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.

Antibody-mediated SARS-CoV-2 entry in cultured cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. Here, we report that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity in vitro. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interaction with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may help expand the viral tropism to otherwise nonpermissive cell types with potential implications for viral transmission and pathogenesis.

Clustering on hierarchical heterogeneous data with prior pairwise relationships.

BACKGROUND: Clustering is a fundamental problem in statistics and has broad applications in various areas. Traditional clustering methods treat features equally and ignore the potential structure brought by the characteristic difference of features. Especially in cancer diagnosis and treatment, several types of biological features are collected and analyzed together. Treating these features equally fails to identify the heterogeneity of both data structure and cancer itself, which leads to incompleteness and inefficacy of current anti-cancer therapies. OBJECTIVES: In this paper, we propose a clustering framework based on hierarchical heterogeneous data with prior pairwise relationships. The proposed clustering method fully characterizes the difference of features and identifies potential hierarchical structure by rough and refined clusters. RESULTS: The refined clustering further divides the clusters obtained by the rough clustering into different subtypes. Thus it provides a deeper insight of cancer that can not be detected by existing clustering methods. The proposed method is also flexible with prior information, additional pairwise relationships of samples can be incorporated to help to improve clustering performance. Finally, well-grounded statistical consistency properties of our proposed method are rigorously established, including the accurate estimation of parameters and determination of clustering structures. CONCLUSIONS: Our proposed method achieves better clustering performance than other methods in simulation studies, and the clustering accuracy increases with prior information incorporated. Meaningful biological findings are obtained in the analysis of lung adenocarcinoma with clinical imaging data and omics data, showing that hierarchical structure produced by rough and refined clustering is necessary and reasonable.

Structure and activity of SLAC1 channels for stomatal signaling in leaves.

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of Arabidopsis thaliana SLAC1 (AtSLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of Brachypodium distachyon SLAC1 (BdSLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in AtSLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in BdSLAC1 for regulatory interaction with the N-terminal extension. This BdSLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.

Laccase-Catalyzed Grafting of Lauryl Gallate on Chitosan To Improve Its Antioxidant and Hydrophobic Properties.

Biografting is a promising and ecofriendly approach to meet various application requirements of products. Herein, a popular green enzyme, laccase, was adopted to graft a hydrophobic phenolic compound (lauryl gallate, LG) onto chitosan (CTS). The resultant chitosan derivate (Lac/LG-CTS) was systematically analyzed by Fourier transform infrared (FTIR), grafting efficiency, scanning probe microscopy (SPM), and X-ray diffraction (XRD). This grafting technique produced a multifunctional chitosan copolymer with remarkably enhanced antioxidant property, hydrophobicity, and moisture barrier property. Furthermore, the swelling capacity and acid solubility of the copolymer film decreased significantly, although the tensile strength and elongation were slightly weakened as compared to those of native chitosan. These results suggest that the Lac/LG-CTS holds great potential as a food-packaging material, preservative agent, or edible coating material.

Microsporidia infection upregulates host energy metabolism but maintains ATP homeostasis.

Microsporidia are a group of obligate intracellular parasites which lack mitochondria and have highly reduced genomes. Therefore, they are unable to produce ATP via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Instead, they have evolved strategies to obtain and manipulate host metabolism to acquire nutrients. However, little is known about how microsporidia modulate host energy metabolisms. Here, we present the first targeted metabolomics study to investigate changes in host energy metabolism as a result of infection by a microsporidian. Metabolites of silkworm embryo cell (BmE) were measured 48 h post infection by Nosema bombycis. Thirty metabolites were detected, nine of which were upregulated and mainly involved in glycolysis (glucose 6-phosphate, fructose 1,6-bisphosphate) and the TCA cycle (succinate, α-ketoglutarate, cis-aconitate, isocitrate, citrate, fumarate). Pathway enrichment analysis suggested that the upregulated metabolites could promote the synthesization of nucleotides, fatty acids, and amino acids by the host. ATP concentration in host cells, however, was not significantly changed by the infection. This ATP homeostasis was also found in Encephalitozoon hellem infected mouse macrophage RAW264.7, human monocytic leukemia THP-1, human embryonic kidney 293, and human foreskin fibroblast cells. These findings suggest that microsporidia have evolved strategies to maintain levels of ATP in the host while stimulating metabolic pathways to provide additional nutrients for the parasite.

[Recent research advances of 1-deoxynojirimycin and its derivatives].

