A mechanism for SARS-CoV-2 RNA capping and its inhibition by nucleotide analog inhibitors.

Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

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.

Genetically Elevated Serum Uric Acid and Renal Function in an Apparently Healthy Population.

BACKGROUND: The association between uric acid and kidney disease has been extensively investigated. Numerous studies have reported the association between circulating levels of uric acid and renal function. OBJECTIVES: To test, by the Mendelian randomization method, whether there is a causal association between circulating levels of uric acid and renal function. METHODS: In 989 participants, estimated glomerular filtration rate (eGFR) was calculated, the circulating level of uric acid was tested, and the uric acid polymorphism (rs11722228) was genotyped. RESULTS: After adjusting for age, gender, smoking history, alcohol intake, antihypertensive medication, body mass index, waist-to-hip ratio, and levels of urea nitrogen and creatinine, a significant allelic difference was found in uric acid levels for each genotype (p < 0.0001). Furthermore, the circulating levels of uric acid were negatively associated with eGFR after adjusting for cardiovascular risk factors and other potential confounders (p < 0.0001). Meanwhile, eGFR was significantly associated with the genotypes of rs11722228 (ß = -0.07; p = 0.02). CONCLUSIONS: Evidence from the Mendelian randomization approach implied a causal relationship between uric acid and renal function in an apparently healthy population.

The relationship between elevated serum uric acid and arterial stiffness in a healthy population.

OBJECTIVE: This study aims to investigate the relationship between serum uric acid and arterial stiffness in a healthy population. METHODS: Among the 979 participants, baPWV was non-invasively measured, the circulating levels of uric acid were tested, and the uric acid polymorphisms (rs2231142 and rs11722228) were genotyped. Then, the Mendelian randomization method was employed to test the relationship between serum uric acid and arterial stiffness in a healthy population. RESULTS: After adjusting for age, gender, antihypertensive medication, body mass index, waist-to-hip ratio, urea nitrogen, creatinine and diabetic mellitus, there was a significant allelic difference in uric acid levels for each genotype (P < 0.0001 for rs2231142; P = 0.007 for rs11722228). However, there were no differences on the potential confounders between the genotypes of rs2231142 and rs11722228 (P > 0.05). The baPWV was significantly associated with circulating levels of uric acid after adjusting for cardiovascular risk factors and other potential confounders (P = 0.002). However, neither the single polymorphism, nor the accumulation of culprit alleles was associated with baPWV (P = 0.92 for rs2231142; P = 0.60 for rs11722228; P for trend = 0.77 for the combined analysis of culprit alleles). CONCLUSION: These results do not support the causal role of circulating levels of uric acid in the development of arterial stiffness.

Predictive accuracy of serum total calcium for both critically high and critically low ionized calcium in critical illness.

BACKGROUND: The accuracy of total calcium and its corrected value for predicting critically high and critically low ionized calcium in critical illness is controversial. The aim of this study was to investigate whether the concentration of total serum calcium, either corrected for albumin or not, could predict critically high or low values in critical illness. METHODS: This report describes a retrospective study using the Medical Information Mart for Intensive Care (MIMIC) III database. Test panels that contained serum albumin, total calcium, and ionized calcium (named ATI panels) with order time intervals of less than one hour were extracted. The predictive accuracy of total calcium, either corrected for albumin or not, was assessed using receiver operating characteristic (ROC) curve analysis. RESULTS: A total of 12 118 ATIs with 103 critically low and 92 critically high ionized calcium results were extracted. The areas under ROC curves (AUCs) of corrected and uncorrected total calcium for predicting critically low ionized calcium were 0.69 (95% CI: 0.61-0.76) and 0.70 (95% CI: 0.63-0.78), respectively. For predicting critically high ionized calcium, the AUCs were 0.98 (95% CI: 0.97-1.00) and 0.97 (95% CI: 0.95-1.00), respectively. With positive predictive values (PPVs) of 0.05 and 0.10, the sensitivities (both corrected and uncorrected) were approximately 0.50 for predicting critically low ionized calcium and 0.95 for predicting critically high ionized calcium. CONCLUSIONS: Total calcium, either corrected for albumin or not, is not a reliable test to predict critically low ionized calcium in critical illness. Total calcium's predictive accuracy for critically high ionized calcium is high.

