Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.

Visible-Light-Promoted Aerobic α-Thiocyanation of Carbonyl Compounds with Ammonium Thiocyanate.

In the present study, we successfully developed an efficient thiocyanation of carbonyl compounds by using low-toxicity and inexpensive ammonium thiocyanate as the thiocyanate source under visible light in air (O2) at room temperature. This unified strategy is very facile for thiocyanation of various carbonyl compound derivatives (ß-keto esters, ß-keto amides, pyrazo-5-ones, isoxazol-5-ones, etc.). More importantly, the reaction proceeded smoothly without the addition of a photocatalyst and strong oxidant, ultimately minimizing the production of chemical waste. Furthermore, this green and sustainable synthetic chemistry can be used in the late-stage functionalization (LSF) of biorelevant compounds, which offers unique opportunities to achieve smooth and clean thiocyanation of drugs under mild reaction conditions.

Enantioselective α-Trifluoromethylthiolation of Carbonyl Compounds with AgSCF3 and Trichloroisocyanuric Acid.

We successfully developed an enantioselective trifluoromethylthiolation of structurally diverse carbonyl compounds. Trichloroisocyanuric acid and AgSCF3 were employed to generate active electrophilic trifluoromethylthio species in situ for asymmetric C-SCF3 bond formation. A broad variety of chiral SCF3-carbon nucleophiles (pyrazolones, ß-keto esters, and ß-keto amides) were obtained in excellent yields with high enantioselectivities (up to 92% ee) by Cinchona alkaloid derived squaramide catalysts. The reaction exhibits high efficiency, good enantioselectivity, and high functional group tolerance, which provided a novel and efficient way for asymmetric synthesis of trifluoromethylthiolated carbonyl compounds.

Association between oxidative balance score and resistant hypertension and arterial stiffness among US adults: A population-based study.

BACKGROUND AND AIMS: Prior studies have established the correlation between oxidative balance score (OBS) and hypertension (HTN). While the association between OBS and resistant hypertension (RHT) as well as arterial stiffness among individuals with hypertension remains undisclosed. METHODS AND RESULTS: In this study, total of 15,910 adults diagnosed with hypertension were enrolled from NHANES 2001-2018. OBS was calculated and categorized into quartiles. Weighted regression model, stratified analyses and restricted cubic spline (RCS) were employed to evaluate the association between OBS and RHT, major adverse cardiovascular events (MACEs) and arterial stiffness in individuals with hypertension. Among enrolled participants, high OBS quartiles consistently demonstrated a negative association with resistant hypertension across all models (all p < 0.05), indicating robust stability. Compared with the lowest OBS quartile, the risk of resistant hypertension in the highest OBS quartile was decreased by 30.8% (95%CI 0.471-0.995, p = 0.049). After dividing OBS into dietary OBS and lifestyle OBS, a significant inverse association with lifestyle OBS and RHT was observed. With regard to MACEs, the inverse association was also found in participants with high OBS. Besides, the potential relation between OBS and arterial stiffness was explored and we found OBS was significantly associated with decreased arterial stiffness (ß for ePWV, -0.014; 95%CI -0.026 to -0.001; p = 0.032). RCS analysis confirmed a nonlinear association between OBS and RHT, MACEs, cardiovascular death and nonfatal MI among participants with hypertension. CONCLUSION: Elevated OBS was negatively associated with the risk of RHT and arterial stiffness among US adults with hypertension.

Nanopore Signatures of Nucleoside Drugs.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

Astrocytes Excessively Engulf Synapses in a Mouse Model of Alzheimer's Disease.

