Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

CAMTA3 repressor destabilization triggers TIR domain protein TN2-mediated autoimmunity in the Arabidopsis exo70B1 mutant.

Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5-TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2-CPK5-CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes.

Structural basis of CXC chemokine receptor 2 activation and signalling.

Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor-chemokine recognition2-4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles.

One-Step Dual-Color Labeling of Viral Envelope and Intraviral Genome with Quantum Dots Harnessing Virus Infection.

Labeling the genome and envelope of a virus with multicolor quantum dots (QDs) simultaneously enables real-time monitoring of viral uncoating and genome release, contributing to our understanding of virus infection mechanisms. However, current labeling techniques require genetic modification, which alters the virus's composition and infectivity. To address this, we utilized the CRISPR/Cas13 system and a bioorthogonal metabolic method to label the Japanese encephalitis virus (JEV) genome and envelopes with different-colored QDs in situ. This technique allows one-step two-color labeling of the viral envelope and intraviral genome with QDs harnessing virus infection. In combination with single-virus tracking, we visualized JEV uncoating and genome release in real time near the endoplasmic reticulum of live cells. This labeling strategy allows for real-time visualization of uncoating and genome release at the single-virus level, and it is expected to advance the study of other viral infection mechanisms.

PC71BM as Morphology Regulator for Highly Efficient Ternary Organic Solar Cells with Bulk Heterojunction or Layer-by-Layer Configuration.

The ternary strategy is one of the effective methods to regulate the morphology of the active layer in organic solar cells (OSCs). In this work, the ternary OSCs with bulk heterojunction (BHJ) or layer-by-layer (LbL) active layers are prepared by using the polymer donor PM6 and the non-fullerene acceptor L8-BO as the main system and the fullerene acceptor PC71BM as the third component. The power conversion efficiencies (PCEs) of BHJ OSCs and LbL OSCs are increased from 17.10% to 18.02% and from 17.20% to 18.20% by introducing PC71BM into the binary active layer, respectively. The in situ UV-vis absorption spectra indicate that the molecular aggregation and crystallization process can be prolonged by introducing PC71BM into the PM6:L8-BO or PM6/L8-BO active layer. The molecular orientation and molecular crystallinity in the active layer are optimized by introducing the PC71BM into the binary BHJ or LbL active layers, which can be confirmed by the experimental results of grazing incidence wide-angle X-ray scattering. This study demonstrates that the third component PC71BM can be used as a morphology regulator to regulate the morphology of BHJ or LbL active layers, thus effectively improving the performance of BHJ and LbL OSCs.

Highly Efficient Organic Solar Cells with the Highly Crystalline Third Component as a Morphology Regulator.

The morphology of the active layer is crucial for highly efficient organic solar cells (OSCs), which can be regulated by selecting a rational third component. In this work, the highly crystalline nonfullerene acceptor BTP-eC9 is selected as the morphology regulator in OSCs with PM6:BTP-BO-4Cl as the main system. The addition of BTP-eC9 can prolong the nucleation and crystallization progress of acceptor and donor molecules, thereby enhancing the order of molecular arrangement. Meanwhile, the nucleation and crystallization time of the donor is earlier than that of the acceptors after introducing BTP-eC9, which is beneficial for obtaining a better vertical structural phase separation. The exciton dissociation, charge transport, and charge collection are promoted effectively by the optimized morphology of the active layer, which improves the short-circuit current density and filling factor. After introducing BTP-eC9, the power conversion efficiencies (PCEs) of the ternary OSCs are improved from 17.31% to 18.15%. The PCE is further improved to 18.39% by introducing gold nanopyramid (Au NBPs) into the hole transport layer to improve photon utilization efficiency. This work indicates that the morphology can be optimized by selecting a highly crystalline third component to regulate the nucleation and crystallization progress of the acceptor and donor molecules.

Brachytherapy in craniopharyngiomas: a systematic review and meta-analysis of long-term follow-up.

