Search

The biosynthesis of brasilane-type sesquiterpenoids (BTSs) attracts much attention owing to their unique skeleton of 5/6 bicyclic structure that contains five Me groups. Here, the crystal structures of a BTS cyclase TaTC6 from Trichoderma atroviride FKI-3849 and its complexes with farnesyl pyrophosphate (FPP) and analogue were reported. These structural information reveal that TaTC6 exploits a hydrophobic pocket to constrain the hydrocarbon region of FPP in a "U-shape" to facilitate the initial C1-C11 bond formation after pyrophosphate ionization. Following, four carbocations of reaction intermediates were molecularly docked into the hydrophobic pocket to reveal critical residues involved in the cyclization cascade. Finally, an S239-stabilized water molecule that is 3.9 Å away from the C8 of the last allyl cation may conduct hydration to quench the reaction cascade. Mutating S239 to alanine led to ca. 40% reduction in activity compared with the wild-type enzyme. The conservation of the residues that constitute the hydrophobic pocket is also discussed. Overall, this study will give an insight into the mechanism of how the active site of STCs constrain the conformation of the flexible FPP and series allylic carbocations for the complicated-ring formation and unusual carbon rearrangement in the biosynthesis of BTSs.

Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.

Since December 2019, A new type of coronavirus pneumonia (coronavirus disease 2019, COVID-19) has become endemic in Wuhan, China. So far, COVID-19 has developed into a global epidemic. The body's immune system plays an important role in the fight against COVID-19. Here, we followed up the clinical data and treatment of two COVID-19 patients diagnosed with acquired immunodeficiency syndrome (AIDS), hoping to be helpful for the subsequent diagnosis and treatment of patients with related diseases.

Enterovirus 71 (EV71) infection causes hand, foot, and mouth disease (HFMD), and even fatal neurological complications. However, the mechanisms underlying EV71 neurological pathogeneses are largely unknown. This study reveals a distinct mechanism by which EV71 induces apoptosis and autophagy in neural cells. EV71 non-structure protein 3D (also known as RNA-dependent RNA polymerase, RdRp) interacts with the peroxisomal protein acyl-CoA oxidase 1 (ACOX1), and contributes to ACOX1 downregulation. Further studies demonstrate that EV71 reduces peroxisome numbers. Additionally, knockdown of ACOX1 or peroxin 19 (PEX19) induces apoptosis and autophagy in neural cells including human neuroblastoma (SK-N-SH) cells and human astrocytoma (U251) cells, and EV71 infection induces neural cell death through attenuating ACOX1 production. Moreover, EV71 infection and ACOX1 knockdown facilitate reactive oxygen species (ROS) production and attenuate the cytoprotective protein deglycase (DJ-1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Heme oxygenase 1 (HO-1) pathway (DJ-1/NRF2/HO-1), which collectively result in ROS accumulation in neural cells. In conclusion, EV71 downregulates ACOX1 protein expression, reduces peroxisome numbers, enhances ROS generation, and attenuates the DJ-1/NRF2/HO-1 pathway, thereby inducing apoptosis and autophagy in neural cells. These findings provide new insights into the mechanism underlying EV71-induced neural pathogenesis, and suggest potential treatments for EV71-associated diseases.

A new antibody diagnostic assay with more rapid and robust properties is demanded to quantitatively evaluate anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity in a large population. Here, we developed a nanometer-scale fluorescent biosensor system consisting of CdSe-ZnS quantum dots (QDs) coupled with the highly sensitive B-cell epitopes of SARS-CoV-2 that could remarkably identify the corresponding antibody with a detection limit of 100 pM. Intriguingly, we found that fluorescence quenching of QDs was stimulated more obviously when coupled with peptides than the corresponding proteins, indicating that the energy transfer between QDs and peptides was more effective. Compared to the traditional enzyme-linked immunosorbent assay (ELISA), the B-cell-epitope-based QD-biosensor could robustly distinguish coronavirus disease 2019 (COVID-19) antibody-positive patients from uninfected individuals with a higher sensitivity (92.3–98.1% positive rates by QD-biosensor vs. 78.3–83.1% positive rates by ELISAs in 207 COVID-19 patients’ sera) in a more rapid (5 min) and labor-saving manner. Taken together, the ‘QD-peptides’ biosensor provided a novel real-time, quantitative, and high-throughput method for clinical diagnosis and home-use tests.

We report herein a facile synthetic method for converting unactivated (hetero)aryl electrophiles into β-fluoroethylated (hetero)arenes via nickel-catalyzed reductive cross-couplings. This coupling reaction features the involvement of FCH2 CH2 radical intermediate rather than β-fluoroethyl manganese species which provides effective solutions to the problematic β-fluoride side eliminations. The practical value of this protocol is further demonstrated by the late-stage modification of several complex ArCl or ArOH-derived bioactive molecules.

