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.

Overexpression of tonoplast Ca2+-ATPase in guard cells synergistically enhances stomatal opening and drought tolerance.

Stomata play a crucial role in plants by controlling water status and responding to drought stress. However, simultaneously improving stomatal opening and drought tolerance has proven to be a significant challenge. To address this issue, we employed the OnGuard quantitative model, which accurately represents the mechanics and coordination of ion transporters in guard cells. With the guidance of OnGuard, we successfully engineered plants that overexpressed the main tonoplast Ca2+-ATPase gene, ACA11, which promotes stomatal opening and enhances plant growth. Surprisingly, these transgenic plants also exhibited improved drought tolerance due to reduced water loss through their stomata. Again, OnGuard assisted us in understanding the mechanism behind the unexpected stomatal behaviors observed in the ACA11 overexpressing plants. Our study revealed that the overexpression of ACA11 facilitated the accumulation of Ca2+ in the vacuole, thereby influencing Ca2+ storage and leading to an enhanced Ca2+ elevation in response to abscisic acid. This regulatory cascade finely tunes stomatal responses, ultimately leading to enhanced drought tolerance. Our findings underscore the importance of tonoplast Ca2+-ATPase in manipulating stomatal behavior and improving drought tolerance. Furthermore, these results highlight the diverse functions of tonoplast-localized ACA11 in response to different conditions, emphasizing its potential for future applications in plant enhancement.

The orphan G protein-coupled receptor GPR149 is a negative regulator of myelination and remyelination.

Myelin sheath, formed by oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells in periphery, plays a critical role in supporting neuronal functions. OLs, differentiated from oligodendrocyte precursor cells (OPCs), are important for myelination during development and myelin repair in CNS demyelinating disease. To identify mechanisms of myelin development and remyelination after myelin damage is of great clinical interest. Here we show that the orphan G protein-coupled receptor GPR149, enriched in OPCs, negatively regulate OPC to OL differentiation, myelination, as well as remyelination. The expression of GPR149 is downregulated during OPCs differentiation into OLs. GPR149 deficiency does not affect the number of OPCs, but promotes OPC to OL differentiation which results in earlier development of myelin. In cuprizone-induced demyelination model, GPR149 deficiency significantly enhances myelin regeneration. Further study indicates that GPR149 may regulate OL differentiation and myelin formation via MAPK/ERK pathway. Our study suggests that deleting or blocking GPR149 might be an intriguing way to promote myelin repair in demyelinating diseases.

Inhibition of MAPK/ERK pathway promotes oligodendrocytes generation and recovery of demyelinating diseases.

Oligodendrocytes (OLs) are the myelinating glia of the central nervous system. Injury to OLs causes myelin loss. In demyelinating diseases, such as multiple sclerosis, the remyelination is hindered principally due to a failure of the oligodendrocyte precursor cells (OPCs) to differentiate into mature OLs. To identify inducers of OPC to OL differentiation, a high-throughput screening based on myelin basic protein expression using neural progenitor cells-derived OPCs has been performed and, PD0325901-an MEK (MAPK kinase) inhibitor-is found to significantly enhance OPC to OL differentiation in a dose- and time-dependent manner. Other MEK inhibitors also display similar effect, indicating blockade of MAPK-ERK signaling is sufficient to induce OPC differentiation into OLs. PD0325901 facilitates the formation of myelin sheaths in OPC-neuron co-culture in vitro. And in experimental autoimmune encephalomyelitis model and cuprizone-induced demyelination model, PD0325901 displays significant therapeutic effect by promoting myelin regeneration. Our results suggest that targeting the MAPK-ERK pathway might be an intriguing way to develop new therapies for demyelinating diseases.

Targeting Gpr52 lowers mutant HTT levels and rescues Huntington's disease-associated phenotypes.

