The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.

SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets.

IMPORTANCE: Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

Exploring gut-lung axis crosstalk in SARS-CoV-2 infection: Insights from a hACE2 mouse model.

Based on the forefront of clinical research, there is a growing recognition that the gut microbiota, which plays a pivotal role in shaping both the innate and adaptive immune systems, may significantly contribute to the pathogenesis of coronavirus disease 2019 (COVID-19). Although an association between altered gut microbiota and COVID-19 pathogenesis has been established, the causative mechanisms remain incompletely understood. Additionally, the validation of the precise functional alterations within the gut microbiota relevant to COVID-19 pathogenesis has been limited by a scarcity of suitable animal experimental models. In the present investigation, we employed a newly developed humanized ACE2 knock-in (hACE2-KI) mouse model, capable of recapitulating critical aspects of pulmonary and intestinal infection, to explore the modifications in the gut microbiota following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Examination of fecal samples using 16S rRNA gene profiling unveiled a notable reduction in species richness and conspicuous alterations in microbiota composition at 6 days postinfection (dpi). These alterations were primarily characterized by a decline in beneficial bacterial species and an escalation in certain opportunistic pathogens. Moreover, our analysis entailed a correlation study between the gut microbiota and plasma cytokine concentrations, revealing the potential involvement of the Lachnospiraceae_NK4A136_group and unclassified_f_Lachnospiraceae genera in attenuating hyperinflammatory responses triggered by the infection. Furthermore, integration of gut microbiota data with RNA-seq analysis results suggested that the increased presence of Staphylococcus in fecal samples may signify the potential for bacterial coinfection in lung tissues via gut translocation. In summary, our hACE2-KI mouse model effectively recapitulated the observed alterations in the gut microbiota during SARS-CoV-2 infection. This model presents a valuable tool for elucidating gut microbiota-targeted strategies aimed at mitigating COVID-19.

Humic acid biosynthesis and bacterial community evolution during aerobic composting of rice straw.

In this study, the effects of inoculum ratio, substrate particle size and aeration rate on humic acid (HA) biosynthesis during aerobic composting of rice straw were investigated, respectively. The contents of total organic carbon, total nitrogen and HA, as well as lignocellulose degradation in the composting were evaluated, respectively. It is found that the maximal HA yield of 356.9 g kg-1 was obtained at an inoculum ratio of 20%, a substrate particle size of 0.83 mm and an aeration rate of 0.3 L·kg-1 DM min-1 in the process of composting. The changes of microbial communities and metabolic functions at different stages of the composting were also analyzed through high-throughput sequencing. The result demonstrates that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the dominant phyla and their relative abundance significantly varied over time (p < 0.05), and Rhizobium, Phenylobacterium, Pseudoxanthomonas and Paenibacillus were positively related to HA content in the compost. Furthermore, the metabolic function profiles of bacterial community indicate that these functional genes in carbohydrate metabolism and amino acid metabolism were involved in lignocellulose biodegradation and HA biosynthesis. This work may be conducive to explore new regulation strategy to improve bioconversion efficiency of agricultural residues to applicable biofertilizers. KEY POINTS: • Temperature, pH, TOC, TN and C/N caused a great influence on humic acids synthesis • The succession of the microbial community during the composting were evaluated • The metabolisms of carbohydrate and amino acids were involved in HA synthesis.

Antiviral activity of theaflavins against Zika virus in vivo and in vitro.

INTRODUCTION: The prevalence and infection of the Zika virus (ZIKV) have recently posed a major threat to global public health security. However, there is currently a lack of specific vaccines and effective antiviral drugs for ZIKV infection. METHODS: Theaflavins TF1 and TF2 were selected by evaluating the anti-Zika virus activity of four kinds of theaflavins in vitro. Subsequently, in vivo, we investigated the effects of TF1 and TF2 on weight, survival, tissue viral load, and cytokines in ZIKV-infected mice. RESULTS: We compared the anti-ZIKV activity of four theaflavins (TFs) in cells and found that TF1 and TF2b significantly inhibited the replication of ZIKV/Z16006 toxic strain in BHK and Vero cells by inhibiting the replication and release of ZIKV, while no similar effects were observed for TF2a and TF3. In vivo assay, we only found that TF2b improved the survival rate of infected mice. In tissues of ZIKV-infected mice, the viral load was higher in spleen and blood, followed by liver, epididymis, and testis, the lowest in muscle. Additionally, TF2b treatment significantly reduced the expression of cytokines (IL-6, IL-1ß, TNF-α) and chemokines (CCL2, CCL5, CXCL10) induced by ZIKV infection. CONCLUSIONS: These findings suggest that TF2b has a potent antiviral effect and can be used as a potential candidate for the treatment of ZIKV infection.

