Molecular recognition of morphine and fentanyl by the human µ-opioid receptor.

Morphine and fentanyl are among the most used opioid drugs that confer analgesia and unwanted side effects through both G protein and arrestin signaling pathways of µ-opioid receptor (µOR). Here, we report structures of the human µOR-G protein complexes bound to morphine and fentanyl, which uncover key differences in how they bind the receptor. We also report structures of µOR bound to TRV130, PZM21, and SR17018, which reveal preferential interactions of these agonists with TM3 side of the ligand-binding pocket rather than TM6/7 side. In contrast, morphine and fentanyl form dual interactions with both TM3 and TM6/7 regions. Mutations at the TM6/7 interface abolish arrestin recruitment of µOR promoted by morphine and fentanyl. Ligands designed to reduce TM6/7 interactions display preferential G protein signaling. Our results provide crucial insights into fentanyl recognition and signaling of µOR, which may facilitate rational design of next-generation analgesics.

CD40 signal rewires fatty acid and glutamine metabolism for stimulating macrophage anti-tumorigenic functions.

Exposure of lipopolysaccharide triggers macrophage pro-inflammatory polarization accompanied by metabolic reprogramming, characterized by elevated aerobic glycolysis and a broken tricarboxylic acid cycle. However, in contrast to lipopolysaccharide, CD40 signal is able to drive pro-inflammatory and anti-tumorigenic polarization by some yet undefined metabolic programming. Here we show that CD40 activation triggers fatty acid oxidation (FAO) and glutamine metabolism to promote ATP citrate lyase-dependent epigenetic reprogramming of pro-inflammatory genes and anti-tumorigenic phenotypes in macrophages. Mechanistically, glutamine usage reinforces FAO-induced pro-inflammatory and anti-tumorigenic activation by fine-tuning the NAD+/NADH ratio via glutamine-to-lactate conversion. Genetic ablation of important metabolic enzymes involved in CD40-mediated metabolic reprogramming abolishes agonistic anti-CD40-induced antitumor responses and reeducation of tumor-associated macrophages. Together these data show that metabolic reprogramming, which includes FAO and glutamine metabolism, controls the activation of pro-inflammatory and anti-tumorigenic polarization, and highlight a therapeutic potential of metabolic preconditioning of tumor-associated macrophages before agonistic anti-CD40 treatments.

Metabolic reprogramming of terminally exhausted CD8+ T cells by IL-10 enhances anti-tumor immunity.

T cell exhaustion presents one of the major hurdles to cancer immunotherapy. Among exhausted CD8+ tumor-infiltrating lymphocytes, the terminally exhausted subset contributes directly to tumor cell killing owing to its cytotoxic effector function. However, this subset does not respond to immune checkpoint blockades and is difficult to be reinvigorated with restored proliferative capacity. Here, we show that a half-life-extended interleukin-10-Fc fusion protein directly and potently enhanced expansion and effector function of terminally exhausted CD8+ tumor-infiltrating lymphocytes by promoting oxidative phosphorylation, a process that was independent of the progenitor exhausted T cells. Interleukin-10-Fc was a safe and highly efficient metabolic intervention that synergized with adoptive T cell transfer immunotherapy, leading to eradication of established solid tumors and durable cures in the majority of treated mice. These findings show that metabolic reprogramming by upregulating mitochondrial pyruvate carrier-dependent oxidative phosphorylation can revitalize terminally exhausted T cells and enhance the response to cancer immunotherapy.

Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

CD36-mediated metabolic adaptation supports regulatory T cell survival and function in tumors.

Depleting regulatory T cells (Treg cells) to counteract immunosuppressive features of the tumor microenvironment (TME) is an attractive strategy for cancer treatment; however, autoimmunity due to systemic impairment of their suppressive function limits its therapeutic potential. Elucidating approaches that specifically disrupt intratumoral Treg cells is direly needed for cancer immunotherapy. We found that CD36 was selectively upregulated in intrautumoral Treg cells as a central metabolic modulator. CD36 fine-tuned mitochondrial fitness via peroxisome proliferator-activated receptor-ß signaling, programming Treg cells to adapt to a lactic acid-enriched TME. Genetic ablation of Cd36 in Treg cells suppressed tumor growth accompanied by a decrease in intratumoral Treg cells and enhancement of antitumor activity in tumor-infiltrating lymphocytes without disrupting immune homeostasis. Furthermore, CD36 targeting elicited additive antitumor responses with anti-programmed cell death protein 1 therapy. Our findings uncover the unexplored metabolic adaptation that orchestrates the survival and functions of intratumoral Treg cells, and the therapeutic potential of targeting this pathway for reprogramming the TME.

Preparing random states and benchmarking with many-body quantum chaos.