The 1-DNJ named 1-deoxynojirimycinis (2R,3R,4R,5S)-2-(hydroxymethyl) piperidine-3,4,5-triol, which is the nature active components existingin mulberryresources including leaves, stems, roots and silkworm larva, silkworm chrysalis, etc.The 1-deoxynojirimycin is a polyhydroxylated piperidine alkaloid, which was first found in Streptomyces as an antibiotic. Then the Japanese researchers isolated it from the mulberry root. 1-DNJ can inhibit postprandial hyperglycemia by suppressing intestinal alpha glucosidase. Therefore, 1-DNJ is often used to treat treating diabetes and complicating disease and to prevent obesity and weight-related disorders. With the development of the researches, 1-deoxynojirimycin and its derivtiv was discovered to possess anti-hyperglycemic, anti-virus, anti-tumor functions and so on. Therefore,based on our current studythe existing knowledge on source, technique preparation process, pharmacokinetics, bioactivties,and in silico target fishing of 1-DNJ were summarized, so that the researchers may use it to explore future perspective of research on 1-DNJ.

Response of soil CO2 efflux to precipitation manipulation in a semiarid grassland.

Soil CO2 efflux (SCE) is an important component of ecosystem CO2 exchange and is largely temperature and moisture dependent, providing feedback between C cycling and the climate system. We used a precipitation manipulation experiment to examine the effects of precipitation treatment on SCE and its dependences on soil temperature and moisture in a semiarid grassland. Precipitation manipulation included ambient precipitation, decreased precipitation (-43%), or increased precipitation (+17%). The SCE was measured from July 2013 to December 2014, and CO2 emission during the experimental period was assessed. The response curves of SCE to soil temperature and moisture were analyzed to determine whether the dependence of SCE on soil temperature or moisture varied with precipitation manipulation. The SCE significantly varied seasonally but was not affected by precipitation treatments regardless of season. Increasing precipitation resulted in an upward shift of SCE-temperature response curves and rightward shift of SCE-moisture response curves, while decreasing precipitation resulted in opposite shifts of such response curves. These shifts in the SCE response curves suggested that increasing precipitation strengthened the dependence of SCE on temperature or moisture, and decreasing precipitation weakened such dependences. Such shifts affected the predictions in soil CO2 emissions for different precipitation treatments. When considering such shifts, decreasing or increasing precipitation resulted in 43 or 75% less change, respectively, in CO2 emission compared with changes in emissions predicted without considering such shifts. Furthermore, the effects of shifts in SCE response curves on CO2 emission prediction were greater during the growing than the non-growing season.

Regional risk assessment for point source pollution based on a water quality model of the Taipu River, China.

Point source pollution is one of the main threats to regional environmental health. Based on a water quality model, a methodology to assess the regional risk of point source pollution is proposed. The assessment procedure includes five parts: (1) identifying risk source units and estimating source emissions using Monte Carlo algorithms; (2) observing hydrological and water quality data of the assessed area, and evaluating the selected water quality model; (3) screening out the assessment endpoints and analyzing receptor vulnerability with the Choquet fuzzy integral algorithm; (4) using the water quality model introduced in the second step to predict pollutant concentrations for various source emission scenarios and analyzing hazards of risk sources; and finally, (5) using the source hazard values and receptor vulnerability scores to estimate overall regional risk. The proposed method, based on the Water Quality Analysis Simulation Program (WASP), was applied in the region of the Taipu River, which is in the Taihu Basin, China. Results of source hazard and receptor vulnerability analysis allowed us to describe aquatic ecological, human health, and socioeconomic risks individually, and also integrated risks in the Taipu region, from a series of risk curves. Risk contributions of sources to receptors were ranked, and the spatial distribution of risk levels was presented. By changing the input conditions, we were able to estimate risks for a range of scenarios. Thus, the proposed procedure may also be used by decisionmakers for long-term dynamic risk prediction.

Anaerobic reduction of graphene oxide induces the release of sorbed organic contaminants and enhances environmental risk.

Graphene oxide (GO) is an oxidized form of graphene-based materials with abundant hydrophilic oxygen-containing functional groups, forming well-dispersed suspensions and serving as pollution carriers. The natural anaerobic environment might alter the sorption behavior of GO, which in turn affects the fate and bioavailability of GO-sorbed organic contaminants. In this study, GO can be reduced by diverse environmental reductants, including sodium sulfide, DL-1,4-dithiothretiol, and L-cysteine, forming aggregates. Meanwhile, the GO-sorbed organic contaminants were released during the reduction process owing to the decreasing oxygen content and sorption sites. The effect of solution chemistry conditions (dissolved humic acid/HA and ionic strength) on the reduction release process was also investigated. HA reduced the release rate of organic contaminants due to its stabilization effect. Adding NaCl did not alter the release rate, while CaCl2 markedly enhanced the release rate. Toxicity tests with Bacillus subtilis indicated that releasing the pre-sorbed organic compound on GO led to a lower survival ratio and enhanced the superoxide dismutase activity. The findings of this study imply that the anaerobic environment could alter the dispersion/aggregation status of GO, affecting the sorption interaction between GO and the organic compounds and consequently influencing the toxicity and risk of pollution in the environment.

