The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation.

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

SMDB: a Spatial Multimodal Data Browser.

Understanding the relationship between fine-scale spatial organization and biological function necessitates a tool that effectively combines spatial positions, morphological information, and spatial transcriptomics (ST) data. We introduce the Spatial Multimodal Data Browser (SMDB, https://www.biosino.org/smdb), a robust visualization web service for interactively exploring ST data. By integrating multimodal data, such as hematoxylin and eosin (H&E) images, gene expression-based molecular clusters, and more, SMDB facilitates the analysis of tissue composition through the dissociation of two-dimensional (2D) sections and the identification of gene expression-profiled boundaries. In a digital three-dimensional (3D) space, SMDB allows researchers to reconstruct morphology visualizations based on manually filtered spots or expand anatomical structures using high-resolution molecular subtypes. To enhance user experience, it offers customizable workspaces for interactive exploration of ST spots in tissues, providing features like smooth zooming, panning, 360-degree rotation in 3D and adjustable spot scaling. SMDB is particularly valuable in neuroscience and spatial histology studies, as it incorporates Allen's mouse brain anatomy atlas for reference in morphological research. This powerful tool provides a comprehensive and efficient solution for examining the intricate relationships between spatial morphology, and biological function in various tissues.

Electrooxidative Rhodium(III)/Chiral Carboxylic Acid-Catalyzed Enantioselective C-H Annulation of Sulfoximines with Alkynes.

The combination of achiral Cp*Rh(III) with chiral carboxylic acids (CCAs) represents an efficient catalytic system in transition metal-catalyzed enantioselective C-H activation. However, this hybrid catalysis is limited to redox-neutral C-H activation reactions and the adopt to oxidative enantioselective C-H activation remains elusive and pose a significant challenge. Herein, we describe the development of an electrochemical Cp*Rh(III)-catalyzed enantioselective C-H annulation of sulfoximines with alkynes enabled by chiral carboxylic acid (CCA) in an operationally friendly undivided cell at room temperature. A broad range of enantioenriched 1,2-benzothiazines are obtained in high yields with excellent enantioselectivities (up to 99 % yield and 98 : 2 er). The practicality of this method is demonstrated by scale-up reaction in a batch reactor with external circulation. A crucial chiral Cp*Rh(III) intermediate is isolated, characterized, and transformed, providing rational support for a Rh(III)/Rh(I) electrocatalytic cycle.

Histone methyltransferase Dot1L recruits O-GlcNAc transferase to target chromatin sites to regulate histone O-GlcNAcylation.

O-GlcNAc transferase (OGT) is the distinctive enzyme responsible for catalyzing O-GlcNAc addition to the serine or threonine residues of thousands of cytoplasmic and nuclear proteins involved in such basic cellular processes as DNA damage repair, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by which OGT regulates gene transcription remains elusive. Using proximity labeling-based mass spectrometry, here, we searched for functional partners of OGT and identified interacting protein Dot1L, a conserved and unique histone methyltransferase known to mediate histone H3 Lys79 methylation, which is required for gene transcription, DNA damage repair, cell proliferation, and embryo development. Although this specific interaction with OGT does not regulate the enzymatic activity of Dot1L, we show that it does facilitate OGT-dependent histone O-GlcNAcylation. Moreover, we demonstrate that OGT associates with Dot1L at transcription start sites and that depleting Dot1L decreases OGT associated with chromatin globally. Notably, we also show that downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal that O-GlcNAcylation of chromatin is dependent on Dot1L.

Molecular cloning, genomic structure, polymorphism analysis and recombinant expression of a α1-antitrypsin like gene from swamp eel, Monopterus albus.

Alpha-1-antitrypsin (AAT) is a highly polymorphic glycoprotein antiprotease, involved in the regulation of human immune response. Beyond some genomic characterization and a few protein characterizations, the function of teleost AAT remains uncertain. In this study we cloned an AAT-like gene from a swamp eel liver identifying four exons and three introns, and the full-length cDNA. The elucidated swamp eel AAT amino acid sequence showed high homology with known AATs from other teleosts. The swamp eel AAT was examined both in ten healthy tissues and in four bacterially-stimulated tissues resulting in up-regulation of swamp eel AAT at different times. Swamp eel AAT transcripts were ubiquitously but unevenly expressed in ten tissues. Further, the mature peptide sequence of swamp eel AAT was subcloned and transformed into E. coli with the recombinant proteins successfully inhibiting bovine trypsin activity. Analysis of recombinant AAT showed equimolar formation of irreversible complexes with proteinases, high stability at pH 7.0-10.0 and temperatures below 55 °C. Serum AAT protein level significantly increased in response to inflammation with AAT anti-sera, and, NF-κB, apolipoprotein A1 and transferrin gene expression were dramatically decreased over 72 h post recombinant AAT injection. Lastly, examination of swamp eel AAT allelic polymorphism identified all alleles in both healthy and diseased stock except allele*g, found only in diseased stock, but without statistical difference between the distribution frequency of allele*g in the two stocks. These results are crucial to our ongoing study of the role of teleost AAT in the innate immune system.