Predictive effect of estimated glomerular filtrate rate by creatinine or cystatin C on mortality in patients with coronary artery disease.

BACKGROUND: Renal dysfunction leads to poor prognosis of patients with coronary artery disease (CAD). Current studies have reported the prognosis or mortality of various diseases using different estimated glomerular filtrate rate (eGFR) formulas, while the performance of these equations is unclear in CAD patients. We aim to evaluate the predict effect of creatinine-based eGFR (eGFRcr), cystatin C-based eGFR (eGFRcys), and both creatinine and cystatin C-based eGFR (eGFRcr-cys) in CAD patients. METHODS: A total of 23,178 patients with CAD were included from CIN-II cohort study. The association of eGFRcr, eGFRcys and eGFRcr-cys with cardiovascular and all-cause mortality was detected by Cox regression analysis. The predictive effect of eGFRcr, eGFRcys and eGFRcr-cys on mortality was assessed. RESULTS: During a median follow up of 4.3 years, totally 2051 patients (8.8%) experience all-cause mortality, of which 1427 patients (6.2%) died of cardiovascular disease. For the detection of cardiovascular mortality among CAD patients, eGFRcr-cys had high discriminatory capacity with area under the curve (AUC) in receiver operator characteristic analysis of 0.730, which was significantly better than eGFRcr (AUC = 0.707, p < 0.001) and eGFRcys (AUC = 0.719, p < 0.001). Similar results were observed in all-cause mortality. Restricted cubic spline showed a U-shaped association between eGFRcr and all outcomes in patients with both reduced and supranormal eGFR levels, while a L-shaped association in eGFRcys and eGFRcr-cys. CONCLUSIONS: Estimated GFR based on both creatinine and cystatin C has highest predictive effect for cardiovascular and all-cause mortality among CAD patients. Meanwhile, supranormal eGFRcr may indicate a higher risk of mortality.

[Application of Photoplethysmography Combined with Deep Learning in Postoperative Monitoring of Flaps].

Objective: Photoelectric volumetric tracing (PPG) exhibits high sensitivity and specificity in flap monitoring. Deep learning (DL) is capable of automatically and robustly extracting features from raw data. In this study, we propose combining PPG with 1D convolutional neural networks (1D-CNN) to preliminarily explore the method's ability to distinguish the degree of embolism and to localize the embolic site in skin flap arteries. Methods: Data were collected under normal conditions and various embolic scenarios by creating vascular emboli in a dermatome artery model and a rabbit dermatome model. These datasets were then trained, validated, and tested using 1D-CNN. Results: As the degree of arterial embolization increased, the PPG amplitude upstream of the embolization site progressively increased, while the downstream amplitude progressively decreased, and the gap between the upstream and downstream amplitudes at the embolization site progressively widened. 1D-CNN was evaluated in the skin flap arterial model and rabbit skin flap model, achieving average accuracies of 98.36% and 95.90%, respectively. Conclusion: The combined monitoring approach of DL and PPG can effectively identify the degree of embolism and locate the embolic site within the skin flap artery.

Humanized mice for investigating SARS-CoV-2 lung infection and associated human immune responses.

There is an urgent need for animal models of coronavirus disease 2019 to study immunopathogenesis and test therapeutic intervenes. In this study, we showed that NOD/SCID IL2rg-/- (NSG) mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-Sequencing identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, IFN stimulation and pulmonary fibrosis, between HL grafts from infected and control NSG-L mice. Furthermore, when infected with SARS-CoV-2, humanized mice with both human immune system (HIS) and autologous HL grafts (HISL mice) had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.

SARS-CoV-2 Virus Culture, Genomic and Subgenomic RNA Load, and Rapid Antigen Test in Experimentally Infected Syrian Hamsters.