Synapse loss is one of the most critical features in Alzheimer's disease (AD) and correlates with cognitive decline. Astrocytes mediate synapse elimination through multiple EGF-like domains 10 (MEGF10) pathways in the developing and adult brain to build the precise neural connectivity. However, whether and how astrocytes mediate synapse loss in AD remains unknown. We here find that the phagocytic receptor MEGF10 of astrocytes is significantly increased in vivo and in vitro, which results in excessive engulfment of synapses by astrocytes in APP/PS1 mice. We also observe that the astrocytic lysosomal-associated membrane protein 1 (LAMP1) is significantly elevated, colocalized with the engulfed synaptic puncta in APP/PS1 mice, and astrocytic lysosomes contain more engulfed synaptic puncta in APP/PS1 mice relative to wild type mice. Together, our data provide evidence that astrocytes excessively engulf synapses in APP/PS1 mice, which is mediated by increased MEGF10 and activated lysosomes. The approach targeting synapse engulfment pathway in astrocytes would be a potent therapy for AD.

Psychometric testing of the 10-item perceived stress scale for Chinese nurses.

BACKGROUND: Nurses bear a lot of stressors at work. The 10-item Perceived Stress Scale (PSS-10) is a widely used self-reported scale for measuring the global perception of stress. However, there is a lack of use of the PSS-10 in Chinese nurses. This study aimed to test the psychometric properties of the PSS-10 among Chinese nurses. METHODS: A total of 708 Chinese nurses completed the PSS-10, the Big Five Inventory (BFI), and the Depression Anxiety and Stress Scale (DASS). Confirmatory factor analysis (CFA) tested the factor structure of the PSS-10. Cronbach's α and test-retest correlation examined the scale reliability. Pearson correlation and hierarchical regression analyses tested the convergent, discriminant and criterion validity of the PSS-10. RESULTS: CFA revealed that a two-factor model fits the structure of the PSS-10 in Chinese nurses (χ2/df = 6.25, p < 0.001; comparative fit index [CFI] = 0.94, non-normal fit index [NNFI] = 0.92, Tucker-Lewis index [TLI] = 0.91, root mean square error of approximation [RMSEA] = 0.08, standardized root mean square residual [SRMR] = 0.05). The scale demonstrated adequate internal consistency (α = 0.86) and test-retest reliability (r = 0.66, p < 0.001), satisfactory convergent and discriminant validity with relations to Big Five personalities, as well as good criterion validity such that the PSS-10 score could explain incremental variance in predicting anxiety, depression and stress. CONCLUSIONS: Our findings suggest that PSS-10 is a reliable and valid measure of perceived stress among Chinese nurses and can be used in future research and practice on stress management and coping in Chinese nurses.

Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap.

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and ß-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Clonal copy-number mosaicism in autoreactive T lymphocytes in diabetic NOD mice.

Concordance for type 1 diabetes (T1D) is far from 100% in monozygotic twins and in inbred nonobese diabetic (NOD) mice, despite genetic identity and shared environment during incidence peak years. This points to stochastic determinants, such as postzygotic mutations (PZMs) in the expanding antigen-specific autoreactive T cell lineages, by analogy to their role in the expanding tumor lineage in cancer. Using comparative genomic hybridization of DNA from pancreatic lymph-node memory CD4+ T cells of 25 diabetic NOD mice, we found lymphocyte-exclusive mosaic somatic copy-number aberrations (CNAs) with highly nonrandom independent involvement of the same gene(s) across different mice, some with an autoimmunity association (e.g., Ilf3 and Dgka). We confirmed genes of interest using the gold standard approach for CNA quantification, multiplex ligation-dependent probe amplification (MLPA), as an independent method. As controls, we examined lymphocytes expanded during normal host defense (17 NOD and BALB/c mice infected with Leishmania major parasite). Here, CNAs found were fewer and significantly smaller compared to those in autoreactive cells (P = 0.0019). We determined a low T cell clonality for our samples suggesting a prethymic formation of these CNAs. In this study, we describe a novel, unexplored phenomenon of a potential causal contribution of PZMs in autoreactive T cells in T1D pathogenesis. We expect that exploration of point mutations and studies in human T cells will enable the further delineation of driver genes to target for functional studies. Our findings challenge the classical notions of autoimmunity and open conceptual avenues toward individualized prevention and therapeutics.