A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli.

Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNATrp (UGG) reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNATrp (UGA) mutants exhibited over fivefold increases in readthrough efficiency.

Aromatic profiles and enantiomeric distributions of volatile compounds during the ripening of Dendropanax dentiger honey.

Dendropanax dentiger honey (DDH) is a specialty herbal honey from China. Previous research on DDH has mostly focused on its composition and potential chemical markers, no studies have been conducted on the changes in aroma characteristics and chiral odorants during its maturation. Therefore, the present study aims to address the missing parts. The proportions and total concentrations of 185 volatile compounds identified in different classes varied with DDHs ripening. Fourteen common odor-active compounds were identified by odor activity values (OAVs) and GC-olfactometry (GC-O) analysis. The aroma profiles of DDHs were observed to vary at different ripening stages, although the dominant aroma characteristic was "fruity" aroma, which became more pronounced with increasing maturity. The enantiomeric contents and distributions of 7 volatile enantiomers were related to specific physicochemical indicators and the maturity of DDHs, among which the enantiomers of linalool oxide A may be a potential indicator to identify its maturity. Furthermore, precise quantification and OAVs calculation showed that the enantiomer (2S, 5S)-linalool oxide A presented the highest concentration (8.83-27.39 ng/mL) and only the enantiomer R-linalool (OAVs: 5.56-6.14) was an important contributor to the aroma profiles of DDHs at different stages of maturity. These results provided a new research idea for quality control and identification of DDHs at different maturity stages.

Pd-Catalyzed Atroposelective C-H Olefination: Diverse Synthesis of Axially Chiral Biaryl-2-carboxylic Acids.

Axially chiral carboxylic acids are important motifs in chiral catalysts and ligands. We herein reported the synthesis of axially chiral carboxylic acids via Pd(II)-catalyzed atroposelective C-H olefination using carboxylic acid as the native directing group. A broad range of axial chiral biaryl-2-carboxylic acids were synthesized in good yields with high enantioselectivities (up to 84% yield with 99% ee). Gram-scale reaction and further transformation reactions also provide a platform for synthetic applications of this method.

Dynamic variation in the aroma characteristics of Rhus chinensis honey at different stages after capping.

The ripening characteristics after capping of honey are favourable for improving its quality. However, research on the variation and formation of aroma characteristics of honey in this process is lacking. Therefore, the present study was carried out with different stages of Rhus chinensis honeys (RCHs) after capping and identified 192 volatiles with varying levels of concentration. "Fruity" was the main aroma characteristic of RCHs at different stages after capping, mainly contributed by (E)-ß-damascenone. Methyl salicylate might be a potential indicator for differentiating RCHs at different stages after capping. The metabolic pathway analyses revealed that the aroma compounds in RCHs undergo transformation at different stages after capping, which subsequently affects its aroma characteristics formation. This work is the first to study the dynamic changes in honey aroma characteristics after capping from multiple perspectives, and the results are of great significance for understanding the aroma characteristics after capping and quality control of honey.

Large herbivore grazing accelerates litter decomposition in terrestrial ecosystems.

Plant litter decomposition is critical for carbon and nutrient cycling globally. However, the effect of large herbivore grazing on litter decomposition and its mechanisms remain less explored. Here, 1203 paired observations and 381 independent experiments were analyzed to determine how litter decomposition and nutrient cycling respond to changes in grazing intensity. Grazing significantly increased litter decomposition rate by 14.08 % and litter carbon release by 5.03 %, and this effect was observed in grasslands and croplands but not in forests. The positive grazing effect was also found under sheep and cattle/yak grazing. Moderate grazing advanced the home-field advantage effect but inhibited under heavy grazing for grazed litters. The grazing effect was larger for high quality litter than for low quality litter. Litter decomposition slowed under >10 years heavy grazing but accelerated under moderate grazing. The effects of large herbivore grazing on litter decomposition were jointly influenced by grazing intensity, livestock type, climate condition, decomposition duration, litter quality, and soil properties. Our results demonstrated that large herbivore grazing accelerates litter decomposition globally and emphasized the significance and importance of grazing intensity on litter decomposition, which should be integrated into terrestrial ecosystem models.

Controlled Synthesis of Large-Area Oriented ZnO Nanoarrays.