The duration of SARS-CoV-2 genomic RNA shedding is much longer than that of infectious SARS-CoV-2 in most COVID-19 patients. It is very important to determine the relationship between test results and infectivity for efficient isolation, contact tracing, and post-isolation. We characterized the duration of viable SARS-CoV-2, viral genomic and subgenomic RNA (gRNA and sgRNA), and rapid antigen test positivity in nasal washes, oropharyngeal swabs, and feces of experimentally infected Syrian hamsters. The duration of viral genomic RNA shedding is longer than that of viral subgenomic RNA, and far longer than those of rapid antigen test (RAgT) and viral culture positivity. The rapid antigen test results were strongly correlated with the viral culture results. The trend of subgenomic RNA is similar to that of genomic RNA, and furthermore, the subgenomic RNA load is highly correlated with the genomic RNA load. IMPORTANCE Our findings highlight the high correlation between rapid antigen test and virus culture results. The rapid antigen test would be an important supplement to real-time reverse transcription-RCR (RT-PCR) in early COVID-19 screening and in shortening the isolation period of COVID-19 patients. Because the subgenomic RNA load can be predicted from the genomic RNA load, measuring sgRNA does not add more benefit to determining infectivity than a threshold determined for gRNA based on viral culture.

Impact of stress hyperglycemia ratio on mortality in patients with critical acute myocardial infarction: insight from american MIMIC-IV and the chinese CIN-II study.

BACKGROUND: Among patients with acute coronary syndrome and percutaneous coronary intervention, stress hyperglycemia ratio (SHR) is primarily associated with short-term unfavorable outcomes. However, the relationship between SHR and long-term worsen prognosis in acute myocardial infarction (AMI) patients admitted in intensive care unit (ICU) are not fully investigated, especially in those with different ethnicity. This study aimed to clarify the association of SHR with all-cause mortality in critical AMI patients from American and Chinese cohorts. METHODS: Overall 4,337 AMI patients with their first ICU admission from the American Medical Information Mart for Intensive Care (MIMIC)-IV database (n = 2,166) and Chinese multicenter registry cohort Cardiorenal ImprovemeNt II (CIN-II, n = 2,171) were included in this study. The patients were divided into 4 groups based on quantiles of SHR in both two cohorts. RESULTS: The total mortality was 23.8% (maximum follow-up time: 12.1 years) in American MIMIC-IV and 29.1% (maximum follow-up time: 14.1 years) in Chinese CIN-II. In MIMIC-IV cohort, patients with SHR of quartile 4 had higher risk of 1-year (adjusted hazard radio [aHR] = 1.87; 95% CI: 1.40-2.50) and long-term (aHR = 1.63; 95% CI: 1.27-2.09) all-cause mortality than quartile 2 (as reference). Similar results were observed in CIN-II cohort (1-year mortality: aHR = 1.44; 95%CI: 1.03-2.02; long-term mortality: aHR = 1.32; 95%CI: 1.05-1.66). In both two group, restricted cubic splines indicated a J-shaped correlation between SHR and all-cause mortality. In subgroup analysis, SHR was significantly associated with higher 1-year and long-term all-cause mortality among patients without diabetes in both MIMIC-IV and CIN-II cohort. CONCLUSION: Among critical AMI patients, elevated SHR is significantly associated with and 1-year and long-term all-cause mortality, especially in those without diabetes, and the results are consistently in both American and Chinese cohorts.

Elevated urine albumin creatinine ratio increases cardiovascular mortality in coronary artery disease patients with or without type 2 diabetes mellitus: a multicenter retrospective study.