Producing quantum states at random has become increasingly important in modern quantum science, with applications being both theoretical and practical. In particular, ensembles of such randomly distributed, but pure, quantum states underlie our understanding of complexity in quantum circuits1 and black holes2, and have been used for benchmarking quantum devices3,4 in tests of quantum advantage5,6. However, creating random ensembles has necessitated a high degree of spatio-temporal control7-12 placing such studies out of reach for a wide class of quantum systems. Here we solve this problem by predicting and experimentally observing the emergence of random state ensembles naturally under time-independent Hamiltonian dynamics, which we use to implement an efficient, widely applicable benchmarking protocol. The observed random ensembles emerge from projective measurements and are intimately linked to universal correlations built up between subsystems of a larger quantum system, offering new insights into quantum thermalization13. Predicated on this discovery, we develop a fidelity estimation scheme, which we demonstrate for a Rydberg quantum simulator with up to 25 atoms using fewer than 104 experimental samples. This method has broad applicability, as we demonstrate for Hamiltonian parameter estimation, target-state generation benchmarking, and comparison of analogue and digital quantum devices. Our work has implications for understanding randomness in quantum dynamics14 and enables applications of this concept in a much wider context4,5,9,10,15-20.

Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

Pharmacological inhibition of Kir4.1 evokes rapid-onset antidepressant responses.

Major depressive disorder, a prevalent and severe psychiatric condition, necessitates development of new and fast-acting antidepressants. Genetic suppression of astrocytic inwardly rectifying potassium channel 4.1 (Kir4.1) in the lateral habenula ameliorates depression-like phenotypes in mice. However, Kir4.1 remains an elusive drug target for depression. Here, we discovered a series of Kir4.1 inhibitors through high-throughput screening. Lys05, the most potent one thus far, effectively suppressed native Kir4.1 channels while displaying high selectivity against established targets for rapid-onset antidepressants. Cryogenic-electron microscopy structures combined with electrophysiological characterizations revealed Lys05 directly binds in the central cavity of Kir4.1. Notably, a single dose of Lys05 reversed the Kir4.1-driven depression-like phenotype and exerted rapid-onset (as early as 1 hour) antidepressant actions in multiple canonical depression rodent models with efficacy comparable to that of (S)-ketamine. Overall, we provided a proof of concept that Kir4.1 is a promising target for rapid-onset antidepressant effects.

Structural basis of ligand recognition and self-activation of orphan GPR52.

GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.

Structural basis of GPBAR activation and bile acid recognition.

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver-bile acid-microbiota-metabolism axis1-3. Here we report the cryo-electron microscopy structures of GPBAR-Gs complexes stabilized by either the high-affinity P3954 or the semisynthesized bile acid derivative INT-7771,3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the Gs-coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily.

Assembly and comparative analysis of the complete mitochondrial genome of Brassica rapa var. Purpuraria.

BACKGROUND: Purple flowering stalk (Brassica rapa var. purpuraria) is a widely cultivated plant with high nutritional and medicinal value and exhibiting strong adaptability during growing. Mitochondrial (mt) play important role in plant cells for energy production, developing with an independent genetic system. Therefore, it is meaningful to assemble and annotate the functions for the mt genome of plants independently. Though there have been several reports referring the mt genome of in Brassica species, the genome of mt in B. rapa var. purpuraria and its functional gene variations when compared to its closely related species has not yet been addressed. RESULTS: The mt genome of B. rapa var. purpuraria was assembled through the Illumina and Nanopore sequencing platforms, which revealed a length of 219,775 bp with a typical circular structure. The base composition of the whole B. rapa var. purpuraria mt genome revealed A (27.45%), T (27.31%), C (22.91%), and G (22.32%). 59 functional genes, composing of 33 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes, were annotated. The sequence repeats, codon usage, RNA editing, nucleotide diversity and gene transfer between the cp genome and mt genome were examined in the B. rapa var. purpuraria mt genome. Phylogenetic analysis show that B. rapa var. Purpuraria was closely related to B. rapa subsp. Oleifera and B. juncea. Ka/Ks analysis reflected that most of the PCGs in the B. rapa var. Purpuraria were negatively selected, illustrating that those mt genes were conserved during evolution. CONCLUSIONS: The results of our findings provide valuable information on the B.rapa var. Purpuraria genome, which might facilitate molecular breeding, genetic variation and evolutionary researches for Brassica species in the future.

Reactive oxygen species (ROS) modulate nitrogen signaling using temporal transcriptome analysis in foxtail millet.