Large-area oriented ZnO nanoarrays (including nanowire, nanorod, and nanotube) on ITO glass substrates are synthesized via the simple hydrothermal, electrodeposition, and electrochemical etching approach. The morphology of ZnO nanoarrays is controlled by adjusting the reaction temperature, reaction time, and current density. The scanning and transmission electron microscopy (SEM and TEM) results indicate the successful preparation of large-area oriented ZnO nanoarrays with different types, and the energy-dispersive X-microanalysis spectrum (EDS) and X-ray diffraction (XRD) results confirm that the composition of the obtained nanoarrays is ZnO. More importantly, the as-prepared ZnO nanotube arrays are observed with about a 40% increase in ultraviolet absorption intensity compared to the ZnO nanowire/nanorod arrays, due to having larger specific surface areas. The as-prepared different types of ZnO nanoarrays have great potential for applications in low-cost and high-performance optoelectronic devices.

DOT1L/H3K79me2 represses HIV-1 reactivation via recruiting DCAF1.

DOT1L mediates the methylation of histone H3 at lysine 79 and, in turn, the transcriptional activation or repression in a context-dependent manner, yet the regulatory mechanisms and functions of DOT1L/H3K79me remain to be fully explored. Following peptide affinity purification and proteomic analysis, we identified that DCAF1-a component of the E3 ligase complex involved in HIV regulation-is associated with H3K79me2 and DOT1L. Interestingly, blocking the expression or catalytic activity of DOT1L or repressing the expression of DCAF1 significantly enhances the tumor necrosis factor alpha (TNF-α)/nuclear factor κB (NF-κB)-induced reactivation of the latent HIV-1 genome. Mechanistically, upon TNF-α/NF-κB activation, DCAF1 is recruited to the HIV-1 long terminal repeat (LTR) by DOT1L and H3K79me2. Recruited DCAF1 subsequently induces the ubiquitination of NF-κB and restricts its accumulation at the HIV-1 LTR. Altogether, our findings reveal a feedback modulation of HIV reactivation by DOT1L-mediated histone modification regulation and highlight the potential of targeting the DOT1L/DCAF1 axis as a therapeutic strategy for HIV treatment.

Directed Evolution of Escherichia coli Nissle 1917 to Utilize Allulose as Sole Carbon Source.

Sugar substitutes are popular due to their akin taste and low calories. However, excessive use of aspartame and erythritol can have varying effects. While D-allulose is presently deemed a secure alternative to sugar, its excessive consumption is not devoid of cellular stress implications. In this study, the evolution of Escherichia coli Nissle 1917 (EcN) is directed to utilize allulose as sole carbon source through a combination of adaptive laboratory evolution (ALE) and fluorescence-activated droplet sorting (FADS) techniques. Employing whole genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats interference (CRISPRi) in conjunction with compensatory expression displayed those genetic mutations in sugar and amino acid metabolic pathways, including glnP, glpF, gmpA, nagE, pgmB, ybaN, etc., increased allulose assimilation. Enzyme-substrate dynamics simulations and deep learning predict enhanced substrate specificity and catalytic efficiency in nagE A247E and pgmB G12R mutants. The findings evince that these mutations hold considerable promise in enhancing allulose uptake and facilitating its conversion into glycolysis, thus signifying the emergence of a novel metabolic pathway for allulose utilization. These revelations bear immense potential for the sustainable utilization of D-allulose in promoting health and well-being.

The effect of colour environments on visual tracking and visual strain during short-term simulation of three gravity states.

This study investigated the effects of nine colour environments on visual tracking accuracy and visual strain during normal sitting (SP), -12° head-down bed (HD) and 9.6° head-up tilt bed (HU). In a standard posture change laboratory study, fifty-four participants performed visual tracking tasks in nine colour environments while in the three postures. Visual strain was measured by means of a questionnaire. The results showed that in all colour environments, the -12° head-down bed rest posture significantly affected visual tracking accuracy and visual strain. During the three postures, the participants' visual tracking accuracy in the cyan environment was significantly higher than that in other colour environments, and their visual strain was the lowest. Overall, the study adds to our understanding of how environmental and postural factors impact on visual tracking and visual strain.

Microglia trigger the structural plasticity of GABAergic neurons in the hippocampal CA1 region of a lipopolysaccharide-induced neuroinflammation model.

It is well-established that microglia-mediated neuroinflammatory response involves numerous neuropsychiatric and neurodegenerative diseases. While the role of microglia in excitatory synaptic transmission has been widely investigated, the impact of innate immunity on the structural plasticity of GABAergic inhibitory synapses is not well understood. To investigate this, we established an inflammation model using lipopolysaccharide (LPS) and observed a prolonged microglial response in the hippocampal CA1 region of mice, which was associated with cognitive deficits in the open field test, Y-maze test, and novel object recognition test. Furthermore, we found an increased abundance of GABAergic interneurons and GABAergic synapse formation in the hippocampal CA1 region. The cognitive impairment caused by LPS injection could be reversed by blocking GABA receptor activity with (-)-Bicuculline methiodide. These findings suggest that the upregulation of GABAergic synapses induced by LPS-mediated microglial activation leads to cognitive dysfunction. Additionally, the depletion of microglia by PLX3397 resulted in a decrease in GABAergic interneurons and GABAergic inhibitory synapses, which blocked the cognitive decline induced by LPS. In conclusion, our findings indicate that excessive reinforcement of GABAergic inhibitory synapse formation via microglial activation contributes to LPS-induced cognitive impairment.