The NLRP3 inflammasome regulates innate immune and inflammatory responses by promoting caspase1-dependent induction of pro-inflammatory cytokines. However, aberrant inflammasome activation causes diverse diseases, and thus inflammasome activity must be tightly controlled. Here, we reveal a molecular mechanism underlying the regulation of NLRP3 inflammasome. NLRP3 interacts with SUMO-conjugating enzyme (UBC9), which subsequently promotes small ubiquitin-like modifier 1 (SUMO1) to catalyze NLRP3 SUMOylation at residue Lys204. SUMO1-catalyzed SUMOylation of NLRP3 facilitates ASC oligomerization, inflammasome activation, and interleukin-1β secretion. Moreover, this study also reveals that SUMO-specific protease 3 (SENP3) is required for the deSUMOylation of NLRP3. Interestingly, SENP3 deSUMOylates NLRP3 to attenuate ASC recruitment and speck formation, the NLRP3 inflammasome activation, as well as IL-1β cleavage and secretion. In conclusion, we reveal that SUMO1-catalyzed SUMOylation and SENP3-mediated deSUMOylation of NLRP3 orchestrate the inflammasome activation.

Most COVID-19 convalescents can build effective anti-SARS-CoV-2 humoral immunity, but it remains unclear how long it can maintain and how efficiently it can prevent the reinfection of the emerging SARS-CoV-2 variants. Here, we tested the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest known epicenter. SARS-CoV-2 immunoglobulins G (IgG) were well maintained in most patients and potently neutralizes the infection of the original strain and the B.1.1.7 variant. However, varying degrees of immune escape was observed on the other tested variants in a patient-specific manner, with individuals showing remarkably broad neutralization potency. The immune escape can be largely attributed to several critical spike mutations. These results suggest that SARS-CoV-2 can elicit long-lasting immunity but escaped by the emerging variants., graphical Abstract

Nationwide prospective surveillance of all-age patients with acute respiratory infections was conducted in China between 2009‒2019. Here we report the etiological and epidemiological features of the 231,107 eligible patients enrolled in this analysis. Children, China operates a national surveillance program for acute respiratory infections and sampled over 200,000 patients between 2009–2019. Here, the authors present results from this program and describe patterns by age, pathogen type, presence of pneumonia, and season.

Most COVID-19 patients can build effective humoral immunity against SARS-CoV-2 after recovery(1, 2). However, it remains unknown how long the protection can maintain and how efficiently it can protect people from the reinfection of the emerging SARS-CoV-2 variants. Here we evaluated the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest epicenter of SARS-CoV-2. We demonstrated that the SARS-CoV-2 immunoglobulin G (IgG) maintains at a high level and potently neutralizes the infection of the original strain (WT) and the B.1.1.7 variant in most patients. However, they showed varying degrees of efficacy reduction against the other variants of concern (P.1, B.1.525, and especially B.1.351) in a patient-specific manner. Mutations in RBD including K417N, E484K, and E484Q/L452R (B.1.617) remarkably impair the neutralizing activity of the convalescents’ sera. Encouragingly, we found that a small fraction of patients’ sera showed broad neutralization potency to multiple variants and mutants, suggesting the existence of broadly neutralizing antibodies recognizing the epitopes beyond the mutation sites. Our results suggest that the SARS-CoV-2 vaccination effectiveness relies more on the timely re-administration of the epitope-updated vaccine than the durability of the neutralizing antibodies.

An unprecedent transition-metal-free protocol for the synthesis of β-ketoamides from β-ketoesters is described. This method involves selective cleaving N P bond of an unusual aminating reagent, [tris(dimethylamino)phosphonio]difluoroacetate (ADFA), which is well known as the difluoromethylene ylide precursor. The process is notable for its operational simplicity and good functional group tolerance.

Activation of the NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome plays a key role in host immune response, which is the first line of defense against cellular stresses and pathogen infections. However, excessive inflammasome activation damages host cells, and therefore it must be precisely controlled. Here, we discover that Cullin1 (CUL1), a key component of the Skp1-Cullin1-F-box E3 ligase, plays a critical role in controlling the NLRP3 inflammasome. CUL1 represses inflammasome assembly in cultured cells, suppresses NLRP3 function in human monocytic cell line macrophages, and attenuates inflammatory responses in mouse model. Detailed studies demonstrate that CUL1 interacts with NLRP3 and promotes NLRP3 ubiquitination, but not protein degradation, to repress the NLRP3 inflammasome activation. Moreover, upon inflammatory stimuli, including ATP and nigericin treatments, CUL1 disassociates from NLRP3 to release the repression of the NLRP3 inflammasome. Thus, this study reveals a distinct and unique mechanism underlying the control of systematic activation of the NLRP3 inflammasome.-Wan, P., Zhang, Q., Liu, W., Jia, Y., Ai, S., Wang, T., Wang, W., Pan, P., Yang, G., Xiang, Q., Huang, S., Yang, Q., Zhang, W., Liu, F., Tan, Q., Zhang, W., Wu, K., Liu, Y., Wu, J. Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation.