Reactive oxygen species (ROS) is a chemically reactive chemical substance containing oxygen and a natural by-product of normal oxygen metabolism. Excessive ROS affect the growth process of crops, which will lead to the decrease of yield. Nitrogen, as a critical nutrient element in plants and plays a vital role in plant growth and crop production. Nitrate is the primary nitrogen source available to plants in agricultural soil and various natural environments. However, the molecular mechanism of ROS-nitrate crosstalk is still unclear. In this study, we used the foxtail millet (Setaria italica L.) as the material to figure it out. Here, we show that excessive NaCl inhibits nitrate-promoted plant growth and nitrogen use efficiency (NUE). NaCl induces ROS accumulation in roots, and ROS inhibits nitrate-induced gene expression in a short time. Surprisingly, low concentration ROS slight promotes and high concentration of ROS inhibits foxtail millet growth under long-term H2O2 treatment. These results may open a new perspective for further exploration of ROS-nitrate signaling pathway in plants.

The interplay of miRNAs and ferroptosis in diseases related to iron overload.

Ferroptosis has been conceptualized as a novel cell death modality distinct from apoptosis, necroptosis, pyroptosis and autophagic cell death. The sensitivity of cellular ferroptosis is regulated at multiple layers, including polyunsaturated fatty acid metabolism, glutathione-GPX4 axis, iron homeostasis, mitochondria and other parallel pathways. In addition, microRNAs (miRNAs) have been implicated in modulating ferroptosis susceptibility through targeting different players involved in the execution or avoidance of ferroptosis. A growing body of evidence pinpoints the deregulation of miRNA-regulated ferroptosis as a critical factor in the development and progression of various pathophysiological conditions related to iron overload. The revelation of mechanisms of miRNA-dependent ferroptosis provides novel insights into the etiology of diseases and offers opportunities for therapeutic intervention. In this review, we discuss the interplay of emerging miRNA regulators and ferroptosis players under different pathological conditions, such as cancers, ischemia/reperfusion, neurodegenerative diseases, acute kidney injury and cardiomyopathy. We emphasize on the relevance of miRNA-regulated ferroptosis to disease progression and the targetability for therapeutic interventions.

Involvement of Fgf2-mediated tau protein phosphorylation in cognitive deficits induced by sevoflurane in aged rats.

OBJECTIVE: Anesthetics have been linked to cognitive alterations, particularly in the elderly. The current research delineates how Fibroblast Growth Factor 2 (Fgf2) modulates tau protein phosphorylation, contributing to cognitive impairments in aged rats upon sevoflurane administration. METHODS: Rats aged 3, 12, and 18 months were subjected to a 2.5% sevoflurane exposure to form a neurotoxicity model. Cognitive performance was gauged, and the GEO database was employed to identify differentially expressed genes (DEGs) in the 18-month-old cohort post sevoflurane exposure. Bioinformatics tools, inclusive of STRING and GeneCards, facilitated detailed analysis. Experimental validations, both in vivo and in vitro, examined Fgf2's effect on tau phosphorylation. RESULTS: Sevoflurane notably altered cognitive behavior in older rats. Out of 128 DEGs discerned, Fgf2 stood out as instrumental in regulating tau protein phosphorylation. Sevoflurane exposure spiked Fgf2 expression in cortical neurons, intensifying tau phosphorylation via the PI3K/AKT/Gsk3b trajectory. Diminishing Fgf2 expression correspondingly curtailed tau phosphorylation, neurofibrillary tangles, and enhanced cognitive capacities in aged rats. CONCLUSION: Sevoflurane elicits a surge in Fgf2 expression in aging rats, directing tau protein phosphorylation through the PI3K/AKT/Gsk3b route, instigating cognitive aberrations.

Convenient Size Analysis of Nanoplastics on a Microelectrode.

Chemical recycling is a promising approach to reduce plastic pollution. Timely and accurate size analysis of produced nanoplastics is necessary to monitor the process and assess the quality of chemical recycling. In this work, a sandwich-type microelectrode sensor was developed for the size assessment of nanoplastics. ß-Mercaptoethylamine was modified on the microelectrode to enhance its surface positive charge density. Polystyrene (PS) nanoplastics were captured on the sensor through electrostatic interactions. Ferrocene was used as an electrochemical beacon and attached to PS via hydrophobic interactions. The results show a nonlinear dependence of the sensor's current response on the PS particle size. The size resolving ability of the microelectrode is mainly attributed to the small size of the electrode and the resulting attenuation of the electric field strength. For mixed samples with different particle sizes, this method can provide accurate average particle sizes. Through an effective pretreatment process, the method can be applied to PS nanoplastics with different surface properties, ensuring its application in evaluating different chemical recycling methods.

Synergistic Interfacial Effect of Ru/Co3O4 Heterojunctions for Boosting Overall Water Splitting.