See Huang and Gitler (doi:10.1093/brain/awy112) for a scientific commentary on this article.Lowering the levels of disease-causing proteins is an attractive treatment strategy for neurodegenerative disorders, among which Huntington's disease is an appealing disease for testing this strategy because of its monogenetic nature. Huntington's disease is mainly caused by cytotoxicity of the mutant HTT protein with an expanded polyglutamine repeat tract. Lowering the soluble mutant HTT may reduce its downstream toxicity and provide potential treatment for Huntington's disease. This is hard to achieve by small-molecule compound drugs because of a lack of effective targets. Here we demonstrate Gpr52, an orphan G protein-coupled receptor, as a potential Huntington's disease drug target. Knocking-out Gpr52 significantly reduces mutant HTT levels in the striatum and rescues Huntington's disease-associated behavioural phenotypes in a knock-in Huntington's disease mouse model expressing endogenous mutant Htt. Importantly, a novel Gpr52 antagonist E7 reduces mutant HTT levels and rescues Huntington's disease-associated phenotypes in cellular and mouse models. Our study provides an entry point for Huntington's disease drug discovery by targeting Gpr52.

Interpretable machine learning models for predicting in-hospital death in patients in the intensive care unit with cerebral infarction.

BACKGROUND: Research on patients with cerebral infarction in the Intensive Care Unit (ICU) is still lacking. Our study aims to develop and validate multiple machine-learning (ML) models using two large ICU databases-Medical Information Mart for Intensive Care version III (MIMIC-III) and eICU Research Institute Database (eRI)-to guide clinical practice. METHODS: We collected clinical data from patients with cerebral infarction in the MIMIC-III and eRI databases within 24 h of admission. The opinion of neurologists and the Least Absolute Shrinkage and Selection Operator regression was used to screen for relevant clinical features. Using eRI as the training set and MIMIC-III as the test set, we developed and validated six ML models. Based on the results of the model validation, we select the best model and perform the interpretability analysis on it. RESULTS: A total of 4,338 patients were included in the study (eRI:3002, MIMIC-III:1336), resulting in a total of 18 clinical characteristics through screening. Model validation results showed that random forest (RF) was the best model, with AUC and F1 scores of 0.799 and 0.417 in internal validation and 0.733 and 0.498 in external validation, respectively; moreover, its sensitivity and recall were the highest of the six algorithms for both the internal and external validation. The explanatory analysis of the model showed that the three most important variables in the RF model were Acute Physiology Score-III, Glasgow Coma Scale score, and heart rate, and that the influence of each variable on the judgement of the model was consistent with medical knowledge. CONCLUSION: Based on a large sample of patients and advanced algorithms, our study bridges the limitations of studies on this area. With our model, physicians can use the admission information of cerebral infarction patients in the ICU to identify high-risk groups among them who are prone to in-hospital death, so that they could be more alert to this group of patients and upgrade medical measures early to minimize the mortality of patients.

Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation.

Background & Aims: Hepatocyte transplantation has emerged as a possible treatment option for end-stage liver disease. However, an important obstacle to therapeutic success is the low level of engraftment and proliferation of transplanted hepatocytes, which do not survive long enough to exert therapeutic effects. Thus, we aimed to explore the mechanisms of hepatocyte proliferation in vivo and find a way to promote the growth of transplanted hepatocytes. Methods: Hepatocyte transplantation was performed in Fah -/- mice to explore the mechanisms of hepatocyte proliferation in vivo. Guided by in vivo regeneration mechanisms, we identified compounds that promote hepatocyte proliferation in vitro. The in vivo effects of these compounds on transplanted hepatocytes were then evaluated. Results: The transplanted mature hepatocytes were found to dedifferentiate into hepatic progenitor cells (HPCs), which proliferate and then convert back to a mature state at the completion of liver repopulation. The combination of two small molecules Y-27632 (Y, ROCK inhibitor) and CHIR99021 (C, Wnt agonist) could convert mouse primary hepatocytes into HPCs, which could be passaged for more than 30 passages in vitro. Moreover, YC could stimulate the proliferation of transplanted hepatocytes in Fah -/- livers by promoting their conversion into HPCs. Netarsudil (N) and LY2090314 (L), two clinically used drugs which target the same pathways as YC, could also promote hepatocyte proliferation in vitro and in vivo, by facilitating HPC conversion. Conclusions: Our work suggests drugs promoting hepatocyte dedifferentiation may facilitate the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy. Impact and implications: Hepatocyte transplantation may be a treatment option for patients with end-stage liver disease. However, one important obstacle to hepatocyte therapy is the low level of engraftment and proliferation of the transplanted hepatocytes. Herein, we show that small molecule compounds which promote hepatocyte proliferation in vitro by facilitating dedifferentiation, could promote the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy.

