High-resolution in situ structures of mammalian respiratory supercomplexes.

Mitochondria play a pivotal part in ATP energy production through oxidative phosphorylation, which occurs within the inner membrane through a series of respiratory complexes1-4. Despite extensive in vitro structural studies, determining the atomic details of their molecular mechanisms in physiological states remains a major challenge, primarily because of loss of the native environment during purification. Here we directly image porcine mitochondria using an in situ cryo-electron microscopy approach. This enables us to determine the structures of various high-order assemblies of respiratory supercomplexes in their native states. We identify four main supercomplex organizations: I1III2IV1, I1III2IV2, I2III2IV2 and I2III4IV2, which potentially expand into higher-order arrays on the inner membranes. These diverse supercomplexes are largely formed by 'protein-lipids-protein' interactions, which in turn have a substantial impact on the local geometry of the surrounding membranes. Our in situ structures also capture numerous reactive intermediates within these respiratory supercomplexes, shedding light on the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. Structural comparison of supercomplexes from mitochondria treated under different conditions indicates a possible correlation between conformational states of complexes I and III, probably in response to environmental changes. By preserving the native membrane environment, our approach enables structural studies of mitochondrial respiratory supercomplexes in reaction at high resolution across multiple scales, from atomic-level details to the broader subcellular context.

Regular transient limb ischemia improves endothelial function and inhibits endothelial cell apoptosis to prevent atherosclerosis in rabbit.

AIMS: Regular transient limb ischemia (RTLI) can prevent atherosclerosis (AS) progression in hypercholesterolemic rabbits. This study aimed to investigate the minimum effective intensity and possible mechanisms of RTLI for preventing atherosclerosis. METHODS: Eighty rabbits were divided into eight groups: normal (N), high cholesterol (H), three RTLI [three RTLI cycles every other day (R3qod), three RTLI cycles daily (R3qd), and six RTLI cycles daily (R6qd), each cycle of RTLI included 5 min of limb ischemia followed by 5 min limb reperfusion], and three correlated sham RTLI [sham ischemia for 30 min once every other day (S3qod), sham ischemia for 30 min once daily (S3qd), and sham ischemia for 60 min once daily (S6qd)]. Rabbits in group N were kept normally, while the others were fed 1% cholesterol diet for 12 weeks. The RTLI and sham RTLI groups were received RTLI or sham RTLI procedure, respectively. The plaque area in the thoracic aorta was determined by oil red O staining, and quantifying the ratio of plaque area to intimal area (PA/IA). Endothelium-dependent and -independent relaxation were also determined. Endothelial cell were isolated from abdominal aorta of rabbits, and the apoptosis ratio was detected using flow cytometry. RESULTS: The PA/IA and early apoptotic cell ratio was significantly lower as well as the endothelium-dependent relaxation response was higher in group R6qd than those in groups H and S6qd, while those in the R3qod group was not significantly different from those in groups H and S3qod, as well as those in the R3qd group showed no significant difference compared to those in groups H and S3qd. CONCLUSIONS: Six cycles of RTLI daily was the optimal effective intensity to prevent AS progression in rabbits. Endothelial function improvement and apoptosis inhibition might contribute to the anti-AS effects.

Associations between non-insulin-based insulin resistance indices and heart failure prevalence in overweight/obesity adults without diabetes mellitus: evidence from the NHANES 2001-2018.

BACKGROUND: The triglyceride glucose (TyG) index and triglyceride-to-high-density lipoprotein cholesterol (TG/HDL-C) ratio are recognized as simple non-insulin-based insulin resistance indices. Our study aimed to explore the relationship between these two indicators and heart failure (HF) in overweight or obesity individuals without diabetes. METHODS: This cross-sectional study selected 13,473 participants from the National Health and Nutrition Examination Survey (NHANES) 2001-2018 dataset. Weighted multivariable logistic regression and subgroup analysis were employed to evaluate the relationships between TyG index, TG/HDL-C ratio, and HF prevalence, respectively. Additionally, smooth curve fitting was utilized to analyze the dose-response relationships. RESULTS: A total of 13,473 obesity or overweight people without diabetes were included in this study through screening, among whom 291 (2.16%) had comorbid HF. The results of multivariable logistic regression suggested that the highest TyG index (OR = 2.4, 95% CI = 1.4-4.2, p = 0.002) and the highest TG/HDL-C ratio (OR = 1.2, 95% CI = 1.1-1.3, p < 0.001) both increased the prevalence of HF, especially in the non-Hispanic population. Dose-response relationships suggested nonlinear relationships between these two indicators and HF. CONCLUSION: Our study demonstrated that elevated TyG index and TG/HDL-C ratio were closely associated with the prevalence of HF, and both exhibited nonlinear relationships with HF prevalence in overweight/obesity adults without diabetes. Based on these findings, additional prospective studies are needed for further validation.

