Mechanism of NMDA receptor channel block by MK-801 and memantine.

The NMDA (N-methyl-D-aspartate) receptor transduces the binding of glutamate and glycine, coupling it to the opening of a calcium-permeable ion channel 1 . Owing to the lack of high-resolution structural studies of the NMDA receptor, the mechanism by which ion-channel blockers occlude ion permeation is not well understood. Here we show that removal of the amino-terminal domains from the GluN1-GluN2B NMDA receptor yields a functional receptor and crystals with good diffraction properties, allowing us to map the binding site of the NMDA receptor blocker, MK-801. This crystal structure, together with long-timescale molecular dynamics simulations, shows how MK-801 and memantine (a drug approved for the treatment of Alzheimer's disease) bind within the vestibule of the ion channel, promote closure of the ion channel gate and lodge between the M3-helix-bundle crossing and the M2-pore loops, physically blocking ion permeation.

Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding.

STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved aa 153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of posttranslational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than serine 358. This study provides a glimpse into the unique architecture of STING and sheds light on the mechanism of c-di-GMP-mediated TBK1 signaling.

NMDA receptor structures reveal subunit arrangement and pore architecture.

N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

DNMT1-mediated epigenetic suppression of FBXO32 expression promoting cyclin dependent kinase 9 (CDK9) survival and esophageal cancer cell growth.

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.

Landscape changes and livelihood outcomes in rural tea farming communities: A case study in Fuding City, Fujian Province, Southeast China.

Landscape changes driven by cash crop plantations have been prevalent in tropical and subtropical regions worldwide in recent decades. Investigating the landscape changes and concluding livelihood outcomes are fundamental to figure out the solutions for rural sustainability. This paper examined the landscape changes which was caused by land use changes in tea plantations as well as investigated the resultant livelihood impacts, based on a case study in Fuding City, Southeast China. A questionnaire survey of 114 rural households in four sampled villages was conducted. Results demonstrated that expansion and intensification of tea plantations were two major proximate causes of landscape changes in recent decade. Our survey indicated that some existing intensively-managed tea plantations had derived from intensification and expansion of tea plantations, respectively. We identified four underlying driving forces of landscape changes, including economic benefit, governmental policies, wildlife destruction on grain crops, and rural return migration. Our study confirmed that landscape changes have significant positive effects on farmers' livelihoods, including increasing employment and incomes, raising living standards, enhancing livelihood assets and livelihood sustainability. Especially, the aged rural populations could have a relatively decent living standard. Meanwhile, the excessive expansion of tea plantations may impair livelihood resilience. Lastly, three policy suggestions based on different time scales have been put forward to promote rural households' livelihood sustainability and resilience.

A dual-channel visual sensing system for recognition of multiple metal ions.

Here, we propose a simple, rapid, and effective colorimetric sensor array for discrimination of metal ions. The sensor array was constructed using two sensing channels, i.e., gold nanoparticles (AuNPs)- Tetramethylbenzidine (TMB)-H2O2 and AuNPs-O-phenylenediamine (OPD)-H2O2 reaction systems. The presence of metal ions with positive charges would lead to the corresponding surface charge change of negatively charged AuNPs, resulting in diverse catalytic performances of citrate-modified AuNPs, accompanied by a substantial colorimetric performance of oxidation products of TMB and OPD. Employing the diversity of colorimetric responses of metal ions to the two sensing channels, nine metal ions including Cr3+, Fe3+, Cu2+, Co2+, Ni2+, Pb2+, Mg2+, K+, and Cd2+ were well distinguished with a discrimination accuracy of 100% at a concentration as low as 50 nM. Further experiment showed that the sensor array was also capable of discriminating and quantifying metal ions at various concentrations, as well as the identification of metal ion mixtures. The feasibility of the sensor array was also verified by the successful identification of the nine metal ions in river water samples.

A colorimetric sensing probe for chromium (III) ion based on domino like reaction.

In this work, a gold nanobipyramid@Ag nanorod (AuNBP@Ag NR)-based sensor platform was developed for the quantitative, visual, and sensitive detection of Cr3+ ions in aqueous solutions. This assay provides quantitative detection of Cr3+, which relies on the absorbance change of AuNBP@Ag NRs due to morphological change of the AuNBP@Ag NRs induced by Cr3+. When AuNBP@Ag NRs and Cr3+ mix, the coordination reaction of the carboxyl groups of citrate and Cr3+ occurs, which leads to the collapse of Ag shell nanorods, similar to the domino effect, and obvious color changes from yellow to pink can be observed by the naked eye. When combined with UV-vis spectrophotometer-based colorimetric detection, a detection limit of 8.7 nM for Cr3+ in ultrapure water was achieved. With the advantages of high sensitivity, selectivity, and performance, we anticipate that the sensor will be helpful for the on-site, quantitative detection of Cr3+ ions in water samples.

Dynamic livelihood impacts of COVID-19 on different rural households in mountainous areas of China.

