A method for structure determination of GPCRs in various states.

G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the ß2-adrenergic receptor (ß2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For ß2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structure‒function relationships and drug development.

Charge-Powered Electrotaxis for Versatile Droplet Manipulation.

Taxis is an instinctive behavior of living organisms to external dangers or benefits. Here, we report a taxis-like behavior associated with liquid droplets on charged substrates in response to the external stimuli, referred to as droplet electrotaxis. Such droplet electrotaxis enables us to use either solid or liquid (such as water) matter, even a human finger, as stimuli to spatiotemporal precisely manipulate the liquid droplets of various physicochemical properties, including water, ethanol with low surface tension, viscous oil, and so on. Droplet electrotaxis also features a flexible configuration that even can manifest in the presence of an additional layer, such as the ceramic with a thickness of ∼10 mm. More importantly, superior to existing electricity-based strategies, droplet electrotaxis can harness the charges generated from diverse manners, including pyroelectricity, triboelectricity, piezoelectricity, and so on. These properties dramatically increase the application scenarios of droplet electrotaxis, such as cell labeling and droplet information recording.

Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.

The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and ß-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance ß-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.

Kallikrein-related peptidase-8 (KLK8) aggravated hypoxia-induced right ventricular hypertrophy by targeting P38 MAPK/P53 signaling pathway.

Right ventricular (RV) hypertrophy and further heart failure are major co-morbidities, resulting in the premature death of patients with hypoxic pulmonary hypertension (HPH). The regulatory effects of kallikrein-related peptidase (KLK) family members on cardiac function have been extensively studied. However, to the best of the authors' knowledge, the regulatory effects of KLK8 on RV hypertrophy caused by HPH have yet to be reported. The aim of the present study was to assess KLK8 expression in the RV tissue of HPH-modeled rats, and to further explore the effects and underlying mechanism of KLK8 in regulating the hypertrophy of hypoxia-induced H9c2 cardiomyocytes. In HPH model rats, increases in the right ventricle hypertrophy index, the right ventricular systolic pressure, cardiac output, as well as pulmonary artery wall thickness were observed. Western blot analysis revealed that KLK8 expression and MAPK/p53 signaling activity were enhanced in the RVs of rats in an RV HPH rat model. In hypoxia-induced H9c2 cardiomyocytes, KLK8 overexpression promoted cardiomyocyte hypertrophy, whereas KLK8 silencing showed the opposite results. KLK8 overexpression increased the expression levels of ventricular hypertrophy markers, including atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain 7, which were blocked upon addition of the p38 MAPK inhibitor, SB202190. Conversely, KLK8 silencing caused a decrease in the expression levels of the ventricular hypertrophy markers, which were further reduced via inhibition of the p38 MAPK/p53 signaling pathway. Taken together, the results of the present study have shown that KLK8 may subtly regulate RV hypertrophy, and therefore KLK8 may be a promising therapeutic target for treating HPH-induced RV hypertrophy.

Structural basis of the ligand binding and signaling mechanism of melatonin receptors.

Melatonin receptors (MT1 and MT2 in humans) are family A G protein-coupled receptors that respond to the neurohormone melatonin to regulate circadian rhythm and sleep. Numerous efforts have been made to develop drugs targeting melatonin receptors for the treatment of insomnia, circadian rhythm disorder, and cancer. However, designing subtype-selective melatonergic drugs remains challenging. Here, we report the cryo-EM structures of the MT1-Gi signaling complex with 2-iodomelatonin and ramelteon and the MT2-Gi signaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT1 and MT2 possess highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be targeted to design subtype-selective drugs. The unique structural motifs in MT1 and MT2 mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. Gi is engaged in the receptor core shared by MT1 and MT2 and presents a conformation deviating from those in other Gi complexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism.

Down-regulation of microRNA-342-5p or Up-regulation of Wnt3a Inhibits Angiogenesis and Maintains Atherosclerotic Plaque Stability in Atherosclerosis Mice.

Evidence has demonstrated that microRNA-342-5p (miR-342-5p) is implicated in atherosclerosis (AS), but little is known regarding its intrinsic regulatory mechanisms. Here, we aimed to explore the effect of miR-342-5p targeting Wnt3a on formation of vulnerable plaques and angiogenesis of AS. ApoE-/- mice were fed with high-fat feed for 16 w to replicate the AS vulnerable plaque model. miR-342-5p and Wnt3a expression in aortic tissues of AS were detected. The target relationship between miR-342-5p and Wnt3a was verified. Moreover, ApoE-/- mice were injected with miR-342-5p antagomir and overexpression-Wnt3a vector to test their functions in serum lipid levels, inflammatory and oxidative stress-related cytokines, aortic plaque stability and angiogenesis in plaque of AS mice. miR-342-5p expression was enhanced and Wnt3a expression was degraded in aortic tissues of AS mice and miR-342-5p directly targeted Wnt3a. Up-regulating Wnt3a or down-regulating miR-342-5p reduced blood lipid content, inflammatory and oxidative stress levels, the vulnerability of aortic tissue plaque and inhibited angiogenesis in aortic plaque of AS mice. Functional studies show that depleting miR-342-5p can stabilize aortic tissue plaque and reduce angiogenesis in plaque in AS mice via restoring Wnt3a.