BACKGROUND: Albuminuria has been suggested as an atherosclerotic risk factor among the general population. However, whether this association will be amplified in patients with coronary artery disease (CAD) is unknown. It is also unknown whether diabetes mellitus confounds the association. We aim to analyse the prognosis of elevated urine albumin creatinine ratio (uACR) in the CAD population with or without type 2 diabetes mellitus (T2DM). METHODS: This multi-center registry cohort study included 5,960 patients with CAD. Patients were divided into T2DM and non-T2DM group, and baseline uACR levels were assessed on three grades (low: uACR < 10 mg/g, middle: 10 mg/g ≤ uACR < 30 mg/g, and high: uACR ≥ 30 mg/g). The study endpoints were cardiovascular mortality and all-cause mortality. RESULTS: During the median follow-up of 2.2 [1.2-3.1] years, 310 (5.2%) patients died, of which 236 (4.0%) patients died of cardiovascular disease. CAD patients with elevated uACR had a higher risk of cardiovascular mortality (middle: HR, 2.32; high: HR, 3.22) than those with low uACR, as well as all-cause mortality. Elevated uACR increased nearly 1.5-fold risk of cardiovascular mortality (middle: HR, 2.33; high: HR, 2.34) among patients without T2DM, and increased 1.5- fold to 3- fold risk of cardiovascular mortality in T2DM patients (middle: HR, 2.49; high: HR, 3.98). CONCLUSIONS: Even mildly increased uACR could increase the risk of cardiovascular mortality in patients with CAD, especially when combined with T2DM.

MHC class I links with severe pathogenicity in C57BL/6N mice infected with SARS-CoV-2/BMA8.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes non-symptomatic infection, mild influenza-like symptoms to pneumonia, severe acute respiratory distress syndrome, and even death, reflecting different clinical symptoms of viral infection. However, the mechanism of its pathogenicity remains unclear. Host-specific traits have a breakthrough significance for studying the pathogenicity of SARS-CoV-2. We previously reported SARS-CoV-2/BMA8, a mouse-adapted strain, was lethal to aged BALB/c mice but not to aged C57BL/6N mice. Here, we further investigate the differences in pathogenicity of BMA8 strain against wild-type aged C57BL/6N and BALB/c mice. METHODS: Whole blood and tissues were collected from mice before and after BMA8 strain infection. Viral replication and infectivity were assessed by detection of viral RNA copies and viral titers; the degree of inflammation in mice was tested by whole blood cell count, ELISA and RT-qPCR assays; the pathogenicity of SARS-CoV-2/BMA8 in mice was measured by Histopathology and Immunohistochemistry; and the immune level of mice was evaluated by flow cytometry to detect the number of CD8+ T cells. RESULTS: Our results suggest that SARS-CoV-2/BMA8 strain caused lower pathogenicity and inflammation level in C57BL/6N mice than in BALB/c mice. Interestingly, BALB/c mice whose MHC class I haplotype is H-2Kd showed more severe pathogenicity after infection with BMA8 strain, while blockade of H-2Kb in C57BL/6N mice was also able to cause this phenomenon. Furthermore, H-2Kb inhibition increased the expression of cytokines/chemokines and accelerated the decrease of CD8+ T cells caused by SARS-CoV-2/BMA8 infection. CONCLUSIONS: Taken together, our work shows that host MHC molecules play a crucial role in the pathogenicity differences of SARS-CoV-2/BMA8 infection. This provides a more profound insight into the pathogenesis of SARS-CoV-2, and contributes enlightenment and guidance for controlling the virus spread.

MiRNA-520a-3p combined with folic acid conjugated Fe2O3@PDA multifunctional nanoagents for MR imagine and antitumor gene-photothermal therapy.

Osteosarcoma (OS) is a primary malignant bone tumor that occurs mainly in adolescents. Researchers are devoting to develop combination therapy methods in a multifunctional nanoplatform for the treatment of osteosarcoma. The results of previous research have shown that up-regulation of miR-520a-3p could induce anticancer effects in osteosarcoma. In order to improve the effect of gene therapy (GT), we attempted to carry miR-520a-3p in a multifunctional vector for comprehensive therapy. Fe2O3is a type of magnetic resonance imaging (MRI) contrast that is widely used as a drug delivery agent. When coated with polydopamine (PDA), it can also be used as a photothermal therapy (PTT) agent (Fe2O3@ PDA). To deliver nanoagents targeted to a tumor site, folic acid (FA) conjugated with Fe2O3@PDA was manufactured as FA-Fe2O3@PDA. FA was chosen as the target molecule to enhance utilization and reduce toxicity of nanoparticles. However, the therapeutic efficacy of FA-Fe2O3-PDA combined with miR-520a-3p has not yet been studied. In this study, we synthesized FA-Fe2O3@PDA-miRNA and investigated the potential of combining PDA regulated PTT and miR-520a-3p regulated GT to kill osteosarcoma cells. The results indicated that down-regulation of interleukin 6 receptor (IL6R) by miR-520a-3p and the photothermal ability of PDA could induce satisfactory anticancer effects in osteosarcoma, and the curative ratio was better than that used alone PTT or GT. Moreover, as a kind ofT2magnetic contrast, miRNA-Fe2O3@PDA-FA can be used for MRI. These findings indicated that miRNA-Fe2O3@PDA-FA is an effective anti-tumor nanovector for PTT combined with GT.