NSUN2-mediated M5c methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections.

5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, negatively regulates type I interferon responses during various viral infections, including SARS-CoV-2. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-ß production. Knockout or knockdown of NSUN2 enhanced type I interferon and downstream ISGs during various viral infection in vitro. And in vivo, the antiviral innate response is more dramatically enhanced in Nsun2+/- mice than in Nsun2+/+ mice. The highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation enhanced cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Zika virus (ZIKV) resulted in a reduction of endogenous NSUN2 levels. Especially, SARS-CoV-2 infection (WT strain and BA.1 omicron variant) also decreased endogenous levels of NSUN2 in COVID-19 patients and K18-hACE2 KI mice, further increasing type I interferon and downstream ISGs. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease during SARS-CoV-2 and various viral infections to boost antiviral responses for effective elimination of viruses.

The Effect of Correlated Colour Temperature on Physiological, Emotional and Subjective Satisfaction in the Hygiene Area of a Space Station.

The hygiene area is one of the most important facilities in a space station. If its environmental lighting is appropriately designed, it can significantly reduce the psychological pressure on astronauts. This study investigates the effect of correlated colour temperature (CCT) on heart rate, galvanic skin response, emotion and satisfaction in the hygiene area of a space station. Forty subjects participated in experiments in a hygiene area simulator with a controlled lighting environment. The lighting conditions included 2700 K, 3300 K, 3600 K, 5000 K and 6300 K; physiological responses (heart rate, galvanic skin response), as well as emotion and satisfaction, were recorded. The results showed that CCT significantly influenced the participants' physiological and subjective responses in the space station hygiene area. 6300 K led to the best emotion and satisfaction levels, the highest galvanic skin response and the lowest heart rate. The opposite was true for 2700 K.

Molecular dynamics simulations of adsorption behavior of DDAH, NaOL and mixed DDAH/NaOL surfactants on muscovite (001) surface in aqueous solution.

The adsorption mechanism of collectors on minerals is of fundamental importance in the research and development of flotation science and processing technology. To examine the effect of cationic dodecylamine hydrochloride (DDAH), anionic sodium oleate (NaOL) and mixed DDAH/NaOL surfactants with different molar ratios on the adsorption behavior on the muscovite (001) surface, the adsorption mechanism of DDAH, NaOL and their mixture on the muscovite (001) surface in neutral aqueous solution was investigated by molecular dynamics (MD) simulations. The results showed that the cationic DDAH molecules absorb on the muscovite (001) surface by electrostatic interactions and hydrogen bonding, whereas the anionic NaOL molecules cannot independently adsorb on the muscovite surface. Based on the analysis of the density distribution profile, radial distribution function (RDF) and interaction energy between surfactant molecules and muscovite surface, and the root mean square displacement (RMSD) of surfactants on water-muscovite interface, individual DDAH surfactant is a superior collector for the muscovite flotation. The molar ratio of DDAH/NaOL surfactants is found to be a key factor in the flotation response of muscovite. No significant adsorption of 1:2, 1:3 and 1:4 mixed DDAH/NaOL surfactants on the muscovite surface can be detected, while an effective adsorption was observed for the DDAH/NaOL mixture in molar ratios of 1:1, 2:1, 3:1 and 4:1. The cationic DDAH surfactant was determined to play a primary role in the adsorption of the mixed surfactants on the muscovite surface, while the anionic NaOL molecules co-adsorb with the DDAH molecules. The additional micro-flotation experiments under neutral aqueous conditions also showed that the flotation recovery of muscovite was the highest in the presence of DDAH surfactant, which was consistent with the findings derived from MD simulations.

Colour schemes to reduce stress response in the hygiene area of a space station: A Delphi study.

This paper aims to explore colour schemes to reduce stress response in the hygiene area of a space station. We conducted a two-stage exploratory Delphi-study with 30 international experts. It was found that the overall environment, stool-urine collection device, garbage collection interface and negative pressure package interface of the hygiene area most affected astronauts' experience. Remarkably, experts have highest visual requirements for the cleanliness of the overall environment and for stool and urine collection devices in the hygiene area. These tend to have low saturation and low blackness colours, while the garbage collection interface and negative pressure package interface have conspicuity and discernibility visual requirements. It was found that experts tend to choose high saturation and high brightness colours.