SMALPs Are Not Simply Nanodiscs: The Polymer-to-Lipid Ratios of Fractionated SMALPs Underline Their Heterogeneous Nature.

Amphipathic styrene-maleic acid (SMA) copolymers directly solubilize biomembranes into SMA-lipid particles, or SMALPs, that are often regarded as nanodiscs and hailed as a native membrane platform. The promising outlook of SMALPs inspires the discovery of many SMA-like copolymers that also solubilize biomembranes into putative nanodiscs, but a fundamental question remains on how much the SMALPs or SMALP analogues truly resemble the bilayer structure of nanodiscs. This unfortunate ambiguity undermines the utility of SMA or SMA-like copolymers in membrane biology because the structure and function of many membrane proteins depend critically on their surrounding matrices. Here, we report the structural heterogeneity of SMALPs revealed through fractionating SMALPs comprised of lipids and well-defined SMAs via size-exclusion chromatography followed by quantitative determination of the polymer-to-lipid (P/L) stoichiometric ratios in individual fractions. Through the lens of P/L stoichiometric ratios, different self-assembled polymer-lipid nanostructures are inferred, such as polymer-remodeled liposomes, polymer-encased nanodiscs, polymer-lipid mixed micelles, and lipid-doped polymer micellar aggregates. We attribute the structural heterogeneity of SMALPs to the microstructure variations amongst individual polymer chains that give rise to their polydisperse detergency. As an example, we demonstrate that SMAs with a similar S/MA ratio but different chain sizes participate preferentially in different polymer-lipid nanostructures. We further demonstrate that proteorhodopsin, a light-driven proton pump solubilized within the same SMALPs is distributed amongst different self-assembled nanostructures to display different photocycle kinetics. Our discovery challenges the native nanodisc notion of SMALPs or SMALP analogues and highlights the necessity to separate and identify the structurally dissimilar polymer-lipid particles in membrane biology studies.

Discovery of the SHP2 allosteric inhibitor 2-((3R,4R)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-(2,3-dichlorophenyl)-3-methylpyrrolo[2,1-f][1,2,4] triazin-4(3H)-one.

The non-receptor protein tyrosine phosphatase (PTP) SHP2 encoded by the PTPN11 gene is a critical regulator in a number of cellular signalling processes and pathways, including the MAPK and the immune-inhibitory programmed cell death PD-L1/PD-1 pathway. Hyperactivation and inactivation of SHP2 is of great therapeutic interest for its association with multiple developmental disorders and cancer-related diseases. In this work, we characterised a potent SHP2 allosteric inhibitor 2-((3 R,4R)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-(2,3-dichlorophenyl)-3-methylpyrrolo[2,1-f][1,2,4]triazin-4(3H)-one (PB17-026-01) by using structure-based design. To study the structure-activity relationship, we compared co-crystal structures of SHP2 bound with PB17-026-01 and its analogue compound PB17-036-01, which is ∼20-fold less active than PB17-026-01, revealing that both of the compounds are bound to SHP2 in the allosteric binding pocket and PB17-026-01 forms more polar contacts with its terminal group. Overall, our results provide new insights into the modes of action of allosteric SHP2 inhibitor and a guide for the design of SHP2 allosteric inhibitor.

Hikikomori: A perspective from bibliometric analysis.

AIMS: Hikikomori is a common phenomenon reported in Japan and many other countries. However, the broad trends of the research publications on hikikomori are unclear. Therefore, this study examined the patterns of research on hikikomori using bibliometric analysis. METHODS: Relevant publications were searched in Web of Science. Bibliometric analyses were performed with CiteSpace, R and VOSviewer. RESULTS: In total, 297 publications on hikikomori met the eligibility criteria. The International Journal of Social Psychiatry (IF = 10.461) published the most papers (K = 17, or 5.7%) on hikikomori. Takahiro A. Kato from Kyushu University (41; 13.8%; H-index = 18) was the most influential author, while Takahiro A. Kato (total link strength [TLS]: 235), Alan R. Teo (TLS: 157), and Masaru Tateno (TLS: 153) separately had the strongest research collaboration with other researchers. Of all countries that published on hikikomori, Japan had the highest number of publications (K = 91). The keywords "United States" and "psychiatric diagnosis" received the most attention between 2013 and 2015, whereas "health" and "autism spectrum disorder" received the most attention in 2021 and 2022. CONCLUSIONS: Peer-reviewed research publications on hikikomori are growing rapidly and the research trends in this field are also changing.