New guidelines for DNA methylome studies regarding 5-hydroxymethylcytosine for understanding transcriptional regulation.

Many DNA methylome profiling methods cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Because 5mC typically acts as a repressive mark whereas 5hmC is an intermediate form during active demethylation, the inability to separate their signals could lead to incorrect interpretation of the data. Is the extra information contained in 5hmC signals worth the additional experimental and computational costs? Here we combine whole-genome bisulfite sequencing (WGBS) and oxidative WGBS (oxWGBS) data in various human tissues to investigate the quantitative relationships between gene expression and the two forms of DNA methylation at promoters, transcript bodies, and immediate downstream regions. We find that 5mC and 5hmC signals correlate with gene expression in the same direction in most samples. Considering both types of signals increases the accuracy of expression levels inferred from methylation data by a median of 18.2% as compared to having only WGBS data, showing that the two forms of methylation provide complementary information about gene expression. Differential analysis between matched tumor and normal pairs is particularly affected by the superposition of 5mC and 5hmC signals in WGBS data, with at least 25%-40% of the differentially methylated regions (DMRs) identified from 5mC signals not detected from WGBS data. Our results also confirm a previous finding that methylation signals at transcript bodies are more indicative of gene expression levels than promoter methylation signals. Overall, our study provides data for evaluating the cost-effectiveness of some experimental and analysis options in the study of DNA methylation in normal and cancer samples.

Viral and Host Transcriptomes in SARS-CoV-2-Infected Human Lung Cells.

Coronaviruses are commonly characterized by a unique discontinuous RNA transcriptional synthesis strategy guided by transcription-regulating sequences (TRSs). However, the details of RNA synthesis in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been fully elucidated. Here, we present a time-scaled, gene-comparable transcriptome of SARS-CoV-2, demonstrating that ACGAAC functions as a core TRS guiding the discontinuous RNA synthesis of SARS-CoV-2 from a holistic perspective. During infection, viral transcription, rather than genome replication, dominates all viral RNA synthesis activities. The most highly expressed viral gene is the nucleocapsid gene, followed by ORF7 and ORF3 genes, while the envelope gene shows the lowest expression. Host transcription dysregulation keeps exacerbating after viral RNA synthesis reaches a maximum. The most enriched host pathways are metabolism related. Two of them (cholesterol and valine metabolism) affect viral replication in reverse. Furthermore, the activation of numerous cytokines emerges before large-scale viral RNA synthesis. IMPORTANCE SARS-CoV-2 is responsible for the current severe global health emergency that began at the end of 2019. Although the universal transcriptional strategies of coronaviruses are preliminarily understood, the details of RNA synthesis, especially the time-matched transcription level of each SARS-CoV-2 gene and the principles of subgenomic mRNA synthesis, are not clear. The coterminal subgenomic mRNAs of SARS-CoV-2 present obstacles in identifying the expression of most genes by PCR-based methods, which are exacerbated by the lack of related antibodies. Moreover, SARS-CoV-2-related metabolic imbalance and cytokine storm are receiving increasing attention from both clinical and mechanistic perspectives. Our transcriptomic research provides information on both viral RNA synthesis and host responses, in which the transcription-regulating sequences and transcription levels of viral genes are demonstrated, and the metabolic dysregulation and cytokine levels identified at the host cellular level support the development of novel medical treatment strategies.

Improvement of Lignocellulolytic Enzyme Production Mediated by Calcium Signaling in Bacillus subtilis Z2 under Graphene Oxide Stress.