[Screening and structure analysis of the aptamer target to Escherichia coli tolC protein].

OBJECTIVE: To screen and characterize the aptamer of Escherichia coli outer member protein tolC. METHODS: By using the recombinant E.coli outer member protein tolC for the screening target, oligonucleotides which were capable of specifically binding to the protein were screened from a random oligonucleotide library through the stematic evolution of ligand by exponential enrichment (SELEX) technique. The binding capacity of ssDNA to the targeted protein from each round was detected by the FITC fluorescence labeling technique.The ssDNA from the last cycle was cloned and sequenced,and the second structure was further analyzed by the DNAMan program. RESULTS: After 12 cycles of selection, 40 clones were selected randomly and sequenced. Although a unique conserved sequence was not obtained among the 23 obtained aptamers by the primary structure analysis,three pairs of aptamers and two pairs of aptamers were found to be identical.Analysis of the secondary structure revealed that the stem-loop and bulge loop were the main motifs,indicating that they might play a key role in the binding of aptamers to the target protein. According to the characteristic of the second structure,23 aptamers were divided into four families,and aptamer 20 bore the greatest affinity. CONCLUSION: Aptamers against E.coli outer member protein tolC were successfully identified by the SELEX method. The results laid a foundation for the investigation of the interference to the drug resistance of E. coli and the underlying mechanisms.

Crystal structure of 2-chloro-1-(6-fluoro-3,4-di-hydro-2H-chromen-2-yl)ethanone.

In the title mol-ecule, C11H10ClFO2, the benzene ring, the F atom and the O atom of the di-hydro-pyran ring are essentially coplanar, with an r.m.s. deviation of 0.007 Å. The di-hydro-pyran ring is in a half-chair conformation. In the crystal, mol-ecules are linked by pairs of weak C-H⋯π hydrogen bonds, forming inversion dimers.

Electrochemical oxidation technique to pharmaceutical pollutants removal.

Human progress in medical science and drug production has improved the growth process and increased human lifespan. Most of the drugs used are to control or prevent common human diseases. These drugs can be produced in different ways such as synthetic, chemical, biological, etc. On the other hand, pharmaceutical companies have a large volume of pharmaceutical effluents and wastewater that enters the environment and harms nature and human life. The main problems of entering the pharmaceutical effluent into the environmental cycle are the creation of drug resistance against the active substance of the drugs and the occurrence of abnormalities in the next generations. Therefore, the process of pharmaceutical wastewater treatment is used to reduce the level of pharmaceutical pollutants in order to enter the pharmaceutical wastewater into the environmental cycle. Until recently, filtration, passing through reverse osmosis and ion exchange resins, cleaning facilities, etc., have been various methods to remove pharmaceutical pollutants. Due to the low efficiency of the usual and old systems, the use of new methods has attracted more attention. In this article, the aim is to investigate the electrochemical oxidation method in order to remove the active ingredient of some commonly used drugs (aspirin, atorvastatin, metformin, metronidazole and ibuprofen) from the wastewater of pharmaceuticals. Therefore, in order to observe the initial conditions of the samples, a cyclic voltammetry diagram with a scanning rate of 100 mV/s has been performed. Next, by using the chronoamperometry process and applying a constant potential, the desired drugs were subjected to the electrochemical process of oxidation. As a result, the re-examined samples were subjected to cyclic voltammetry test to determine the conditions of sample oxidation peaks as well as the removal efficiency of the samples by examining the surface under the initial and final voltammetry graph. The results indicate that this method for removing selected drugs has a high removal efficiency of about 70% and 100% for atorvastatin samples. Therefore, this method is accurate, reproducible (RSD 2%), efficient, easy and economical and can be used in drug manufacturing industries. This method is used in a wide range of drug concentration. This means that by increasing the concentration of the drug, without the need to change the equipment used and the applied potential, by spending more time in the oxidation process, it is possible to remove very high amounts of the drug (more than 1000 ppm).

Research and development of field theory-based three-dimensional risk assessment. Part II: Regional overall risk.