Gender differences in brain response to infant emotional faces.

Infant emotional stimuli can preferentially engage adults' attention and provide valuable information essential for successful interaction between adults and infants. Exploring the neural processes of recognizing infant stimuli promotes better understandings of the mother-infant attachment mechanisms. Here, combining task-functional magnetic resonance imaging (Task-fMRI) and resting-state fMRI (rs-fMRI), we investigated the effects of infants' faces on the brain activity of adults. Two groups including 26 women and 25 men were recruited to participate in the current study. During the task-fMRI, subjects were exposed to images of infant emotional faces (including happy, neutral, and sad) randomly. We found that the brains of women and men reacted differently to infants' faces, and these differential areas are in facial processing, attention, and empathetic networks. The rs-fMRI further showed that the connectivity of the default-mode network-related regions increased in women than in men. Additionally, brain activations in regions related to emotional networks were associated with the empathetic abilities of women. These differences in women might facilitate them to more effective and quick adjustments in behaviors and emotions during the nurturing infant period. The findings provide special implications and insights for understanding the neural processing of reacting to infant cues in adults.

NPRL2 reduces the niraparib sensitivity of castration-resistant prostate cancer via interacting with UBE2M and enhancing neddylation.

In this study, we explored the regulatory effects of nitrogen permease regulator 2-like (NPRL2) on niraparib sensitivity, a PARP inhibitor (PARPi) in castrate-resistant prostate cancer (CRPC). Data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) program were retrospectively examined. Gene-set enrichment analysis (GSEA) was conducted between high and low NRPL2 expression prostate adenocarcinoma (PRAD) cases in TCGA. CCK-8 assay, Western blot analysis of apoptotic proteins, and flow cytometric analysis of apoptosis were applied to test niraparib sensitivity. Immunofluorescent (IF) staining and co-immunoprecipitation (co-IP) were conducted to explore the proteins interacting with NPRL2. Results showed that the upregulation of a canonical protein-coding transcript of NPRL2 (ENST00000232501.7) is associated with an unfavorable prognosis. Bioinformatic analysis predicts a physical interaction between NPRL2 and UBE2M, which is validated by a following Co-IP assay. This interaction increases NPRL2 stability by reducing polyubiquitination and proteasomal degradation. Depletion of NPRL2 or UBE2M significantly increases the niraparib sensitivity of CRPC cells and enhances niraparib-induced tumor growth inhibition in vivo. NPRL2 cooperatively enhances UBE2M-mediated neddylation and facilitates the degradation of multiple substrates of Cullin-RING E3 ubiquitin ligases (CRLs). In conclusion, this study identified a novel NPRL2-UBE2M complex in modulating neddylation and niraparib sensitivity of CRPC cells. Therefore, targeting NPRL2 might be considered as an adjuvant strategy for PARPi therapy.

Dual-mode microstrip leaky-wave antenna for pattern diversity application.

We present a dual-mode microstrip leaky-wave antenna (LWA), which is characterized by radiating a single beam or dual symmetrical beam. Different from the conventional single-mode microstrip LWA, the designed antenna is based on the odd mode and the even mode of the employed shorted microstrip line. The working principle of these two modes is fully studied by analyzing the electric-field distributions and their dispersion curves. Additionally, it can be found that the odd mode and the even mode are capable of realizing different radiation patterns. Furthermore, a mode translator is particularly designed in order to efficiently excite the concerned two modes. Finally, the presented antenna is simulated, fabricated, and measured, and the simulated results agree well with the measurements. The tested far-field results show that the designed dual-mode LWA realized single-beam scanning from 39° to 64° when the frequency ranges from 4.5 to 5.0 GHz and dual symmetrical beam scanning from 44° to 60° as the frequency varies from 4.5 to 4.9 GHz, respectively.