An increase in exoenzyme production can be enhanced by environmental stresses such as graphene oxide (GO) stress, but the link between the two events is still unclear. In this work, the effect of GO as an environmental stress factor on exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) biosynthesis was investigated in Bacillus subtilis Z2, and a plausible mechanism by which cytosolic Ca2+ regulates lignocellulolytic enzyme production in B. subtilis Z2 subjected to GO stress was proposed. The filter paper-hydrolyzing (FPase [representing total cellulase]), carboxymethylcellulase (CMCase [representing endoglucanase]), and ß-glucosidase activities and extracellular protein concentration of the wild-type strain under 10 µg/mL GO stress were 1.37-, 1.64-, 1.24-, and 1.16-fold those of the control (without GO stress), respectively. Correspondingly, the transcription levels of lignocellulolytic enzyme genes, cytosolic Ca2+ level, and biomass concentration of B. subtilis were all increased. With lignocellulolytic enzyme from B. subtilis used to hydrolyze alkali-pretreated rice straw, the released reducing sugar concentration reached 265.53 mg/g, and the removal rates of cellulose, hemicellulose, and lignin were 52.4%, 30.1%, and 7.5%, respectively. Furthermore, transcriptome data revealed that intracellular Ca2+ homeostasis played a key role in regulating the levels of gene transcription related to the synthesis of lignocellulolytic enzymes and exoenzymes. Finally, the use of Ca2+ inhibitors (LaCl3 and EDTA) and deletion of spcF (a calmodulin-like protein gene) further demonstrated that the overexpression of those genes was regulated via calcium signaling in B. subtilis subjected to GO stress. IMPORTANCE To effectively convert lignocellulose into fermentable sugars, high lignocellulolytic enzyme loading is needed. Graphene oxide (GO) has been shown to promote exoenzyme (lignocellulolytic enzyme, amylase, peptidase, and protease) production in some microorganisms; however, the regulatory mechanism of the biosynthesis of lignocellulolytic enzymes under GO stress remains unclear. In this work, the lignocellulolytic enzyme production of B. subtilis under GO stress was investigated, and the potential mechanism by which B. subtilis enhanced lignocellulolytic enzyme production through the calcium signaling pathway under GO stress was proposed. This work revealed the role of calcium signaling in the production of enzymes under external environmental stress and provided a direction to facilitate lignocellulolytic enzyme production by B. subtilis.

pH-Dependent Chemical Transformations of Humic-Like Substances and Further Cognitions Revealed by Optical Methods.

Humic-like substances (HULIS) are macromolecular complex groups in water-soluble organic compounds (WSOC). pH is a crucial factor that influences the chemical transformations of HULIS in atmospheric particles, but this has been rarely investigated, especially under varying pH conditions. This study attempted to unveil the chemical transformation mechanisms of HULIS under a range of pH conditions using optical methods. The pH-dependent light absorption and fluorescence properties of HULIS were comprehensively analyzed; the acidity coefficient (pKa) of HULIS in relation to chemical structures was determined, and the hypothetical chemical transformation mechanisms of HULIS with increasing pH were analyzed by optical characterizations. The results suggested that pH greatly impacted the light absorption and fluorescence efficiencies of HULIS in both winter and summer seasons, and pKa was an important inflection point. The pKa of HULIS ranged from 3.5 to 8.0 in winter and 6.4 to 10.0 in summer. The acidic/basic groups were identified as -OH or -NH2 substituted quinolines, carboxylic aromatics, and pyridines. The pH-sensitive species accounted for about 6% and 21% of HULIS-C (carbon concentrations of HULIS) in winter and summer, respectively. The varying optical spectra with increasing pH might result from charge transfer or complex reactions with HULIS deprotonation.

SARS-CoV-2 Aerosol Exhaled by Experimentally Infected Cynomolgus Monkeys.

We analyzed size of severe acute respiratory coronavirus 2 (SARS-CoV-2) aerosol particles shed by experimentally infected cynomolgus monkeys. Most exhaled particles were small, and virus was mainly released early during infection. By postinfection day 6, no virus was detected in breath, but air in the isolator contained large quantities of aerosolized virus.

Highly Pathogenic Avian Influenza A(H5N8) Virus in Swans, China, 2020.

In October 2020, highly pathogenic avian influenza A(H5N8) viruses were detected in 2 dead swans in Inner Mongolia, China. Genetic analysis showed that the H5N8 isolates belong to clade 2.3.4.4b and that the isolates cluster with the H5N8 viruses isolated in Eurasia in the fall of 2020.