For three-dimensional (3D) risk assessments, the scalar fields of risk can be clarified so that the risk value at any point in 3D space can be obtained. Notably, the 3D risk function can be used to calculate the surface integral to reveal the overall risk level in a certain area. As a result, a novel field theory-based 3D risk assessment method called the regional overall risk assessment (RORA) is proposed in this study. The regional overall risk (ROR) is introduced to describe the overall risk level of the assessed area. The corresponding definition and algorithm of the ROR are determined. The selection rule of the surfaces, which are used to create the surface integrals and compute the ROR, is also provided and discussed. To demonstrate the effectiveness of the RORA, the 3D risk caused by biomass gasification stations is utilized to conduct a case study. For the assessed area (Huangtukan Village and Yanjia Village), values of ROR are 29.5787 and 39.3858, respectively. The results represent accurate overall risk levels of the assessed areas and can provide effective guidance for risk prevention in the assessed areas, including land-use planning and safety planning. Moreover, the validity and availability of the proposed RORA is verified by a sensitivity analysis. The prospects and limitations of the RORA are also analyzed and discussed in this work.

LINC00346 regulates NLRP1-mediated pyroptosis and autophagy via binding to microRNA-637 in vascular endothelium injury.

Endothelial injury and dysfunction contributes to atherosclerosis. LINC00346 plays a key role in vascular endothelial cell injury, however, the specific mechanism remains unclear. This study intends to further explore the relationship between LINC00346 and vascular endothelial injury. Circulating LINC00346 was significantly elevated in patients with coronary artery disease and had high diagnostic value for coronary artery disease. In cell experiments, we found that LINC00346 expression was significantly increased in the oxidized low-density lipoprotein (ox-LDL) intervention group, and LINC00346 knockdown delayed ox-LDL induced human umbilical vein endothelial cell (HUVEC) endothelial-to-mesenchymal transition. In addition, knockdown of LINC00346 mitigated ox-LDL-induced NOD-like receptor protein 1 (NLRP1)-mediated inflammasome formation and pyroptosis, but had no significant effect on NLRP3. By observing the number of autophagosome and detecting intracellular autophagic flux, we found that LINC00346 knockdown inhibited the ox-LDL-induced increase in intracellular autophagy level. Dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA-pull down assay were performed to confirm the inter-molecular interaction. LINC00346 acted as microRNA-637 sponge to up-regulate the expression of NLRP1. Up-regulation of microRNA-637 alleviated NLRP1-mediated pyroptosis in HUVEC and reduced intracellular autophagosome and autolysosome formation. Finally, we explored whether pyropotosis and autophagy interact with each other. We found that inhibition of intracellular autophagy could alleviate NLRP1-mediated pyroptosis. In conclusion, LINC00346 inhibited the activation of NLRP1-mediated pyroptosis and autophagy via binding to microRNA-637, therefore mitigating vascular endothelial injury.

Highly Efficient and para-Selective C-H Functionalization of Polystyrene Providing a Versatile Platform for Diverse Applications.

Postpolymerization modification of polystyrene (PS) can afford numerous value-added materials with different functions and applications, but it has been hampered by the lack of efficient methods. We report herein a highly efficient and para-selective conversion of the C-H bonds of the aromatic ring of PS into diverse functional groups using a combination of thianthrenation and thio-Suzuki-Miyaura coupling reaction. Notably, the thianthrenation efficiency of PS is as high as 99% and the degree of thianthrenation can be conveniently controlled using stoichiometric tuning of the amount of thianthrene-S-oxide added, resulting in 24-99 mol % thianthrenation. In the subsequent thio-Suzuki-Miyaura coupling reaction, 18 functionalized PS containing various functional groups (-CH2OH, -OMe, -SMe, -OTBS, -CH3, -NHBoc, -OCOMe, -CHO, -COMe, -Si(Me)3, etc.) were successfully prepared with a high degree of functionalization (64-99 mol %). The obtained functionalized PS can be readily converted into diverse functional materials, including solid-phase synthesis resins, aggregation-induced emission fluorophores, as well as ionomer binders and ion-exchange membranes for energy conversion devices. This method imparts diverse functionality onto PS with extremely high efficiency and selectivity, providing a versatile platform to transform existing commodity PS plastics into high-performance materials.

Direct Access to Bridged Polycyclic Skeletons by Merging Oxidative C-H Annulation and Cascade [4 + 2] Cycloaddition.