Spermine alleviates experimental autoimmune encephalomyelitis via regulating T cell activation and differentiation.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease which causes demyelination, axonal damage and even disability. Th1 and Th17 cells, more precisely, the IFNγ/IL17a double producing CD4+ T cells, have been known to play critical roles in the pathogenesis of MS and EAE, a mouse model of MS. Polyamines not only regulate the immune system, but also are essential for the normal function of the central nervous system (CNS). In this study, we demonstrate that the supplementation of spermine (SPM), a biogenic polyamine, significantly suppresses EAE progression in both preventative and therapeutic ways. Further study suggests that spermine significantly reduces IFNγ+/IL17a-, IFNγ-/IL17a+ and IFNγ+/IL17a+ cells in periphery, and thus reducing the infiltration of these pathogenic cells into the CNS. In vitro, spermine has been shown to suppress the activation and proliferation of CD4+ T cells and also significantly impede the polarization of T effector cells in a dose-dependent manner, accompanied by the inhibition of ERK phosphorylation. Consistently, a number of MEK/ERK inhibitors (including PD0325901, FR180204 and selumetinib) have been found to mimic the effects of spermine in inhibiting CD4+ T cell activation and T effector cell differentiation. Collectively, spermine alleviates EAE progression by inhibiting CD4+ T cells activation and T effector cell differentiation in a MAPK/ERK-dependent manner, suggesting this pathway might be a target to develop effective therapies for MS.

A Genome-Scale Metabolic Model of Methanoperedens nitroreducens: Assessing Bioenergetics and Thermodynamic Feasibility.

Methane is an abundant low-carbon fuel that provides a valuable energy resource, but it is also a potent greenhouse gas. Therefore, anaerobic oxidation of methane (AOM) is an essential process with central features in controlling the carbon cycle. Candidatus 'Methanoperedens nitroreducens' (M. nitroreducens) is a recently discovered methanotrophic archaeon capable of performing AOM via a reverse methanogenesis pathway utilizing nitrate as the terminal electron acceptor. Recently, reverse methanogenic pathways and energy metabolism among anaerobic methane-oxidizing archaea (ANME) have gained significant interest. However, the energetics and the mechanism for electron transport in nitrate-dependent AOM performed by M. nitroreducens is unclear. This paper presents a genome-scale metabolic model of M. nitroreducens, iMN22HE, which contains 813 reactions and 684 metabolites. The model describes its cellular metabolism and can quantitatively predict its growth phenotypes. The essentiality of the cytoplasmic heterodisulfide reductase HdrABC in the reverse methanogenesis pathway is examined by modeling the electron transfer direction and the specific energy-coupling mechanism. Furthermore, based on better understanding electron transport by modeling, a new energy transfer mechanism is suggested. The new mechanism involves reactions capable of driving the endergonic reactions in nitrate-dependent AOM, including the step reactions in reverse canonical methanogenesis and the novel electron-confurcating reaction HdrABC. The genome metabolic model not only provides an in silico tool for understanding the fundamental metabolism of ANME but also helps to better understand the reverse methanogenesis energetics and its thermodynamic feasibility.