OBJECTIVE: Brachytherapy has been indicated as an alternative option for treating cystic craniopharyngiomas (CPs). The potential benefits of brachytherapy for CPs have not yet been clarified. The purpose of this work was to conduct a meta-analysis to analyze the long-term efficacy and adverse reactions profile of brachytherapy for CPs. MATERIALS AND METHODS: The relevant databases were searched to collect the clinical trials on brachytherapy in patients with CPs. Included studies were limited to publications in full manuscript form with at least 5-year median follow-up, and adequate reporting of treatment outcomes and adverse reactions data. Stata 12.0 was used for data analysis. RESULTS: According to the inclusion and exclusion criteria, a total of 6 clinical trials involving 266 patients with CPs were included in this meta-analysis. The minimum average follow-up was 5 years. The results of the meta-analysis showed that 1-year, 2-3 years and 5 years progression free survival rates (PFS) are 75% (95%CI: 66-84%), 62% (95%CI: 52-72%) and 57% (95%CI: 22-92%), respectively. At the last follow-up, less than 16% of patients with visual outcomes worser than baseline in all included studies. While, for endocrine outcomes, less than 32% of patients worser than baseline level. CONCLUSION: In general, based on the above results, brachytherapy should be considered as a good choice for the treatment of CP.

Placental DNA methylation analysis of selective fetal growth restriction in monochorionic twins reveals aberrant methylated CYP11A1 gene for fetal growth restriction.

Fetal growth restriction (FGR) is associated with increased susceptibility to perinatal morbidity and mortality. Evidence suggests that epigenetic changes play critical roles in the regulation of fetal growth. We sought to present a comprehensive analysis of the associations between placental DNA methylation and selective fetal growth restriction (sFGR), which is a severe complication of monochorionic twin pregnancies, characterized by one fetus experiencing restricted growth. Genome-wide methylation analysis was performed on 24 placental samples obtained from 12 monochorionic twins with sFGR (Cohort 1) using Illumina Infinium MethylationEPIC BeadChip. Integrative analysis of our EPIC data and two previous placental methylation studies of sFGR (a total of 30 placental samples from 15 sFGR twins) was used to identify convincing differential promoter methylation. Validation analysis was performed on the placentas from 15 sFGR twins (30 placental samples), 15 FGR singletons, and 14 control singletons (Cohort 2) using pyrosequencing, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry (IHC). A globe shift toward hypomethylation was identified in the placentas of growth-restricted fetuses compared with the placentas of normal fetuses in monochorionic twins, including 5625 hypomethylated CpGs and 452 hypermethylated CpGs, especially in the regions of CpG islands, gene-body and promoters. The analysis of pathways revealed dysregulation primarily in steroid hormone biosynthesis, metabolism, cell adhesion, signaling transduction, and immune response. Integrative analysis revealed a differentially methylated promoter region in the CYP11A1 gene, encoding a rate-limiting enzyme of steroidogenesis converting cholesterol to pregnenolone. The CYP11A1 gene was validated to have hypomethylation and higher mRNA expression in sFGR twins and FGR singletons. In conclusion, our findings suggested that the changes in placental DNA methylation pattern in sFGR may have functional implications for differentially methylated genes and regulatory regions. The study provides reliable evidence for identifying abnormally methylated CYP11A1 gene in the placenta of sFGR.

Construction of an Angular Tricyclic Benzofuran Skeleton Using the C-H Activation Strategy.

A protocol for the construction of an angular tricyclic benzofuran skeleton based on the C-H activation strategy has been established. Different phthalide lactones on this skeleton can be easily assembled with various side chains by using C-H activation with aldehydes and subsequent reduction. This skeleton provides a versatile and crucial motif for the total synthesis of naturally occurring angular tricyclic benzofurans and their derivatives. Based on this protocol, the improved total syntheses of daldinin A and annullatin D were achieved in yields of 17.3 and 7.6%, respectively.

Exogenous hydrogen sulfide prevents necroptosis by inhibiting p38MAPK pathway activation in JEG-3 trophoblast cells: A role in preeclampsia.

Setting 7 subsection in abstract Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high-glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study was aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. DESIGN: This is an in vitro experimental study. PARTICIPANTS: A total of 10 pregnant women with severe preeclampsia (PE) and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. METHODS: The expression and localization of necrotic proteins were assayed in human placenta samples and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). RESULTS: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine-ß-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. LIMITATIONS: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of preeclampsia. CONCLUSIONS: we proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1, and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against CER-induced necroptosis via the p38MAPK pathway.

Novel insights into antimony mobilization in different high- antimony aquifers from the molecular signatures of dissolved organic matter.