One-step synthesis of phytic acid-assisted hydrochar boost selective sorption and in situ passivation of lanthanum.

The rare earth metal element lanthanum (La) possesses carcinogenic, genotoxic, and accumulative properties, necessitating urgent development of an efficient and cost-effective method to remove La. However, current sorbents still encounter challenges such as poor selectivity, low sorption capacity, and high production costs. This study therefore proposes a promising solution: the creation of phytic acid-assisted sludge hydrochars (P-SHCs) to eliminate La from water and soil environments. This method harnesses phytic acid's exceptional binding ability and the economical hydrothermal carbonization process. P-SHCs exhibit robust sorption affinity, fast sorption kinetics, and excellent sorption selectivity for La when compared with pristine hydrochars (SHCs). This advantage arises from the remarkable binding ability of phosphate functional groups (polyphosphates) on P-SHCs, forming P-O-La complexes. Moreover, P-SHCs demonstrate sustained sorption efficiency across at least five cycles, with a slight decrease attributed to the loss of phosphorus species and mass during recycling. Furthermore, P-SHCs demonstrated superior economic feasibility, with a higher estimated cost-benefit ratio than that of other sorbents. Our study further validates the exceptional passivation capability of P-SHCs, showcasing relative stabilization efficiency ranging from 37.6 % to 79.6 % for La contamination. Additionally, acting as soil passivation agents, P-SHCs foster the enrichment of specific soil microorganisms such as Actinobacteria and Proteobacteria, capable of solubilizing phosphorus and resisting heavy metals. These findings present novel ideas and technical support for employing P-SHCs in combatting environmental pollution stemming from rare earth metals.

[Comparative study of transmission and reflection hyperspectral imaging technology for potato damage detection].

The randomly placed damage parts of potato will affect the detection accuracy, this paper used transmission and reflection hyperspectral imaging technology to acquire potato images of three directions(the damage part facing to the camera, back to the camera, side to the camera), and then processed the comparative study for damage detection. Independent component (IC) analysis was used to analyze the transmission and reflection hyperspectral images and to extract the features, the resulting char acteristics were used for the secondary IC analysis of the reflected images and the variable selection of the transmittance and re flectance spectroscopy. Finally, the potato injury qualitative recognition model was established based on the reflection images, the reflectance spectral and the transmittance spectral; Further optimization was done for high recognition accuracy of model, and secondary variable selection was carried out for the transmission spectrum by the Sub-window Permutation Analysis(SPA) and the optimal model for damage identification of potato randomly placed was established. The results of experiments show that the accuracy of the identification model based on the reflection image and the reflection spectrum is low, wherein the potato bruise based on the reflection images falls into the lowest recognition accuracy of 43. 10% when it is side to the camera; The accuracy of the model for identification based on the transmittance spectroscopy information is the highest, the recognition accuracy with the damage part facing and back to the camera is 100%t, and 99. 53% when it is side to the camera. The accuracy of the optimal model for identification based on the 3 kinds of transmittance spectroscopy information of randomly placed potato is 97. 39%. Then the application of transmission hyperspectral imaging technology could detect potato injury in any orientation, and the research can provide technical support for the online detection of potato quality.

Selective SARS-CoV2 BA.2 escape of antibody Fc/Fc-receptor interactions.

The number of mutations in the omicron (B.1.1.529) BA.1 variant of concern led to an unprecedented evasion of vaccine induced immunity. However, despite rise in global infections, severe disease did not increase proportionally and is likely linked to persistent recognition of BA.1 by T cells and non-neutralizing opsonophagocytic antibodies. Yet, the emergence of new sublineage BA.2, which is more transmissible than BA.1 despite relatively preserved neutralizing antibody responses, has raised the possibility that BA.2 may evade other vaccine-induced responses. Here, we comprehensively profiled the BNT162b2 vaccine-induced response to several VOCs, including omicron BA.1 and BA.2. While vaccine-induced immune responses were compromised against both omicron sublineages, vaccine-induced antibody isotype titers, and non-neutralizing Fc effector functions were attenuated to the omicron BA.2 spike compared to BA.1. Conversely, FcγR2a and FcγR2b binding was elevated to BA.2, albeit lower than BA.1 responses, potentially contributing to persistent protection against severity of disease.

Structural and functional characteristics of the SARS-CoV-2 Omicron subvariant BA.2 spike protein.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. Here, we have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and an animal model with previously prevalent variants. BA.2 S can fuse membranes slightly more efficiently than Omicron BA.1, but still less efficiently than other previous variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces, leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility of the Omicron subvariants.

The weathering process of polyethylene microplastics in the paddy soil system: Does the coexistence of pyrochar or hydrochar matter?