BACKGROUND: The outbreak of COVID-19 pandemic has brought about severe negative livelihood consequences for rural households worldwide. However, the heterogeneity and dynamics of livelihood impacts have been under-researched. There is also lacking a livelihood assessment of the pandemic based on a whole pandemic cycle. This study aimed to investigate the dynamic and heterogeneous livelihood impacts of COVID-19 pandemic for rural households in 2020 based on a case study of Southeast China. METHODS: The pandemic in China had experienced a complete cycle from initial outbreak, to intermediate recovery and finally new normal stage in 2020. We conducted face-to-face interviews with 95 rural households randomly drawn from 2 rural villages in Xunwu County, Jiangxi Province, Southeast China. The sampled households are interviewed with a questionnaire through face-to-face surveys in February and March, 2021 to evaluate the overall livelihood impacts of the pandemic during 2020. The survey collected data on demographic and economic characteristics, governmental control measures, and effects of the COVID-19 on agricultural production, employment, income, education, and daily life. In-depth interviews are also conducted to clarify the livelihood impacts of COVID-19 on villages. RESULTS: Results showed that the pandemic tremendously caused substantially negative livelihood impacts, including decreasing household income, and disorders in daily lives. The average income loss of all survey households is 6,842 RMB, accounting for 13.01% of the total household income in 2020. Containment measures also resulted in a series of disturbances in daily lives, such as rising food price additional expenditures, travel restrictions, party restrictions, closure of schools and deceasing living standards. There is remarkable household heterogeneity in the livelihood impacts. Results also revealed that the livelihood strategies of rural households to cope with the threat of COVID-19 were different in various pandemic stages. CONCLUSION: Our findings have illustrated the severity and heterogeneity of livelihood impacts on rural households induced by COVID-19 pandemic. The dynamics of livelihood impacts is also highlighted in the study. Several policy suggestion was proposed to mitigate these negative consequences of the pandemic.

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor.

Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein-coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs.

Author Correction: Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism.

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.

In-situ stabilisation followed by ex-situ composting for treatment and disposal of heavy metals polluted sediments.

An innovative in-situ stabilisation treatment followed by ex-situ sediment composting was tested for its ability to treat and dispose of heavy-metal-polluted sediments in a river near the Chinese Pearl Delta. First, polluted sediments were treated in-situ to stabilise the heavy metals. Then the treated sediments were dredged, dewatered and sent for high temperature aerobic composting (HTAC) treatment. Finally, the compost products were used as a fertiliser for river bank plants. The stabilisation efficiency of heavy metals during the process was investigated and the results are as follows: (1) using in-situ stabilisation, the extraction concentrations of Cu, Zn and Pb were reduced by 65.0%, 82.2% and 90.0%, respectively, which are much lower than the national standard given in the Identification Standard for Hazardous Waste (GB5085.3-1996); (2) chemical fraction analysis showed that heavy metals were further stabilized during the HTAC treatment; (3) the concentrations of Cu, Zn and Pb in rainwater leachate through the river bank met the level of class V in the Environmental Quality Standards for Surface Water in China (GB3838-2002). Therefore, using this new process, the toxicities of heavy metals in sediments were reduced markedly.

[Effects of airborne pollution on cardiopulmonary function of healthy person].

OBJECTIVE: To understand the effects of airborne pollution on cardiopulmonary function in healthy person. METHODS: 15 healthy workers were selected from heavily polluted area as the experimental group (EG) and 15 healthy workers were selected from relatively clean area as control group (CG). The blood pressure were measured with sphygmomanometer and the vital capacity (VC) were detected with FHL-II type spirometer at rest status. Cardiopulmonary functions in different exercise states were measured by using the cardiopulmonary exercise testing (CPET). RESULTS: It showed that heart rate of EG was more higher than those of the CG at rest status (P < 0.01), and VC was more lower than those of the CG (P < 0.05). While the load increased to AT, the results of VO2, VO2/kg, O2P and METS in CG were more higher than these indexes in EG (P < 0.05, P < 0.01) . While the load increased to VO2 max, the results of VO2 max, VO2 max/kg, O2P, METS and DT in CG were more higher than these indexes in EG (P < 0.01), and the recovery of heart rate in EG was slow in comparison with CG. CONCLUSION: Airborne pollution could have adverse effects on the cardiopulmonary function, reserve ability and function potential. The chronic adverse effects of airborne pollution on the health could be easy to reflect at static state. Therefore CPET could roundly evaluate the damage of airborne pollution to cardiorespiratory function.

Cellular tolerance at the µ-opioid receptor is phosphorylation dependent.

Phosphorylation of the µ-opioid receptor (MOR) is known as a key step in desensitization and internalization but the role in the development of long-term tolerance at the cellular level is not known. Viral expression of wild type (exWT) and mutant MORs, where all phosphorylation sites on the C-terminus (Total Phosphorylation Deficient (TPD)) were mutated to alanine, were examined in locus coeruleus neurons in a MOR knockout rat. Both receptors activated potassium conductance similar to endogenous receptors in wild type animals. The exWT receptors, like endogenous receptors, acutely desensitized, internalized and, after chronic morphine treatment, displayed signs of tolerance. However, TPD receptors did not desensitize or internalize with agonist treatment. In addition the TPD receptors did not develop cellular tolerance following chronic morphine treatment. Thus C-terminal phosphorylation is necessary for the expression of acute desensitization, trafficking and one sign of long-term tolerance to morphine at the cellular level.

The conformational states of talin autoinhibition complex and its activation under forces.

Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement.

Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP ß-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion.

The activation of heterodimeric (α/ß) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin ß cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) - a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic "pull-push" process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic "steric clash" model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.