Silicon Nitride as a Biomedical Material: An Overview.

Silicon nitride possesses a variety of excellent properties that can be specifically designed and manufactured for different medical applications. On the one hand, silicon nitride is known to have good mechanical properties, such as high strength and fracture toughness. On the other hand, the uniqueness of the osteogenic/antibacterial dualism of silicon nitride makes it a favorable bioceramic for implants. The surface of silicon nitride can simultaneously inhibit the proliferation of bacteria while supporting the physiological activities of eukaryotic cells and promoting the healing of bone tissue. There are hardly any biomaterials that possess all these properties concurrently. Although silicon nitride has been intensively studied as a biomedical material for years, there is a paucity of comprehensive data on its properties and medical applications. To provide a comprehensive understanding of this potential cornerstone material of the medical field, this review presents scientific and technical data on silicon nitride, including its mechanical properties, osteogenic behavior, and antibacterial capabilities. In addition, this paper highlights the current and potential medical use of silicon nitride and explains the bottlenecks that need to be addressed, as well as possible solutions.

Non-binary Encoded Nucleic Acid Barcodes Directly Readable by a Nanopore.

A large collection of unique molecular barcodes is useful in the simultaneous sensing or screening of molecular analytes. Though the sequence of DNA has been widely applied to encode for molecular barcodes, decoding of these barcodes is normally assisted by sequencing. We here demonstrate a barcode system based solely on self-assembly of synthetic nucleic acids and direct nanopore decoding. Each molecular barcode is composed of "n" distinct information nodes in a non-binary manner and can be sequentially scanned and decoded by a Mycobacterium smegmatis porin A (MspA) nanopore. Nanopore events containing step-shaped features were consistently reported. 14 unique information nodes were developed which in principle could encode for 14n unique molecular barcodes in a barcode containing "n" information nodes. These barcode probes were adapted to detect different antibody proteins or cancer-related microRNAs, suggesting their immediate application in a wide variety of sensing applications.

A Nanopore-Based Saccharide Sensor.

Saccharides play critical roles in many forms of cellular activities. Saccharide structures are however complicated and similar, setting a technical hurdle for direct identification. Nanopores, which are emerging single molecule tools sensitive to minor structural differences between analytes, can be engineered to identity saccharides. A hetero-octameric Mycobacterium smegmatis porin A nanopore containing a phenylboronic acid was prepared, and was able to clearly identify nine monosaccharide types, including D-fructose, D-galactose, D-mannose, D-glucose, L-sorbose, D-ribose, D-xylose, L-rhamnose and N-acetyl-D-galactosamine. Minor structural differences between saccharide epimers can also be distinguished. To assist automatic event classification, a machine learning algorithm was developed, with which a general accuracy score of 0.96 was achieved. This sensing strategy is generally suitable for other saccharide types and may bring new insights to nanopore saccharide sequencing.

Microscopic Screening of Cyclodextrin Channel Blockers by DiffusiOptoPhysiology.

Blockers of pore-forming toxins (PFTs) limit bacterial virulence by blocking relevant channel proteins. However, screening of desired blockers from a large pool of candidate molecules is not a trivial task. Acknowledging its advantages of low cost, high throughput, and multiplicity, DiffusiOptoPhysiology (DOP), an emerging nanopore technique that visually monitors the states of individual channel proteins without using any electrodes, has shown its potential use in the screening of channel blockers. By taking different α-hemolysin (α-HL) mutants as model PFTs and different cyclodextrins as model blockers, we report direct screening of pore blockers solely by using fluorescence microscopy. Different combinations of pores and blockers were simultaneously evaluated on the same DOP chip and a single-molecule resolution is directly achieved. The entire chip is composed of low-cost and biocompatible materials, which is fully disposable after each use. Though only demonstrated with cyclodextrin derivatives and α-HL mutants, this proof of concept has also suggested its generality to investigate other pore-forming proteins.