We report a step-economic strategy for the direct synthesis of bridged polycyclic skeletons by merging oxidative C-H annulation and cascade cycloaddition. In the protocol, spiro[cyclopentane-1,3'-indoline]-2,4-dien-2'-ones were first synthesized by oxidative C-H annulation of ethylideneoxindoles with alkynes. Subsequent cascade [4 + 2] cycloaddition with dienophiles gave the bridged bicyclo[2.2.1]quinolin-2(1H)-ones and enabled the one-pot construction of two quaternary carbon centers and three C-C bonds. Mechanistic investigations of the latter suggest a cascade ring-opening, 1,5-sigmatropic rearrangement, and [4 + 2] cycloaddition process.

Dendrobium officinale polysaccharide promotes M1 polarization of TAMs to inhibit tumor growth by targeting TLR2.

Promoting M1 polarization of tumor-associated macrophages (TAMs) is an effective pathway for malignant tumor therapy. In this study, we aimed to demonstrate whether homogeneous Dendrobium officinale polysaccharide (DOP) could promote M1 polarization of TAMs to inhibit tumor growth, and how it promoted. Results exhibited that DOP could inhibit the tumor growth and promote the M1 polarization of TAMs in tumor-bearing mice. Macrophage depletion and replenishment experiment clearly proved that the inhibitory effect of DOP on tumor growth is dependent on promoting M1 polarization of TAMs. Moreover, we found that DOP could reach tumor microenvironment (TME) and directly bind to TAMs to promote its M1 polarization via targeting toll-like receptor 2 (TLR2) after oral administration. These results clarified that DOP could remarkably inhibit the tumor growth of tumor-bearing mice via directly targeting the TLR2 of TAMs to promote its M1 polarization.

Inhibition of progesterone receptor membrane component-1 exacerbates neonatal hypoxic-ischemic cerebral damage in male mice.

This study investigated the expression of progesterone receptor membrane component 1 (pgrmc1) in the brains of male and female mice, and the effect of inhibiting pgrmc1 on neonatal hypoxic-ischemic (HI) cerebral injury in male mice. A mouse model of neonatal HI brain injury was established, and AG205, a specific antagonist of pgrmc1, was injected into the left lateral cerebral ventricle 1 h before HI. Histological staining, behavior testing, Western blots, and quantitative PCR (qPCR) were employed to evaluate pgrmc1 expression, brain damage, neurological function, and molecular mechanisms. Results demonstrated that the mRNA and protein levels of pgrmc1 increased significantly in the cortex and hippocampus 72 h after HI without sex differences. The inhibition of pgrmc1 exacerbated the neonatal brain damage in the acute stage of HI in male mice as seen in the increase in brain water content, infarction area, and neuronal death. Inhibition of pgrmc1 also aggravated the neurological dysfunction and anxiety induced by HI brain injury. In addition, inhibition of pgrmc1 activated the NF-kB signaling and NF-κB-mediated cytokines, and inhibited BDNF/PI3K/AKT pathway in the brains of the newborn HI mice. The results indicated that pgrmc1 inhibition exacerbated the brain damage in newborn male mice subjected to HI by activating IκBα/NFκB signaling and inhibiting BDNF/PI3K/Akt pathway.

N-(4-Fluoro-benz-yl)-3-nitro-pyridine-2,6-diamine.

In the title compound, C(12)H(11)FN(4)O(2), the pyridine ring is connected to a benzene ring by a -CH(2)-NH(2)- chain. The nitro group is twisted out of the pyridine ring plane [torsion angle O-N-C-C = 10.41 (10)°]. An intramolecular N-H⋯O hydrogen bond occurs. The fluoro-benzene ring is disordered over two positions [occupancy ratio = 0.59 (3):0.41 (3)]. Inter-molecular N-H⋯O and N-H⋯N hydrogen bonds stabilize the crystal structure.

[Effects of ß-amyloid-induced microglial inflammatory supernatant on neuronal apoptosis].