Two series of [1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine-based energetic materials were synthesized effectively by using monosubstituted tetrazine or tetrazine-based fused rings as starting materials. Among them, compound 5 exhibits excellent insensitivity toward external stimuli (IS = 43 J and FS > 360 N) and a very good calculated detonation performance (Dv = 9408 m s−1 and P = 37.8 GPa) that are comparable to the current secondary-explosive benchmark, CL-20, which strongly suggests 5 as a secondary explosive. The azo compound 10 has a remarkable measured density of 1.91 g cm−3 at 20 °C, excellent thermal stability (Td = 305 °C), and very good calculated detonation performance (Dv = 9200 m s−1 and P = 34.8 GPa), which outperforms all current heat-resistant explosives. Compound 10 has a significant potential as a heat-resistant explosive. Compounds 14, 17 and 19 are very sensitive (IS ≤ 2 J and FS ≤ 100 J) but exhibit excellent calculated detonation performance (Dv ≥ 8690 m s−1 and P ≥ 30.2 GPa) which are very high values among current azide-containing primary explosives. These attractive features suggest strong possibilities for applications as primary explosives. A detailed study based on X-ray diffraction is used to illustrate the relationship between weak interactions and sensitivity of energetic materials. Attempts were made to design next-generation fused ring energetic materials for different applications as an alternating kind or site of the substituent group(s).

A mild method for efficient access to fluoroethylated (hetero)arenes via Suzuki-type coupling has been developed. This base metal Ni-catalyzed method features selective transformation of the more susceptible C–X bond of 1-fluoro-2-haloethanes (FCH2CH2X, X = I or Br) and leaves the vicinal fluorine intact. Preliminary mechanistic studies illustrate the intermediacy of fluoroethyl radicals in the radical-rebound oxidative addition and Ni–B transmetalation as the rate-limiting step within the catalytic cycle. The practical value of this protocol is further demonstrated by late-stage modification of several complex bioactive molecules.

National-based prospective surveillance of all-age patients with acute diarrhea was conducted in China between 2009‒2018. Here we report the etiological, epidemiological, and clinical features of the 152,792 eligible patients enrolled in this analysis. Rotavirus A and norovirus are the two leading viral pathogens detected in the patients, followed by adenovirus and astrovirus. Diarrheagenic Escherichia coli and nontyphoidal Salmonella are the two leading bacterial pathogens, followed by Shigella and Vibrio parahaemolyticus. Patients aged, Diarrhoea is a major cause of morbidity and mortality in China. Here, the authors present results from a large sentinel surveillance scheme from 217 hospitals in all 31 provinces in mainland China, including ~150,000 patients with acute diarrhoea and covering years 2009-2018.

The ubiquitin-proteasome system (UPS) is essential for the degradation of damaged proteins, which takes place in the proteasome. Upon activation by cytokines, the catalytic subunits of the proteasome are replaced by distinct isoforms resulting in the formation of an immunoproteasome (iProteasome). iProteasome generates peptides used by major histocompatibility complex class I (MHC-I) for antigen presentation and is essential for immune responses. HIV-1 is the causative agent of AIDS, and HIV-1-specific cytotoxic T lymphocytes (CTLs) provide immune responses limiting viral replication. This study identifies a distinct mechanism by which HIV-1 promotes immune evasion. The viral protein negative regulatory factor (Nef) interacts with a component of iProteasome, LMP7, attenuating iProteasome formation and protein degradation function, and thus repressing the MHC-I antigen presentation activity of MHC-I. Therefore, HIV-1 targets LMP7 to inhibit iProteasome activation, and LMP7 may be used as the target for the development of anti-HIV-1/AIDS therapy., The proteasome is a major protein degradation machinery with essential and diverse biological functions. Upon induction by cytokines, proteasome subunits β1, β2, and β5 are replaced by β1i/LMP2, β2i/MECL-1, and β5i/LMP7, resulting in the formation of an immunoproteasome (iProteasome). iProteasome-degraded products are loaded onto the major histocompatibility complex class I (MHC-I), regulating immune responses and inducing cytotoxic T lymphocytes (CTLs). Human immunodeficiency virus type 1 (HIV-1) is the causal agent of AIDS. HIV-1-specific CTLs represent a critical immune mechanism limiting viral replication. HIV-1 negative regulatory factor (Nef) counteracts host immunity, particularly the response involving MHC-I/CTL. This study identifies a distinct mechanism by which Nef facilitates immune evasion via suppressing the function of iProteasome and MHC-I. Nef interacts with LMP7 on the endoplasmic reticulum (ER), downregulating the incorporation of LMP7 into iProteasome and thereby attenuating its formation. Moreover, Nef represses the iProteasome function of protein degradation, MHC-I trafficking, and antigen presentation.

