low stability of the friction experiment of the AP cladding layer are analyzed and discussed. The ideas and methods for improving the laser irradiation to achieve both high wear resistance and high strength of the AP cladding layer are proposed

Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1

Resumo Background: Melatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. Objective: Demonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes. Methods: In vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats. Results: In vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059. Conclusion: melatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1