Characterization of a novel sucrose phosphorylase from Paenibacillus elgii and its use in biosynthesis of α-arbutin.

α-Arbutin, a naturally occurring glycosylated derivative of hydroquinone (HQ), effectively inhibits melanin biosynthesis in epidermal cells. It is widely recognized as a fourth-generation whitening agent within the cosmetic industry. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Sucrose phosphorylase (SPase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. In this study, a previously reported SPase known for its effectiveness in synthesizing α-arbutin, was used as a probe sequence to identify a novel SPase from Paenibacillus elgii (PeSP) in the protein database. The sequence similarity between PeSP and the probe was 39.71%, indicating a degree of novelty. Subsequently, the gene encoding PeSP was coexpressed with the molecular chaperone pG-Tf2 in Escherichia coli, significantly improving PeSP's solubility. Following this, PeSP was characterized and employed for α-arbutin biosynthesis. The specific activity of co-expressed PeSP reached 169.72 U/mg, exhibited optimal activity at 35℃ and pH 7.0, with a half-life of 3.6 h under the condition of 35℃. PeSP demonstrated excellent stability at pH 6.5-8.5 and sensitivity to high concentrations of metal ions. The kinetic parameters Km and kcat/Km were determined to be 14.50 mM and 9.79 min- 1·mM- 1, respectively.The reaction conditions for α-arbutin biosynthesis using recombinant PeSP were optimized, resulting in a maximum α-arbutin concentration of 52.60 g/L and a HQ conversion rate of 60.9%. The optimal conditions were achieved at 30℃ and pH 7.0 with 200 U/mL of PeSP, and by combining sucrose and hydroquinone at a molar ratio of 5:1 for a duration of 25 h.

Edaravone alleviates lung damage in mice with hypoxic pulmonary hypertension by increasing nitric oxide synthase 3 expression.

This study is to determine the regulation of nitric oxide synthase 3 (NOS3) by edaravone in mice with hypoxic pulmonary hypertension (HPH). C57BL/6J mice were reared in a hypoxic chamber. HPH mice were treated with edaravone or edaravone + L-NMMA (a NOS inhibitor). Lung tissue was collected for histological assessment, apoptosis analysis, and detection of malondialdehyde, superoxide dismutase, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and NOS3. The levels of serum TNF-α and IL-6 were also measured. Immunohistochemistry was used to visualize the expression of α-smooth muscle actin (SMA) in pulmonary arterioles. Edaravone treatment improved hemodynamics, inhibited right ventricular hypertrophy, increased NOS3 expression, and reduced pathological changes, pulmonary artery wall thickness, apoptotic pulmonary cells, oxidative stress, and the expression of TNF-α, IL-6, and α-SMA in HPH mice. L-NMMA treatment counteracted the lung protective effects of edaravone. In conclusion, edaravone might reduce lung damage in HPH mice by increasing the expression of NOS3.

High-brightness and high-efficiency diode laser module based on double wavelength division multiplexing external cavities.

In this Letter, a new, to the best of our knowledge, external cavity structure based on double wavelength division multiplexing external cavities is proposed and demonstrated. The electro-optical conversion efficiency is improved and the brightness of the spectral beam combining diode lasers is enhanced. One wavelength division multiplexing external cavity is placed on the rear-side of the laser emitters to provide the strong optical feedback for wavelength locking and the other wavelength division multiplexing external cavity is placed on the front-side of laser emitters to combine three emitter beams to one beam. A maximum output power of up to 7.5 W is obtained and the brightness of the laser diode is 100 MW cm-2 sr-1 with an electro-optical conversion efficiency of 46.5%. Compared with a standard cavity for spectral beam combining, the use of double wavelength division multiplexing external cavities results in an electro-optical conversion efficiency improvement of 6.5%. The whole structure provides a new technology to achieve high-brightness and high electro-optical conversion efficiency for a laser diode source.