During January–February 2020, coronavirus disease (COVID-19) and tuberculosis were diagnosed for 3 patients in Wuhan, China. All 3 patients had COVID-19 pneumonia. One severely ill patient died after acute respiratory distress syndrome developed. Clinicians and public health officials should be aware of underlying chronic infections such as tuberculosis in COVID-19 patients.

Background Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K+, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions. Results We show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K+ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages. Conclusions We have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.

Summary: The immune system is not only required for preventing threats exerted by pathogens but also essential for developing immune tolerance to avoid tissue damage. This study identifies a distinct mechanism by which MYSM1 suppresses innate immunity and autoimmunity. The expression of MYSM1 is induced upon DNA virus infection and by intracellular DNA stimulation. MYSM1 subsequently interacts with STING and cleaves STING K63-linked ubiquitination to suppress cGAS-STING signaling. Notably, Mysm1-deficient mice exhibit a hyper-inflammatory response, acute tissue damage, and high mortality upon virus infection. Moreover, in the PBMCs of patients with systemic lupus erythematosus (SLE), MYSM1 production decreases, while type I interferons and pro-inflammatory cytokine expressions increase. Importantly, MYSM1 treatment represses the production of IFNs and pro-inflammatory cytokines in the PBMCs of SLE patients. Thus, MYSM1 is a critical repressor of innate immunity and autoimmunity and is thus a potential therapeutic agent for infectious, inflammatory, and autoimmune diseases.

The ongoing outbreak of coronavirus disease 2019 (COVID-19) has spread rapidly on a global scale. Although it is clear that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted through human respiratory droplets and direct contact, the potential for aerosol transmission is poorly understood1-3. Here we investigated the aerodynamic nature of SARS-CoV-2 by measuring viral RNA in aerosols in different areas of two Wuhan hospitals during the outbreak of COVID-19 in February and March 2020. The concentration of SARS-CoV-2 RNA in aerosols that was detected in isolation wards and ventilated patient rooms was very low, but it was higher in the toilet areas used by the patients. Levels of airborne SARS-CoV-2 RNA in the most public areas was undetectable, except in two areas that were prone to crowding; this increase was possibly due to individuals infected with SARS-CoV-2 in the crowd. We found that some medical staff areas initially had high concentrations of viral RNA with aerosol size distributions that showed peaks in the submicrometre and/or supermicrometre regions; however, these levels were reduced to undetectable levels after implementation of rigorous sanitization procedures. Although we have not established the infectivity of the virus detected in these hospital areas, we propose that SARS-CoV-2 may have the potential to be transmitted through aerosols. Our results indicate that room ventilation, open space, sanitization of protective apparel, and proper use and disinfection of toilet areas can effectively limit the concentration of SARS-CoV-2 RNA in aerosols. Future work should explore the infectivity of aerosolized virus.

Real time fluorescent quantitative PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load in patient throats and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, early treat, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 nucleic acid from 77 clinical throat swab samples, including 63 suspected outpatients with fever and 14 supposed convalescents who were about to discharge after treatment, and compared with RT-PCR in terms of the diagnostic accuracy. In this double-blind study, we tested, surveyed subsequently and statistically analyzed 77 clinical samples. According to our study, 26 samples from COVID-19 patients with RT-PCR negative were detected as positive by ddPCR. No FPRs of RT-PCR and ddPCR were observed. The sensitivity, specificity, PPV, NPV, NLR and accuracy were improved from 40% (95% CI: 27–55%), 100% (95% CI: 54–100%), 100%, 16% (95% CI: 13–19%), 0.6 (95% CI: 0.48–0.75) and 47% (95% CI: 33–60%) for RT-PCR to 94% (95% CI: 83–99%), 100% (95% CI: 48–100%), 100%, 63% (95% CI: 36–83%), 0.06 (95% CI: 0.02–0.18) and 95% (95% CI: 84–99%) for ddPCR, respectively. Moreover, 14 (42.9 %) convalescents still carry detectable SARS-CoV-2 after discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the current standard RT-PCR. It also suggests that the current clinical practice that the convalescent after discharge continues to be quarantined for at least 2 weeks is completely necessary which can prevent potential viral transmission.