OBJECTIVE: To study the effects of neuronal apoptosis under the induction of ß-amyloid inflammatory supernatant. METHODS: The neurons were intervened by ß-amyloid-induced inflammatory supernatant at the concentration of Aß1-42 at 125 nmol/L. And the expressions of Bcl-2, caspase-3, PARP and neuronal apoptosis were detected. RESULTS: The caspase-3 (14.2 ± 1.8), Bcl-2 (10.6 ± 0.8) positive cells of microglia inflammatory supernatant stimulated group were significantly elevated than the control (2.2 ± 0.6, 5.0 ± 0.3, P < 0.01). Nuclear chromatin was uniform yellow-green fluorescent. And there was significant difference of neuronal apoptosis between microglia inflammatory supernatant group and Aß1-42 directly stimulated group. CONCLUSION: Neuronal apoptosis is induced by caspase-3 in ß-amyloid inflammatory supernatant. One of the important causes is chronic inflammatory process of activated microglia by Aß.

Information fusion and multi-classifier system for miner fatigue recognition in plateau environments based on electrocardiography and electromyography signals.

BACKGROUND AND OBJECTIVE: Human factors are important contributors to accidents, especially human error induced by fatigue. In this study, field tests and analyses were conducted on physiological indexes extracted from electrocardiography (ECG) and electromyography (EMG) signals in miners working under the extreme conditions of a plateau environment. To provide insights into models for fatigue classification and recognition based on machine learning, multi-modal feature information fusion and miner fatigue identification based on ECG and EMG signals as physiological indicators were studied. METHODS: Fifty-five miners were randomly selected as field test subjects, and characteristic signals were extracted from 110 groups of ECG and EMG signals as the basic signals for fatigue analysis. We conducted principal component analysis (PCA) and grey relational analysis (GRA) on the measurement indicators. Support vector machine (SVM), random forest (RF) and extreme gradient boosting (XG-Boost) machine learning models were used for fatigue classification based on multi-modal information fusion. The area under the receiver operating characteristic (ROC) curve and the confusion matrix were used to evaluate the performance of the recognition models. RESULTS: The ECG and EMG signals showed obvious changes with fatigue. The results of fatigue model identification showed that PCA feature fusion was superior to GRA feature fusion for all three machine learning approaches, and XG-Boost achieved the best performance, with a recognition accuracy of 89.47%, a sensitivity and specificity of 100%, and an AUC of 0.90. The SVM model also showed good recognition performance (89.47% accuracy, AUC=0.89). The worst performance was that of the RF model, with a recognition accuracy of only 78.95%. CONCLUSIONS: This study shows that the physiological indexes of ECG and EMG exhibit obvious, regular changes with fatigue and that it is feasible to use SVM, RF and XG-Boost models for miner fatigue identification. The PCA fusion technique can improve the identification accuracy more than the GRA method. XG-Boost classification yields the best accuracy and robustness. This study can serve as a reference for clinical research on the identification of human fatigue at high altitudes and for the clinical study of acute mountain sickness and human acclimatization to high altitudes.

Evaluation of machine learning-driven automated Kleihauer-Betke counting: A method comparison study.

INTRODUCTION: The Kleihauer-Betke (KB) test is the diagnostic standard for the quantification of fetomaternal hemorrhage (FMH). Manual analysis of KB slides suffers from inter-observer and inter-laboratory variability and low efficiency. Flow cytometry provides accurate quantification of FMH with high efficiency but is not available in all hospitals or at all times. We have developed an automated KB counting system that uses machine learning to identify and distinguish fetal and maternal red blood cells (RBCs). In this study, we aimed to evaluate and compare the accuracy, precision, and efficiency of the automated KB counting system with manual KB counting and flow cytometry. METHODS: The ratio of fetal RBCs of the same blood sample was quantified by manual KB counting, automated KB counting, and flow cytometry, respectively. Forty patients were enrolled in this comparison study. RESULTS: Comparing the automated KB counting system with flow cytometry, the mean bias in measuring the ratio of fetal RBCs was 0.0048%, with limits of agreement ranging from -0.22% to 0.23%. Using flow cytometry results as a benchmark, results of automated KB counting were more accurate than those from manual counting, with a lower mean bias and narrower limits of agreement. The precision of automated KB counting was higher than that of manual KB counting (intraclass correlation coefficient 0.996 vs 0.79). The efficiency of automated KB counting was 200 times that of manual counting by the certified technologists. CONCLUSION: Automated KB counting provides accurate and precise FMH quantification results with high efficiency.