Adsorption of REEs from Aqueous Solution by EDTA-Chitosan Modified with Zeolite Imidazole Framework (ZIF-8).

Chitosan (CS) modified with ethylenediamine tetraacetic acid (EDTA) was further modified with the zeolite imidazole framework (ZIF-8) by in situ growth method and was employed as adsorbent for the removal of rare-earth elements (REEs). The material (EDTA-CS@ZIF-8) and ZIF-8 and CS were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), and nitrogen adsorption/desorption experiments (N2- Brunauer-Emmet-Teller (BET)). The effects of adsorbent dosage, temperature, the pH of the aqueous solution, contact time on the adsorption of REEs (La(III), Eu(III), and Yb(III)) by EDTA-CS@ZIF-8 were studied. Typical adsorption isotherms (Langmuir, Freundlich, and Dubinin-Radushkevich (D-R)) were determined for the adsorption process, and the maximal adsorption capacity was estimated as 256.4 mg g-1 for La(III), 270.3 mg g-1 for Eu(III), and 294.1 mg g-1 for Yb(III). The adsorption kinetics results were consistent with the pseudo-second-order equation, indicating that the adsorption process was mainly chemical adsorption. The influence of competing ions on REE adsorption was also investigated. After multiple cycles of adsorption/desorption behavior, EDTA-CS@ZIF-8 still maintained high adsorption capacity for REEs. As a result, EDTA-CS@ZIF-8 possessed good adsorption properties such as stability and reusability, which have potential application in wastewater treatment.

Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin†A (MspA) Nanopore-Trap.

Recent developments concerning large protein nanopores suggest a new approach to structure profiling of native folded proteins. In this work, the large vestibule of Mycobacterium smegmatis porin A (MspA) and calmodulin (CaM), a Ca2+ -binding protein, were used in the direct observation of the protein structure. Three conformers, including the Ca2+ -free, Ca2+ -bound, and target peptide-bound states of CaM, were unambiguously distinguished. A disease related mutant, CaM D129G was also discriminated by MspA, revealing how a single amino acid replacement can interfere with the Ca2+ -binding capacity of the whole protein. The binding capacity and aggregation effect of CaM induced by different ions (Mg2+ /Sr2+ /Ba2+ /Ca2+ /Pb2+ /Tb3+ ) were also investigated and the stability of MspA in extreme conditions was evaluated. This work demonstrates the most systematic single-molecule investigation of different allosteric conformers of CaM, acknowledging the high sensing resolution offered by the MspA nanopore trap.

Multi-Color Fluorescent Carbon Dots: Graphitized sp2 Conjugated Domains and Surface State Energy Level Co-Modulate Band Gap Rather Than Size Effects.

Four types of carbon dots (CDs) with various color (blue, green, yellow, and red) emissions have been synthesized under solvent-free conditions from citric acid and different nitrogen sources (DMF, urea, ethanamide, and formamide). By detailed characterization and comparison, it is confirmed that the graphitized sp2 conjugated domain and surface functional groups such as C-O and C=N play synergetic roles in adjusting the fluorescence properties. Notably, the size effect is not the dominant mechanism to achieve multi-color fluorescence emissions in this work. The structural configuration of the carbon dots further influences the energy band structure, as demonstrated in simplified energy level diagrams. An absorption peak at approximately 560 nm appears in the visible light region for red-emitting CDs, assigned to an n→π* transition of the aromatic structure, thus introducing a new surface state energy level, resulting in a reduction in the energy of electron transition and the expansion into the visible region of the UV/Vis spectrum. Taking advantage of the diverse absorption and emission properties, different CDs/TiO2 binary composites are obtained for photocatalytic degradation of organic dyes, and it is found that the absorption range in terms of visible light and the band gap of the carbon dots make a difference to the photocatalytic performance of the composites.