Background There’s an outbreak of a novel coronavirus (SARS-CoV-2) infection since December 2019, first in China, and currently with more than 80 thousand confirmed infection globally in 29 countries till March 2, 2020. Identification, isolation and caring for patients early are essential to limit human-to-human transmission including reducing secondary infections among close contacts and health care workers, preventing transmission amplification events. The RT-PCR detection of viral nucleic acid test (NAT) was one of the most quickly established laboratory diagnosis method in a novel viral pandemic, just as in this COVID-19 outbreak. Methods 4880 cases that had respiratory infection symptoms or close contact with COVID-19 patients in hospital in Wuhan, China, were tested for SARS-CoV-2 infection by use of quantitative RT-PCR (qRT-PCR) on samples from the respiratory tract. Positive rates were calculated in groups divided by genders or ages. Results The positive rate was about 38% for the total 4880 specimens. Male and older population had a significant higher positive rates. However, 57% was positive among the specimens from the Fever Clinics. Binary logistic regression analysis showed that age, not gender, was the risk factor for SARS-CoV-2 infection in fever clinics. Conclusions Therefore, we concluded that viral NAT played an important role in identifying SARS-CoV-2 infection.

During host–virus co-evolution, cells develop innate immune systems to inhibit virus invasion, while viruses employ strategies to suppress immune responses and maintain infection. Here, we reveal that Zika virus (ZIKV), a re-emerging arbovirus causing public concerns and devastating complications, restricts host immune responses through a distinct mechanism. ZIKV nonstructural protein 5 (NS5) interacts with the host retinoic acid-inducible gene I (RIG-I), an essential signaling molecule for defending pathogen infections. NS5 subsequently represses K63-linked polyubiquitination of RIG-I, attenuates the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3), and inhibits the expression and production of interferon-β (IFN-β), thereby restricting the RIG-I signaling pathway. Interestingly, we demonstrate that the methyltransferase (MTase) domain of NS5 is required for the repression of RIG-I ubiquitination, IRF3 activation, and IFN-β production. Detailed studies further reveal that the conservative active site D146 of NS5 is critical for the suppression of the RIG-I signaling. Therefore, we uncover an essential role of NS5 conservative site D146 in ZIKV-mediated repression of innate immune system, illustrate a distinct mechanism by which ZIKV evades host immune responses, and discover a potential target for anti-viral infection.

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27–55%), 100% (95% CI: 54–100%), 100%, 16% (95% CI: 13–19%), 0.6 (95% CI: 0.48–0.75) and 47% (95% CI: 33–60%) for RT-PCR to 94% (95% CI: 83–99%), 100% (95% CI: 48–100%), 100%, 63% (95% CI: 36–83%), 0.06 (95% CI: 0.02–0.18), and 95% (95% CI: 84–99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5–12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.

Circulating in China and 75 other countries and territories, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public health. However, efforts to identify effectively supportive therapeutic drugs and treatments has been hampered by our limited understanding of host immune response for this fatal disease. To characterize the transcriptional signatures of host inflammatory response to SARS-CoV-2 infection, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients. Our results reveal distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release such as CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B. Furthermore, SARS-CoV-2 induced activation of apoptosis and P53 signaling pathway in lymphocytes may be the cause of patients’ lymphopenia. The transcriptome dataset of COVID-19 patients would be a valuable resource for clinical guidance on anti-inflammatory medication and understanding the molecular mechansims of host response. Funding: This study was supported by Special Fund for COVID-19 Research of Wuhan University, National Science and Technology Major Project (#2018ZX10733403), China NSFC grants (#81672008, 31871316), Hubei Natural Science Foundation (#2018CFA035), Basic Scientific Research Foundation of Central Universities (#2042019gf0026), Ministry of Science and Technology of China, the National Mega Project on Major Infectious Disease Prevention (#2017ZX10103005) and National Key Research and Development Program of China (#2018YFE0204500). Declaration of Interest: The authors declare no competing interests. Ethical Approval: This study was approved by the Ethics Committee of the Zhongnan Hospital of Wuhan University. The RNA-seq analyses of BALF samples and PBMC were performed on existing samples collected during standard diagnostic tests, posing no extra burden to patients.