The crucial role of the fluorescent components of dissolved organic matter (DOM) in controlling antimony (Sb) mobilization in groundwater has been confirmed. However, the molecular signatures contributing to Sb enrichment in DOM remain unknown. This study aims to investigate the origins and molecular compositions of DOM in different high-Sb aquifers (Sb-mining and no-Sb-mining aquifer), as well as compare different molecular signatures of DOM and mechanisms for Sb migration. The findings showed that Sb concentrations in Sb-mining aquifer exhibited a positive correlation with lignin- and tannin-like molecules characterized by high O/C and low H/C ratios, indicating an increased abundance of aromatic components with higher Humification Index and SUV-absorbance at 254 nm, compared to no-Sb-mining aquifer. Correspondingly, the complexation and competitive adsorption were considered as the predominate formation mechanisms on Sb enrichment in Sb-mining aquifer. In addition, high abundances of bioreactivity DOM may facilitated the migration of Sb via electron transfer and competitive adsorption in native no-Sb-mining aquifer. The outcomes of this investigation offer novel insights into the mechanism on Sb enrichment influenced by DOM at the molecule level.

Novel PdPtCu Nanozymes for Reprogramming Tumor Microenvironment to Boost Immunotherapy Through Endoplasmic Reticulum Stress and Blocking IDO-Mediated Immune Escape.

Breaking immunosuppressive tumor microenvironment (TME) has unique effects on inhibiting tumor growth and recurrence. Here, an endoplasmic reticulum (ER) targeted PdPtCu nanozyme (PNBCTER ) is prepared to boost immunotherapy. First, PNBCTER has three kinds of enzyme activities, including catalase (CAT), glutathione oxidase (GSHOx), and peroxidase (POD)-like activities, which can reshape the TME. Second, PNBCTER kills tumor cells by photodynamic therapy (PDT) and photothermal therapy (PTT). Third, guided by TER , PNBCTER not only realizes the combination therapy of PDT, PTT and chemodynamic therapy (CDT), but also damages the ER of tumor cells and actives antitumor immune response, which breaks through the immune blockade of TME. Finally, the NLG919 blocks the tryptophan/kynurenine immune escape pathway and reverses the immunosuppressive TME. The strategy that reshaping the TME by enzyme catalysis and breaking immunosuppression provides a novel way for the application of combination therapy in tumor.

Progress in research on the role played by myeloid-derived suppressor cells in liver diseases.

Myeloid-derived suppressor cells (MDSCs) refer to a group of immature myeloid cells with potent immunosuppressive capacity upon activation by pathological conditions. Because of their potent immunosuppressive ability, MDSCs have garnered extensive attention in the past few years in the fields of oncology, infection, chronic inflammation and autoimmune diseases. Research on MDSCs in liver diseases has gradually increased, and their potential therapeutic roles will be further explored. This review presents a summary of the involvement and the role played by MDSCs in liver diseases, thus identifying their potential targets for the treatment of liver diseases and providing new directions for liver disease-related research.

A novel rat model of sarcopenic obesity based on aging and high-fat diet consumption.

Sarcopenic obesity (SO) is defined as a combination of obesity and sarcopenia, leading to serious health consequences. However, a lack of suitable animal models has hampered research into this disorder. 12-month-old Sprague-Dawley rats were given a high fat content (HFD, SO group) or standard diet (DC groups) for 28 weeks (until 20 months of age). In addition, 2-month-old rats were fed a standard diet as an age control (YC group) until they reached 10 months of age. At the end of the intervention, quadriceps development in the rats was monitored using magnetic resonance examinations and MR spectroscopy. Age-related changes in muscle mass and strength, histopathology, HFD-induced adiposity, and metabolic disturbances were compared between the three groups. Comparing with DC group, rats of SO (20 months, and fed by high-fat diet) exhibited a more prominent loss of muscle mass and strength, a more pronounced decline in myofibre number, IFM, increase in myocyte apoptosis accompanied with increased visceral fat, remarkable glycolipid metabolic disorders, and insulin resistance. However, DC group rats (20 months with standard diet) only showed a decline in quadriceps cross-sectional area/body weight, forelimb grip strength, myofibre cross-sectional area and number, and intermyofibrillar mitochondria number (IFM), increased myocyte apoptosis, without significant metabolic disorder compared with YC group rats. After verifying, SO animal model was successfully set up by HFD induced obesity concomitant with aging-related sarcopenia.

Silver-Promoted Radical Cascade Aryldifluoromethylation/Cyclization of 2-Allyloxybenzaldehydes for the Synthesis of 3-Aryldifluoromethyl-Containing Chroman-4-one Derivatives.