White matter pathology in alzheimer's transgenic mice with chronic exposure to low-level ambient fine particulate matter.

BACKGROUND: Air pollution, especially fine particulate matter (PM), can cause brain damage, cognitive decline, and an increased risk of neurodegenerative disease, especially alzheimer's disease (AD). Typical pathological findings of amyloid and tau protein accumulation have been detected in the brain after exposure in animal studies. However, these observations were based on high levels of PM exposure, which were far from the WHO guidelines and those present in our environment. In addition, white matter involvement by air pollution has been less reported. Thus, this experiment was designed to simulate the true human world and to discuss the possible white matter pathology caused by air pollution. RESULTS: 6 month-old female 3xTg-AD mice were divided into exposure and control groups and housed in the Taipei Air Pollutant Exposure System (TAPES) for 5 months. The mice were subjected to the Morris water maze test after exposure and were then sacrificed with brain dissection for further analyses. The mean mass concentration of PM2.5 during the exposure period was 13.85 µg/m3. After exposure, there was no difference in spatial learning function between the two groups, but there was significant decay of memory in the exposure group. Significantly decreased total brain volume and more neuronal death in the cerebral and entorhinal cortex and demyelination of the corpus callosum were noted by histopathological staining after exposure. However, there was no difference in the accumulation of amyloid or tau on immunohistochemistry staining. For the protein analysis, amyloid was detected at significantly higher levels in the cerebral cortex, with lower expression of myelin basic protein in the white matter. A diffuse tensor image study also revealed insults in multiple white matter tracts, including the optic tract. CONCLUSIONS: In conclusion, this pilot study showed that even chronic exposure to low PM2.5 concentrations still caused brain damage, such as gross brain atrophy, cortical neuron damage, and multiple white matter tract damage. Typical amyloid cascade pathology did not appear prominently in the vulnerable brain region after exposure. These findings imply that multiple pathogenic pathways induce brain injury by air pollution, and the optic nerve may be another direct invasion route in addition to olfactory nerve.

Polyphenol and Anthocyanin Composition and Activity of Highland Barley with Different Colors.

In this research, the composition of free phenols, bound phenols, and anthocyanins and their in vitro antioxidant activity and in vitro α-glucosidase inhibiting activity were observed in different barley colors. The outcomes revealed that the contents of total phenols (570.78 mg/100 gDW), total flavonoids (47.08 mg/100 gDW), and anthocyanins (48.07 mg/100 g) were the highest in purple barley. Furthermore, the structure, composition, and concentration of phenolics differed depending on the colors of barley. The types and contents of bound total phenolic acids and flavonoids were greater than those of free total phenolic acids and flavonoids. The main phenolic acids in blue barley were cinnamic acid polyphenols, whereas in black, yellow, and purple barley, benzoic acid polyphenols were the main phenolic acids, and the main types of flavonoids in black and blue barley were chalcones and flavanones, respectively, whereas flavonol was the main type of flavonoid in yellow and purple barley. Moreover, cornflower pigment-3-glucoside was the major anthocyanin in blue, yellow, and purple barley, whereas the main anthocyanin in black barley was delphinidin-3-glucoside. The dark color of barley indicated richness in the anthocyanins. In addition, the free polyphenol fractions had stronger DPPH and ABTS radical scavenging capacity as compared to the bound ones. In vitro α-glucosidase-inhibiting activity was greater in bound polyphenols than in free polyphenols, with differences between different varieties of barley. Purple barley phenolic fractions had the greatest ABTS radical scavenging and iron ion reduction capacities, as well as the highest α-glucosidase-inhibiting activity. The strongest DPPH radical scavenging capacity was found in yellow barley, while the strongest in vitro α-glucosidase-inhibiting activity was found in anthocyanins isolated from black barley. Furthermore, in different colors of barley, there was a strong association between the concentration of specific phenolic compounds and antioxidant and α-glucosidase-inhibiting activities. The outcomes of this study revealed that all colored barley seeds tested were high in phenolic compounds, and had a good antioxidant impact and α-glucosidase-inhibiting activity. As a result, colored barley can serve as an antioxidant and hypoglycemic food. Polyphenols extracted from purple barley and anthocyanins extracted from black barley stand out among them.

Alteration of the Catalytic Reaction Trajectory of a Vicinal Oxygen Chelate Enzyme by Directed Evolution.