Additional file 2: Fig. S1. Full Western blots used for Fig. 1b, c, d, e. (a) Full Western blots used for Fig. 1b. (b) Full Western blots used for Fig. 1c. (c) Full Western blots used for Fig. 1d. (d) Full Western blots used for Fig. 1e. Fig. S2. Full Western blots used for Fig. 2b, d, e, f, g, h, i, j. (a) Full Western blots used for Fig. 2b. (b) Full Western blots used for Fig. 2d. (c) Full Western blots used for Fig. 2e. (d) Full Western blots used for Fig. 2f. (e) Full Western blots used for Fig. 2g. (f) Full Western blots used for Fig. 2h. (g) Full Western blots used for Fig. 2i. (h) Full Western blots used for Fig. 2j.Fig. S3. Full Western blots used for Fig. 3a, b, c, d, e, f, h, j, k. (a) Full Western blots used for Fig. 3a. (b) Full Western blots used for Fig. 3b. (c) Full Western blots used for Fig. 3c. (d) Full Western blots used for Fig. 3d. (e) Full Western blots used for Fig. 3e. (f) Full Western blots used for Fig. 3f. (g) Full Western blots used for Fig. 3h. (h) Full Western blots used for Fig. 3j. (i) Full Western blots used for Fig. 3k. Fig. S4. Full Western blots used for Fig. 4a, b, c, d, e, f, g. (a) Full Western blots used for Fig. 4a. (b) Full Western blots used for Fig. 4b. (c) Full Western blots used for Fig. 4c. (d) Full Western blots used for Fig. 4d. (e) Full Western blots used for Fig. 4e. (f) Full Western blots used for Fig. 4f. (g) Full Western blots used for Fig. 4g. Fig. S5. Full Western blots used for Fig. 5a, b, c, d, e, f. (a) Full Western blots used for Fig. 5a. (b) Full Western blots used for Fig. 5b. (c) Full Western blots used for Fig. 5c. (d) Full Western blots used for Fig. 5d. (e) Full Western blots used for Fig. 5e. (f) Full Western blots used for Fig. 5f. Fig. S6. Full Western blots used for Fig. 6a, b, c, d, e, f, g, h, j, k, l, m, n, o, q. (a) Full Western blots used for Fig. 6a. (b) Full Western blots used for Fig. 6b. (c) Full Western blots used for Fig. 6c. (d) Full Western blots used for Fig. 6d. (e) Full Western blots used for Fig. 6e. (f) Full Western blots used for Fig. 6f. (g) Full Western blots used for Fig. 6g. (h) Full Western blots used for Fig. 6h. (i) Full Western blots used for Fig. 6j. (j) Full Western blots used for Fig. 6k. (k) Full Western blots used for Fig. 6l. (l) Full Western blots used for Fig. 6m. (m) Full Western blots used for Fig. 6n. (n) Full Western blots used for Fig. 6o. (o) Full Western blots used for Fig. 6q. Fig. S7. Full Western blots used for Fig. 8a, b, c, d, e. (a) Full Western blots used for Fig. 8a. (b) Full Western blots used for Fig. 8b. (c) Full Western blots used for Fig. 8c. (d) Full Western blots used for Fig. 8d. (e) Full Western blots used for Fig. 8e. Fig. S8. Full Western blots used for Fig. 9b, d, f, g, j. (a) Full Western blots used for Fig. 9b. (b) Full Western blots used for Fig. 9d. (c) Full Western blots used for Fig. 9f. (d) Full Western blots used for Fig. 9g. (e) Full Western blots used for Fig. 9j.

Three classes of nitrogen-rich energetic compounds 7-9 (combination of tetrazolyl, dinitromethyl and furoxan), 12-17 (combination of tetrazolyl, (1,2,4-oxadiazol-5-yl)nitroamino and furoxan) and 20-22 (combination of dinitromethyl, (1,2,4-oxadiazol-5-yl)nitroamino and furoxan) were obtained by selected reactions with 3,4-dicyano-furoxan. All the new compounds were thoroughly characterized by IR, NMR [1H, 13C{1H}], elemental analysis, and differential scanning calorimetry (DSC). Compounds 7, 13, 20 and 22 were also further characterized with single-crystal X-ray diffraction studies. Heats of formation and detonation performances for nine compounds were determined using Gaussian 03 and EXPLO5 v6.01 programs, showing that 15 as a secondary explosive is superior to 1,3,5-trinitrotriazacyclohexane (RDX) and 20 is a promising green primary explosive.

A novel copper/amine co-catalyzed formal regioselective [3 + 2] cycloaddition reaction of an O-acyl oxime with α,β-unsaturated aldehydes is developed. This novel transformation provided an efficient protocol for the assembly of multifunctionalized pyrroles with good yields and diverse functional group tolerance.