A convenient silver-promoted radical cascade aryldifluoromethylation/cyclization of 2-allyloxybenzaldehydes has been developed. Experimental studies disclosed that the addition of aryldifluoromethyl radicals in situ produced from easily accessible gem-difluoroarylacetic acids to unactivated double bonds in 2-allyloxybenzaldehyde was an effective route to access a series of 3-aryldifluoromethyl-containing chroman-4-one derivatives in moderate to good yields under mild reaction conditions.

Trajectories of intrusive and deliberate rumination in young and middle-aged stroke survivors: A growth mixture modeling approach.

This study aimed to explore the relationship between intrusive and deliberate rumination, to identify distinct trajectories of intrusive and deliberate rumination, and to examine their predictors in young and middle-aged stroke survivors. This study employed a longitudinal design in which 200 young and middle-aged stroke survivors were investigated at 1-week pre-discharge (T0), 1 month (T1), 3 months (T2), and 6 months (T3) post-discharge. The Event-Related Rumination Inventory, Simplified Coping Style Questionnaire, and Perceived Social Support Scale were used for data collection. The results showed that intrusive rumination was positively correlated with deliberate rumination at T0 and T1, and negatively correlated with deliberate rumination at T3. Growth mixture modeling identified three classes of intrusive rumination: Stable-low, Declined, and Elevated group, and two classes of deliberate rumination: High-level and Low-level group. Furthermore, number of children or dysfunctions, type of stroke, family history of stroke, negative coping, and social support were found to predict intrusive rumination. These findings can help healthcare providers timely intervene on survivors in the Elevated and Stable-low intrusive rumination groups, and the Low-level deliberate rumination group.

Molecular mechanism and diagnostic marker investigation of endoplasmic reticulum stress on periodontitis.

PURPOSE: The aim of this study was to reveal the biological function of endoplasmic reticulum stress (ERS)-related genes (ERSGs) in periodontitis, and provide potential ERS diagnostic markers for clinical therapy of periodontitis. METHODS: The differentially expressed ERSGs (DE-ERSGs) were reveled based on periodontitis-related microarray dataset in Gene Expression Omnibus (GEO) database and 295 ERS in previous study, followed by a protein-protein interaction network construction. Then, the subtypes of periodontitis were explored, followed by validation with immune cell infiltration and gene set enrichment. Two machine learning algorithms were used to reveal potential ERS diagnostic markers of periodontitis. The diagnostic effect, target drug and immune correlation of these markers were further evaluated. Finally, a microRNA(miRNA)-gene interaction network was constructed. RESULTS: A total of 34 DE-ERSGs were revealed between periodontitis samples and control, followed by two subtypes investigated. There was a significant difference of ERS score, immune infiltration and Hallmark enrichment between two subtypes. Then, totally 7 ERS diagnostic markers including FCGR2B, XBP1, EDEM2, ATP2A3, ERLEC1, HYOU1 and YOD1 were explored, and the v the time-dependent ROC analysis showed a reliable result. In addition, a drug-gene network was constructed with 4 up-regulated ERS diagnostic markers and 24 drugs. Finally, based on 32 interactions, 5 diagnostic markers and 20 miRNAs, a miRNA-target network was constructed. CONCLUSIONS: Up-regulated miR-671-5p might take part in the progression of periodontitis via stimulating the expression of ATP2A3. ERSGs including XBP1 and FCGR2B might be novel diagnostic marker for periodontitis.

Epidemic threshold of a COVID-19 model with gaussian white noise and semi-Markov switching.

In this study, we investigate the COVID-19 propagation dynamics using a stochastic SIQR model with Gaussian white noise and semi-Markovian switching, focusing on the impacts of Gaussian white noise and semi-Markovian switching on the propagation dynamics of COVID-19. It is suggested that the fate of COVID-19 is entirely determined by the basic reproduction number R0, under mild extra conditions. By making sensitivity analysis on R0, we found that the effect of quarantine rate on R0 was more significant compared to transmission rate. Our results demonstrate that: (i) The presence of Gaussian white noise, while reducing the basic reproduction number R0 of COVID-19, also poses more challenges for the prediction and control of COVID-19 propagation. (ii) The conditional holding time distribution has a significant effect on the kinetics of COVID-19. (iii) The semi-Markov switching and Gaussian white noise can support irregular recurrence of COVID-19 outbreaks.