Manipulation of a Nonconductive Droplet in an Aqueous Fluid with AC Electric Fields: Droplet Dewetting, Oscillation, and Detachment.

Electrowetting (EW) is an effective method for droplet manipulation in microfluidics. In traditional EW, a conductive droplet is actuated, which spreads on a solid substrate. Recently, we considered an opposite phenomenon of droplet actuation in EW: inducing nonconductive droplet dewetting and detaching from the substrate. An oil/water system is used in which the oil droplet (nonconductive) is actuated on a flat substrate in surrounding water (conductive) by EW. In this work, alternating current (AC) electric fields are applied to EW, and the transient dynamics of droplet dewetting, oscillation, and detachment with the AC signals are investigated. The droplet is not in contact with electrodes, and it dances freely on the substrate. Experiments are performed in a wide range of voltages and AC frequencies. To demonstrate the droplet dynamics, we divide the full process of droplet manipulation into three distinguishable periods, that is, an initiating period, a steady oscillation period, and a detaching condition. Transient droplet dewetting is considered in the initiating period, and we obtain the distribution of the contact line friction factor. In steady oscillation, the oscillation resonance is verified from the oscillating amplitude of the contact line. Different periodical features are found for the droplet dancing at the resonance frequencies and departure from resonance. The droplet is detached at high voltages, and we provide a map for the detachable and nondetachable zones. The voltage is the dominant factor determining the droplet detachment; however, the AC frequency has notable influences on the critical voltage. The detachment is promoted when the AC frequency is within the region of the oscillation resonance (e.g., 20 < f < 75 Hz). In this region, the detaching process is not monotonic but instead, the droplet rebounds by several times before it is completely detached.

Actuation of a Nonconductive Droplet in an Aqueous Fluid by Reversed Electrowetting Effect.

Manipulation of a conductive droplet by electrowetting has been a popular topic in microfluidics whereby wettability of the droplet on a solid surface is increased by applying a voltage between the conductive droplet and the insulated surface. However, the opposite phenomenon, e.g., decreasing the wettability of a nonconductive droplet and increasing its contact angle (CA) by the reversed electrowetting (REW) effect, has been scarcely reported. Such a process involves not only the transient dynamics of droplet dewetting but also a critical condition for droplet detachment from the adhesive surface. In this work, actuation of a nonconductive droplet in an aqueous surrounding fluid by REW is studied experimentally. Silicone oil is used for the actuated droplet, and filtered water is used as the surrounding fluid. The solid substrate is made of a glass substrate coated with an indium tin oxide (ITO) film and then deposited by a dielectric layer of Parylene C. Potential difference is applied between the substrate and the surrounding fluid, eliminating the disturbance from the top needle on the motion of the droplet. Three different regimes are identified in the full range of operation. An underactuated regime occurs at low applied voltages, in which the CA of the droplet shows a monotonic increase with the increase of voltage (V). The friction coefficient of the contact line decreases with V before the CA saturation (Vs) but shows little change when V > Vs. At high voltages, the contact line of the sessile droplet is contracted excessively by REW. The droplet shows oscillation, and it refers to the overactuated regime. A combined time scale is proposed, and it verifies that the viscous dissipation of the contact line and liquid inertia show comparable contributions in the droplet dynamics. At sufficiently high voltages, the droplet is rejected completely from the surface. A critical equation for the threshold voltage of droplet detachment is built, and its validity is confirmed by experimental results.

Thermoelectricity in Heterogeneous Nanofluidic Channels.

Ionic fluids are essential to energy conversion, water desalination, drug delivery, and lab-on-a-chip devices. Ionic transport in nanoscale confinements and complex physical fields still remain elusive. Here, a nanofluidic system is developed using nanochannels of heterogeneous surface properties to investigate transport properties of ions under different temperatures. Steady ionic currents are observed under symmetric temperature gradients, which is equivalent to generating electricity using waste heat (e.g., electronic chips and solar panels). The currents increase linearly with temperature gradient and nonlinearly with channel size. Contributions to ion motion from temperatures and channel properties are evaluated for this phenomenon. The findings provide insights into the study of confined ionic fluids in multiphysical fields, and suggest applications in thermal energy conversion, temperature sensors, and chip-level thermal management.