A mild and efficient NBS promoted intramolecular oxidative cyclization/aromatization of β-tetralone oximes has been explored. Under the optimized conditions, fused α-carbolines containing pentacyclic rings were obtained in moderate to good yields. Furthermore, various benzo[5,6]chromeno[2,3-b]indoles were successfully synthesized in moderate yields from β-tetralones using slightly modified conditions. We proposed a possible reaction pathway based on the experimental results.

A wide variety of multi-substituted 3-fluoroalkenyloxindole ring-fused 3-trifluoromethyloxindoles were obtained in good yields by the reaction of indoline-2,3-diones with difluoromethylene phosphab...

A general method was developed for the synthesis of 1-difluoroalkyl isoquinolines via the palladium-catalyzed radical cascade difluoroalkylation-cyclization of vinyl isocyanides with bromodifluoroacetic derivatives. The difluoroalkylated cyclization products were readily converted to various other valuable gem-difluoro-containing compounds.

A series of monosubstituted 1,2,4,5-tetrazine-based energetic materials was effectively synthesized and fully characterized with IR, multinuclear nuclear magnetic resonance (NMR), and elemental analyses. Heats of formation and detonation performances were determined using Gaussian 03 and EXPLO5 v6.01 programs, which show that 5 and 9 as secondary explosives have detonation velocities superior to the current secondary-explosive benchmark, triaminotrinitrobenzene (TATB). Importantly, compounds 2, 5, and 9 were first characterized with single-crystal X-ray diffraction and Hirshfeld surface calculations, and some intermolecular weak hydrogen bonds (Het-H-N/O) among these compounds illustrate the relationship between these weak interactions and excellent sensitivity of energetic materials. This design method for next-generation energetic materials by incorporating intermolecular weak hydrogen bonds may be of future importance.

Dengue virus (DENV) infection causes several diseases ranging from dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome characterized by endothelial dysfunction, vascular leakage, and shock. Here, we identify a potential mechanism by which DENV induces tissue injury and vascular leakage by promoting the activation of interleukin (IL)-1β. DENV facilitates IL-1β secretion in infected patients, mice, human peripheral blood mononuclear cells (PBMCs), mouse bone marrow-derived macrophages (BMDMs), and monocyte-differentiated macrophages (THP-1) via activating the NLRP3 inflammasome. The accumulated data suggest that IL-1β probably induces vascular leakage and tissue injury in interferon-alpha/beta receptor 1 deficient C57BL/6 mice (IFNAR -/- C57BL/6), whereas IL-1 receptor antagonist (IL-1RA) alleviates these effects of IL-1β. Finally, administration of recombinant IL-1β protein results in vascular leakage and tissue injury in C57BL/6 mice. Together, the accumulated results demonstrate that IL-1β contributes to DENV-associated pathology and suggest that IL-1RA acts as a potential agent for the treatment of DENV-associated diseases.

Proteasome is major protein degradation machinery and plays essential roles in diverse biological functions. Upon cytokine inductions, proteasome subunits β1, β2, and β5 are replaced by β1i/LMP2, β2i/MECL-1, and β5i/LMP7, leading to the formation of immunoproteasome. Immunoproteasome-degraded products are loaded onto the major histocompatibility complex class I (MHC-I) to regulate immune responses and induce cytotoxic-T-lymphocytes (CTLs). Human immunodeficiency virus type 1 (HIV-1) is the causal agent of acquired immunodeficiency syndrome (AIDS). HIV-1-specific CTLs represent critical immune responses to limit viral replication. HIV-1 negative regulatory factor (Nef) counteracts host immunity, especially the MHC-I/CTL. This study reveals a distinct mechanism by which Nef facilitates immune evasion through attenuating the functions of immunoproteasome and MHC-I. Nef interacts with LMP7 on the endoplasmic reticulum (ER) to down-regulate the incorporation of LMP7 into immunoproteasome, and thereby attenuating immunoproteasome formation. Moreover, Nef represses immunoproteasome protein degradation function, MHC-I trafficking, and antigen presentation activity.ImportanceUbiquitin-proteasome system (UPS) is essential for degradation of damaged proteins, which takes place in proteasome. Upon cytokine inductions, proteasome catalytic activities are replaced by distinct isoforms resulting in formation of immunoproteasome. Immunoproteasome generates peptides for MHC-I antigen presentation and plays important roles in immune responses. HIV-1 is the agent of AIDS, and HIV-1-specific CTLs represent immune responses to limit viral replication. This study reveals a distinct mechanism by which HIV-1 promotes immune evasion. Viral protein Nef interacts with immunoproteasome component LMP7 to attenuate immunoproteasome formation and protein degradation function, and repress MHC-I antigen presentation activity. Therefore, HIV-1 targets LMP7 to inhibit immunoproteasome activation and LMP7 may be used as a target for the development of anti-HIV-1/AIDS therapy.