This study is based on a particular test site to simulate the weathering process of microplastics (MPs) in paddy soil. A substantial amount of plastic waste, especially MPs, inevitably accumulates in agricultural soil due to the high consumption and short average use of plastics. Recently, MP pollution has become a global environmental concern. However, insight into the soil weathering process of MPs in paddy soil, particularly in the presence of biochar, is lacking. In this study, the physicochemical properties of polyethylene (PE) MPs were determined through a 24-week weathering system conducted in paddy soil, paddy soil with pyrochar, or hydrochar. Moreover, the sorption of original and weathered PE MPs toward three typical pollutants (cadmium/Cd, bisphenol A/BPA, and dimethyl phthalate/DMP) was investigated. The surface of PE MPs was fractured, 1.1-fold rougher, yellow-colored (11.7 units), and 1.8-fold more oxidized after paddy soil weathering. In addition, the crystallinity, negative charge, and stronger hydrophilicity of weathered PE MPs increased compared to original PE MPs. Weathering in a pyrochar or hydrochar system caused fissures, extensive destruction of amorphous areas, and accelerated chemical or bio-oxidation processes for PE MPs, resulting in a more noticeable change in roughness (1.4-2.2-fold), yellow color (12.7-13.7), crystallinity (1.2-1.5-fold), and oxygen content (2.5-3.6-fold). Weathered PE MPs facilitated the sorption with Cd and BPA, attributed to larger specific surface area, abundant polar functional groups, and increased negatively charged sites. However, sorption of DMP to PE MPs was highly influenced by their hydrophobicity, resulting in decreased hydrophobic partition sorption on weathered PE MPs. Overall, paddy soil weathering affected the properties of PE MPs and enhanced sorption of Cd and BPA but reduced sorption of DMP. The coexistence of biochar exacerbated the paddy soil weathering effect. The insight gained from this study assists in better understanding the weathering process of PE MPs in agricultural soils.

Characterizing the Proliferation Patterns of Representative Microsporidian Species Enlightens Future Studies of Infection Mechanisms.

BACKGROUND: Microsporidia are a group of pathogens that infect all kinds of animals, such as humans, silkworms, honeybees, and shrimp; they, therefore, pose a severe threat to public health and the economy. There are over 1500 species of microsporidia that have been reported, among which Encephalitozoon hellem and Nosema bombycis are the representative zoonotic and insect-infecting species, respectively. Investigating their cell infection patterns is of great significance for understanding their infection mechanisms. METHODS: Specific probes were designed for the ribosomal RNA sequences of microsporidia. Fluorescence in situ hybridization (FISH) was used to trace the proliferation cycle of the pathogens in different cells. RESULTS: Here, two rRNA large subunit gene (LSUrRNA) probes specifically labeling N. bombycis were obtained. The life cycle of N. bombycis in silkworm cells and E. hellem in three kinds of host cells was graphically drawn. N. bombycis meronts were first observed at 30 hours post-infection (hpi), and they began merogony. Sporonts were observed at 42 hpi, and the first entire proliferation cycle was completed at 48 hpi. The proliferation cycle of E. hellem in RK13 and HEK293 epithelial cells was almost the same, completing the first life cycle after 24 hpi, but it was significantly delayed to 32 hpi in RAW264.7. CONCLUSIONS: Specific FISH probes were established for labeling microsporidia in multiple host cells. The proliferation characteristics of representative zoonotic and insect-infecting microsporidian species were clarified. This study provides an experimental pattern for future analyses of microsporidian infection mechanisms.

Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike.

The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.

Innate and Adaptive Immune Responses Against Microsporidia Infection in Mammals.

Microsporidia are obligate intracellular and eukaryotic pathogens that can infect immunocompromised and immunocompetent mammals, including humans. Both innate and adaptive immune systems play important roles against microsporidian infection. The innate immune system can partially eliminate the infection by immune cells, such as gamma delta T cell, natural killer cells (NKs), macrophages and dendritic cells (DCs), and present the pathogens to lymphocytes. The innate immune cells can also prime and enhance the adaptive immune response via surface molecules and secreted cytokines. The adaptive immune system is critical to eliminate microsporidian infection by activating cytotoxic T lymphocyte (CTL) and humoral immune responses, and feedback regulation of the innate immune mechanism. In this review, we will discuss the cellular and molecular responses and functions of innate and adaptive immune systems against microsporidian infection.

Structural properties of the peroxiredoxin AhpC2 from the hyperthermophilic eubacterium Aquifex aeolicus.

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H2O2), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H2O2 in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8 Šresolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys49, Cys212, and Cys218. The peroxidatic cysteine CP49 was found to be hyperoxidized to the sulfonic acid (SO3H) form, while Cys212 forms an intra-monomer disulfide bond with Cys218. Mutagenesis experiments indicate that Cys212 and Cys218 play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.