The vicinal oxygen chelate (VOC) metalloenzyme superfamily catalyzes a highly diverse set of reactions with the mechanism characterized by the bidentate coordination of vicinal oxygen atoms to metal ion centers, but there remains a lack of a platform to steer the reaction trajectories, especially for o-quinone metabolizing pathways. Herein, we present the directed-evolution-enabled bifunctional turnover of ChaP, which is a homotetramer and represents an unprecedented VOC enzyme class. Unlike the ChaP catalysis of extradiol-like o-quinone cleavage and concomitant α-keto acid decarboxylation, a group of ChaP variants (CVs) catalyze intradiol-like o-quinone deconstruction and CO2 liberation from the resulting o-hydroxybenzoic acid scaffolds with high regioselectivity. Enzyme crystal structures, labeling experiments and computational simulations corroborated that the D49L mutation allows the metal ion to change its coordination with the tyrosine phenoxy atoms in different monomers, thereby altering the reaction trajectory with the regiospecificity further improved by the follow-up replacement of the Y92 residue with any of alanine, glycine, threonine, and serine. The study highlights the unpredicted catalytic versatility and enzymatic plasticity of VOC enzymes with biotechnological significance.

Environmentally Benign Nanoantibiotics with a Built-in Deactivation Switch Responsive to Natural Habitats.

The massive use of antibiotics in healthcare and agriculture has led to their artificial accumulation in natural habitats, which risks the structure and function of the microbial communities in ecosystems, threatens food and water security, and accelerates the development of resistome. Ideally, antibiotics should remain fully active in clinical services while becoming deactivated rapidly once released into the environment, but none of the current antibiotics meet this criterion. Here, we show a nanoantibiotic design that epitomizes the concept of carrying a built-in "OFF" switch responsive to natural stimuli. The environmentally benign nanoantibiotics consist of cellulose backbones covalently grafted with hydrophilic polymer brushes that by themselves are antimicrobially inactive. In their nanostructured forms in services, these cellulose-based polymer molecular brushes are potent killers for both Gram-positive and Gram-negative bacteria, including clinical multidrug-resistant strains; after services and being discharged into the environment, they are shredded into antimicrobially inactive pieces by cellulases that do not exist in the human body but are abundant in natural habitats. This study illuminates a new concept of mitigating the environmental footprints of antibiotics with rationally designed nanoantibiotics that can be dismantled and disabled by bioorthogonal chemistry occurring exclusively in natural habitats.

[Mechanisms for establishment of the placental defensive barrier].

Placenta serves as a temporary fetal organ, which mediates maternal-fetal crosstalk and intrauterine fetal growth. Placental defensive barrier is a fundamental physiological function, which balances maternal immune tolerance to the fetus and resistance to pathogens. This review summarizes the latest research progress on the mechanisms of placental barrier formation from the view of placental development. Recent discoveries have shed light on the cellular and molecular properties of placental defensive mechanisms in syncytiotrophoblast, including autophagy, exosome mediated anti-pathogenic pathways, cell-cell junctions and cytoskeleton networks. We also present an overview of placental barrier dysfunction and its implications in intrauterine TORCH infections.

Nrf2⁻ARE Signaling Acts as Master Pathway for the Cellular Antioxidant Activity of Fisetin.

Fisetin, a dietary flavonoid, is reported to have cellular antioxidant activity with an unclear mechanism. In this study, we investigated the effect of fisetin on the nuclear factor, erythroid 2-like 2 (Nrf2) signaling pathway in HepG2 cells to explore the cellular antioxidant mechanism. Fisetin upregulated the mRNA expression of heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and NAD(P)H quinone oxidoreductase-1 (NQO1), and induced the protein of HO-1 but had no significant effect on the protein of GCLC, GCLM and NQO1. Moreover, nuclear accumulation of Nrf2 was clearly observed by immunofluorescence analysis and western blotting after fisetin treatment, and an enhanced luciferase activity of antioxidant response element (ARE)-regulated transactivation was obtained by dual-luciferase reporter gene assays. In addition, fisetin upregulated the protein level of Nrf2 and downregulated the protein level of Kelch-like ECH-associated protein 1 (Keap1). However, fisetin had no significant effect on Nrf2 mRNA expression. When protein synthesis was inhibited with cycloheximide (CHX), fisetin prolonged the half-life of Nrf2 from 15 min to 45 min. When blocking Nrf2 degradation with proteasome inhibitor MG132, ubiquitinated proteins were enhanced, and fisetin reduced ubiquitination of Nrf2. Taken together, fisetin translocated Nrf2 into the nucleus and upregulated the expression of downstream HO-1 gene by inhibiting the degradation of Nrf2 at the post-transcriptional level. These data provide the molecular mechanism to understand the cellular antioxidant activity of fisetin.