One of the fundamental reactions of the innate immune responses to pathogen infection is the release of pro-inflammatory cytokines, including IL-1β, processed by the NLRP3 inflammasome. STING is essential for innate immune responses and inflammasome activation. Here we reveal a distinct mechanism by which STING regulates the NLRP3 inflammasome activation, IL-1β secretion, and inflammatory responses in human cell lines, mice primary cells, and mice. Interestingly, upon HSV-1 infection and cytosolic DNA stimulation, STING binds to NLRP3 and promotes the inflammasome activation through two approaches. First, STING recruits NLRP3 and promotes NLRP3 translocation to the endoplasmic reticulum, thereby facilitating the inflammasome formation. Second, STING interacts with NLRP3 and removes K48- and K63-linked polyubiquitination of NLRP3, thereby promoting the inflammasome activation. Collectively, we demonstrate that the cGAS-STING-NLRP3 signaling is essential for host defense against DNA virus infection.

Dengue virus (DENV) infection causes serious clinical symptoms, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular permeability change is the main feature of the diseases, and the abnormal expression of proinflammatory cytokines is the important cause of vascular permeability change. However, the mechanism underlying vascular permeability induced by DENV has not been fully elucidated. Here, we reveal a distinct mechanism by which DENV infection promotes NLRP3 inflammasome activation and interleukin-1 beta (IL-1β) release to induce endothelial permeability and vascular leakage in mice. DENV M protein interacts with NLRP3 to facilitate NLRP3 inflammasome assembly and activation, which induce proinflammatory cytokine IL-1β activation and release. Notably, M can induce vascular leakage in mouse tissues by activating the NLRP3 inflammasome and IL-1β. More importantly, inflammatory cell infiltration and tissue injuries are induced by M in wild-type (WT) mouse tissues, but they are not affected by M in NLRP3 knockout (NLRP3(−/−)) mouse tissues. Evans blue intensities in WT mouse tissues are significantly higher than in NLRP3(−/−) mouse tissues, demonstrating an essential role of NLRP3 in M-induced vascular leakages in mice. Therefore, we propose that upon DENV infection, M interacts with NLRP3 to facilitate inflammasome activation and IL-1β secretion, which lead to the induction of endothelial permeability and vascular leakage in mouse tissues. The important role of the DENV-M-NLRP3-IL-1β axis in the induction of vascular leakage provides new insights into the mechanisms underlying DENV pathogenesis and DENV-associated DHF and DSS development. IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen, and infections by this virus are prevalent in over 100 tropical and subtropical countries or regions, with approximately 2.5 billion people at risk. DENV infection induces a spectrum of clinical symptoms, ranging from classical dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Therefore, it is important to understand the mechanisms underlying DENV pathogenesis. In this study, we reveal that the DENV membrane protein (M) interacts with the host NLRP3 protein to promote NLRP3 inflammasome activation, which leads to the activation and release of a proinflammatory cytokine, interleukin-1 beta (IL-1β). More importantly, we demonstrate that M protein can induce vascular permeability and vascular leakage and that NLRP3 is required for M-induced vascular leakage in mouse tissues. Collectively, this study reveals a distinct mechanism underlying DENV pathogeneses and provides new insights into the development of therapeutic agents for DENV-associated diseases.

Human hepatitis B virus core protein (HBc) is a structural protein of the hepatitis B virus (HBV) and contributes to HBV regulation of host-cell transcription. However, the mechanisms of transcriptional regulation remain poorly characterized. To dissect the function of HBc, a yeast two-hybrid was performed to identify HBc-binding proteins, and the C-terminal of BRG1/hBRM-associated factors 200 (BAF200C) was identified. Then, the existence of HBc interactions with BAF200C and full-length BAF200 was confirmed via co-immunoprecipitation assays in 293T, HepG2 and HepG2-NTCP cells. Furthermore, we show that the binding between HBc and BAF200 was of vital importance to HBc mediated downregulation of interferon-induced transmembrane protein 1 (IFITM1) expression, and the mechanisms for the downregulation were disclosed as follows. Basal level of IFITM1 expression depends on BAF200, rather than the JAK&ndash, STAT1 pathway. The interaction of HBc with BAF200 disturbs the stability of the polybromo-associated BAF (PBAF) complex and results in the suppression of IFTM1 transcription. Finally, the antiviral effects of IFITM1 on cell proliferation and HBV replication were found to be partially restored when HBc was co-transfected with BAF200. Collectively, our findings indicate that HBc plays a role in HBV resistance against the antiviral activities of IFN&alpha, providing details about HBV evasion of host innate immunity.