The Aurora Kinase Inhibitor CYC116 Promotes the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in applications such as regenerative medicine, cardiac disease modeling, and in vitro drug evaluation. However, hPSC-CMs are immature, which limits their applications. During development, the maturation of CMs is accompanied by a decline in their proliferative capacity. This phenomenon suggests that regulating the cell cycle may facilitate the maturation of hPSC-CMs. Aurora kinases are essential kinases that regulate the cell cycle, the role of which is not well studied in hPSC-CM maturation. Here, we demonstrate that CYC116, an inhibitor of Aurora kinases, significantly promotes the maturation of CMs derived from both human embryonic stem cells (H1 and H9) and iPSCs (induced PSCs) (UC013), resulting in increased expression of genes related to cardiomyocyte function, better organization of the sarcomere, increased sarcomere length, increased number of mitochondria, and enhanced physiological function of the cells. In addition, a number of other Aurora kinase inhibitors have also been found to promote the maturation of hPSC-CMs. Our data suggest that blocking aurora kinase activity and regulating cell cycle progression may promote the maturation of hPSC-CMs.

Response of the Anaerobic Methanotrophic Archaeon Candidatus "Methanoperedens nitroreducens" to the Long-Term Ferrihydrite Amendment.

Anaerobic methanotrophic (ANME) archaea can drive anaerobic oxidation of methane (AOM) using solid iron or manganese oxides as the electron acceptors, hypothetically via direct extracellular electron transfer (EET). This study investigated the response of Candidatus "Methanoperedens nitroreducens TS" (type strain), an ANME archaeon previously characterized to perform nitrate-dependent AOM, to an Fe(III)-amended condition over a prolonged period. Simultaneous consumption of methane and production of dissolved Fe(II) were observed for more than 500 days in the presence of Ca. "M. nitroreducens TS," indicating that this archaeon can carry out Fe(III)-dependent AOM for a long period. Ca. "M. nitroreducens TS" possesses multiple multiheme c-type cytochromes (MHCs), suggesting that it may have the capability to reduce Fe(III) via EET. Intriguingly, most of these MHCs are orthologous to those identified in Candidatus "Methanoperedens ferrireducens," an Fe(III)-reducing ANME archaeon. In contrast, the population of Ca. "M. nitroreducens TS" declined and was eventually replaced by Ca. "M. ferrireducens," implying niche differentiation between these two ANME archaea in the environment.

Effects of Short-Term Unsupervised Exercise, Based on Smart Bracelet Monitoring, on Body Composition in Patients Recovering from Breast Cancer.

OBJECTIVE: We aimed to investigate the effects of exercise, monitored and managed using smart bracelets, on body composition, and quality of life in breast cancer survivors. METHODS: A before-and-after study was conducted in 109 patients who were in the recovery phase of breast cancer and attended the Breast Surgery Department of the Cancer Hospital of Fudan University up to December 2017. Patients were advised to adhere to at least 150 minutes of moderate-intensity exercise per week, and a smart bracelet was issued to each participant to record their daily exercise data for 3 months. Bioelectrical impedance analysis was used to observe the effects of short-term unsupervised exercise intervention on body composition in patients recovering from breast cancer. Patients completed the Functional Assessment of Cancer Therapy-Breast to assess health-related quality of life. RESULTS: Weight, body mass index (BMI), body fat mass (BFM), fat mass index (FMI), percent body fat (PBF), arm circumference (AC), arm muscle circumference (AMC), and visceral fat area (VFA) were lower than baseline after exercising for 3 months based on data from the wearable devices (P < .05). The only significant improvement was found in the "additional concerns about breast cancer" category among the quality-of-life assessments (P < .05). The average walking time was negatively associated with BFM, PBF, and FMI, while the average calorie consumption due to running was positively associated with fat free mass (FFM). CONCLUSION: In this study, we demonstrated that short-term exercise may be beneficial for postoperative breast cancer survivors. A wearable device could help patients track physical data easily and promote a healthier and more positive life.

Guard cell endomembrane Ca2+-ATPases underpin a 'carbon memory' of photosynthetic assimilation that impacts on water-use efficiency.