[Effect of different concentrations of calcitonin gene-related peptide on the long-term potentiation in hippocampus of mice].

The purpose of this study was to explore the effects of different concentrations of calcitonin gene-related peptide (CGRP) on long-term potentiation (LTP) in the hippocampus of mice. C57BL/6J mice (30 days old) were randomly divided into control group, three CGRP groups, and CGRP + CGRP8-37 group (10 mice for each group). Different concentrations of CGRP (50, 100 and 200 nmol/L) were given to the hippocampal slices of mice. The presynaptic release of neurotransmitters and the induction of LTP were measured by extracellular field recording techniques. The result showed that different concentrations of CGRP did not affect the presynaptic release of neurotransmitters, but 100 and 200 nmol/L CGRP increased the amplitude of LTP induced in the hippocampus of mice. This facilitation effect of CGRP was blocked by its specific antagonist CGRP8-37. These results suggest that CGRP dose-dependently facilitates the induction of LTP in the hippocampus of mice through its specific receptor.

[Effect of bilateral injection of calcitonin gene-related peptide into amygdala on learning and memory of mice].

The aim of the present study was to explore the effects of different doses of calcitonin gene-related peptide (CGRP) injected into the central nucleus of amygdala on cognitive function, learning and memory of mice. C57BL/6J mice (30 days old) were randomly divided into control, sham, and three CGRP groups (10 mice for each group). Three doses of CGRP (200, 400 and 800 ng) were bilaterally administered into the central nucleus of the amygdala. Open field test was used to assess cognitive function. Novel object recognition and Morris water maze test were used to evaluate learning and memory of the mice. The results of open field test showed that 800 ng CGRP significantly increased the locomotive score. The results of novel objective recognition test showed that 400 ng CGRP significantly increased the recognition index. Compared with control group, 400 and 800 ng CGRP groups showed significantly shortened latency period and increased crossing times. Simultaneously, the latency periods of 400 and 800 ng CGRP groups were shorter than that of 200 ng CGRP group. These results suggest that bilateral injection of CGRP into amygdala dose-dependently enhances the learning and memory function of mice.

A Usual G-Protein-Coupled Receptor in Unusual Membranes.

G-protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and constitute about 50% of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge-interaction-directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid- and polymer-based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non-biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H(+)] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin-conserved ERY sequence motif that helps break the cytoplasmic "ionic lock". This study unveils a novel design concept of non-biological membranes to reconstitute and harness GPCR functions in synthetic systems.

High-resolution In-situ Structures of Mammalian Mitochondrial Respiratory Supercomplexes in Reaction within Native Mitochondria.

Mitochondria play a pivotal role in ATP energy production through oxidative phosphorylation, which occurs within the inner membrane via a series of respiratory complexes. Despite extensive in-vitro structural studies, revealing the atomic details of their molecular mechanisms in physiological states remains a major challenge, primarily because of the loss of the native environment during purification. Here, we directly image porcine mitochondria using an in-situ cryo-electron microscopy approach. This enables us to determine the structures of various high-order assemblies of respiratory supercomplexes in their native states, achieving up to 1.8-Å local resolution. We identify four major supercomplex organizations: I1III2IV1, I1III2IV2, I2III2IV2, and I2III4IV2, which can potentially expand into higher-order arrays on the inner membranes. The formation of these diverse supercomplexes is largely contributed by 'protein-lipids-protein' interactions, which in turn dramatically impact the local geometry of the surrounding membranes. Our in-situ structures also capture numerous reactive intermediates within these respiratory supercomplexes, shedding light on the dynamic processes of the ubiquinone/ubiquinol exchange mechanism in complex I and the Q-cycle in complex III. By comparing supercomplex structures from mitochondria treated under distinct conditions, we elucidate how conformational changes and ligand binding states interplay between complexes I and III in response to environmental redox alterations. Our approach, by preserving the native membrane environment, enables structural studies of mitochondrial respiratory supercomplexes in reaction at high resolution across multiple scales, spanning from atomic-level details to the broader subcellular context.