Low-cost bifunctional electrocatalysts capable of efficiently driving the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) are needed for the growth of a green hydrogen economy. Herein, a Ru/Co3O4 heterojunction catalyst rich in oxygen vacancies (VO) and supported on carbon cloth (RCO-VO@CC) is prepared via a solid phase reaction (SPR) strategy. A RuO2/Co9S8@CC precursor (ROC@CC) is first prepared by loading Co9S8 nanosheets onto CC, following the addition of RuO2 nanoparticles (NPs). After the SPR process in an Ar atmosphere, Ru/Co3O4 heterojunctions with abundant VO are formed on the CC. The compositionally optimized RCO-VO@CC electrocatalyst with a Ru content of 0.55 wt.% exhibits very low overpotential values of 11 and 253 mV at 10 mA cm-2 for HER and OER, respectively, in 1 m KOH. Further, a low cell voltage of only 1.49 V is required to achieve a current density of 10 mA cm-2. Density functional theoretical calculations verify that the outstanding bifunctional electrocatalytic performance originates from synergistic charge transfer between Ru metal and VO-rich Co3O4. This work reports a novel approach toward a high-efficiency HER/OER electrocatalyst for energy storage and conversion.

Microbially Inspired Calcium Carbonate Precipitation Pathway Integrated Polyelectrolyte Capsules (MICPC) for Biomolecules Release.

Complexation between oppositely charged polyelectrolytes offers a facile single-step strategy for assembling functional micro-nano carriers for efficient drug and vaccine delivery. However, the stability of the delivery system within the physiological environment is compromised due to the swelling of the polyelectrolyte complex, driven by the charge shielding effect, and consequently leads to uncontrollable burst release, thereby limiting its potential applications. In a pioneering approach, cellular pathway-inspired calcium carbonate precipitation pathways are developed that are integrated into polyelectrolyte capsules (MICPC). These innovative capsules are fabricated at the interface of all-aqueous microfluidic droplets, resulting in a precisely controllable and sustained release profile in physiological conditions. Unlike single-step polyelectrolyte assembly capsules which always perform rapid burst release, the MICPC exhibits a sustainable and tunable release pattern, releasing biomolecules at an average rate of 3-10% per day. Remarkably, the degree of control over MICPC's release kinetics can be finely tuned by adjusting the quantity of synthesized calcium carbonate particles within the polyelectrolyte complex. This groundbreaking work not only deepens the insights into polyelectrolyte complexation but also significantly enhances the overall stability of these complexes, opening up new avenues for expanding the range of applications involving polyelectrolyte complex-related materials.

Cross-reaction mediated by distinct key amino acid combinations in the complementary-determining region (CDR) of a monoclonal antibody.

In immunology, cross-reaction between antigens and antibodies are commonly observed. Prior research has shown that various monoclonal antibodies (mAbs) can recognize a broad spectrum of epitopes related to influenza viruses. However, existing theories on cross-reactions fall short in explaining the phenomena observed. This study explored the interaction characteristics of H1-74 mAb with three peptides: two natural peptides, LVLWGIHHP and LPFQNI, derived from the hemagglutinin (HA) antigen of the H1N1 influenza virus, and one synthetic peptide, WPFQNY. Our findings indicate that the complementarity-determining region (CDR) of H1-74 mAb comprised five antigen-binding sites, containing eight key amino acid residues from the light chain variable region and 16 from the heavy chain variable region. These critical residues formed distinct hydrophobic or hydrophilic clusters and functional groups within the binding sites, facilitating interaction with antigen epitopes through hydrogen bonding, salt bridge formation, and π-π stacking. The study revealed that the formation of the antibody molecule led to the creation of binding groups and small units in the CDR, allowing the antibody to attach to a variety of antigen epitopes through diverse combinations of these small units and functional groups. This unique ability of the antibody to bind with antigen epitopes provides a new molecular basis for explaining the phenomenon of antibody cross-reaction.

Immunomodulation of cuproptosis and ferroptosis in liver cancer.

According to statistics, the incidence of liver cancer is increasing yearly, and effective treatment of liver cancer is imminent. For early liver cancer, resection surgery is currently the most effective treatment. However, resection does not treat the disease in advanced patients, so finding a method with a better prognosis is necessary. In recent years, ferroptosis and cuproptosis have been gradually defined, and related studies have proved that they show excellent results in the therapy of liver cancer. Cuproptosis is a new form of cell death, and the use of cuproptosis combined with ferroptosis to inhibit the production of hepatocellular carcinoma cells has good development prospects and is worthy of in-depth discussion by researchers. In this review, we summarize the research progress on cuproptosis combined with ferroptosis in treating liver cancer, analyze the value of cuproptosis and ferroptosis in the immune of liver cancer, and propose potential pathways in oncotherapy with the combination of cuproptosis and ferroptosis, which can provide background knowledge for subsequent related research.