Stomata of most plants close to preserve water when the demand for CO2 by photosynthesis is reduced. Stomatal responses are slow compared with photosynthesis, and this kinetic difference erodes assimilation and water-use efficiency under fluctuating light. Despite a deep knowledge of guard cells that regulate the stoma, efforts to enhance stomatal kinetics are limited by our understanding of its control by foliar CO2. Guided by mechanistic modelling that incorporates foliar CO2 diffusion and mesophyll photosynthesis, here we uncover a central role for endomembrane Ca2+ stores in guard cell responsiveness to fluctuating light and CO2. Modelling predicted and experiments demonstrated a delay in Ca2+ cycling that was enhanced by endomembrane Ca2+-ATPase mutants, altering stomatal conductance and reducing assimilation and water-use efficiency. Our findings illustrate the power of modelling to bridge the gap from the guard cell to whole-plant photosynthesis, and they demonstrate an unforeseen latency, or 'carbon memory', of guard cells that affects stomatal dynamics, photosynthesis and water-use efficiency.

Optimized Protocol for OnGuard2 Software in Studying Guard Cell Membrane Transport and Stomatal Physiology.

Stomata are key innovation in plants that drives the global carbon and water cycle. In the past few decades, many stomatal models have been developed for studying gas exchange, photosynthesis, and transpirational characteristics of plants, but they provide limited information on stomatal mechanisms at the molecular and cellular levels. Quantitative mathematical modeling offers an effective in silico approach to explore the link between microscopic transporter functioning and the macroscopic stomatal characteristics. As a first step, a dynamic system model based on the guard cell membrane transport system was developed and encoded in the OnGuard software. This software has already generated a wealth of testable predictions and outcomes sufficient to guide phenotypic and mutational studies. It has a user-friendly interface, which can be easily accessed by researchers to manipulate the key elements and parameters in the system for guard cell simulation in plants. To promote the adoption of this OnGuard application, here we outline a standard protocol that will enable users with experience in basic plant physiology, cell biology, and membrane transport to advance quickly in learning to use it.

Foccα6, a truncated nAChR subunit, positively correlates with spinosad resistance in the western flower thrips, Frankliniella occidentalis (Pergande).

Nicotinic acetylcholine receptors (nAChRs), a molecular target for spinosyns and neonicotinoids, mediate rapid cholinergic transmission in insect central nervous system by binding acetylcholine. Previous studies have shown that mutations in nAChRs contribute to the high level of resistance to these two classes of insecticides. In this study, we identified nine nAChR subunits from a transcriptome of the western flower thrips, Frankliniella occidentalis, including α1-7, ß1, and ß2. Exon 4 of α4 and exons 3 and 8 of α6 each have two splicing variants, respectively. In addition, altered or incorrect splicing leads to truncated forms of α3, α5, and α6 subunits. The abundance of every nAChRs in both spinosad susceptible and resistant strains was highest in the 1st instar nymph. Significantly more truncated forms of α6 subunit were detected in spinosad resistant strains, whereas, hardly any full-length form was found in the two highly resistant F. occidentalis strains (resistance ratio >104-fold). Under laboratory conditions, spinosad resistance was positively correlated with truncated α6 transcripts. The correlation was later confirmed under the field conditions using five field strains. As the molecular target of spinosad, the percentage of truncated nAChR α6 subunits can be used as a diagnostic tool to detect and quantify spinosad resistance in the field.

Development of Near-Isogenic Lines in a Parthenogenetically Reproduced Thrips Species, Frankliniella occidentalis.

Although near-isogenic lines (NILs) can standardize genetic backgrounds among individuals, it has never been applied in parthenogenetically reproduced animals. Here, through multiple rounds of backcrossing and spinosad screening, we generated spinosad resistant NILs in the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), with a haplo-diploid reproduction system. The resultant F. occidentalis NIL-R strain maintained a resistance ratio over 30,000-fold, which was comparable to its parental resistant strain, Spin-R. More importantly, F. occidentalis NIL-R shared 98.90% genetic similarity with its susceptible parental strain Ivf03. By developing this toolset, we are able to segregate individual resistance and facilitate the mechanistic study of insecticide resistances in phloem-feeding arthropods, a group of devastating pest species reproducing sexually as well as asexually.