Chikungunya fever (CF) is a mosquito-borne viral disease caused by Chikungunya virus (CHIKV) that is being increasingly reported in previously non-endemic areas, including Guangdong, China. Despite the low mortality rate associated with CF, some patients may develop chronic joint pain that persists for months or years. Moreover, due to the similarities in symptomatology between CHIKV and other arboviruses, clinical diagnosis without laboratory confirmation is inaccurate. The CHIKV envelope protein E1, critical for viral entry and assembly, is a promising diagnostic and therapeutic target. In this study, we expressed the CHIKV E1 protein in Escherichia coli and developed 16 monoclonal antibodies (mAbs) against E1 to establish an enzyme immunoassay (EIA). The EIA specifically detects CHIKV E1 with no cross-reactivity to other major human-pathogenic arboviruses, including dengue virus (DENV). We evaluated its performance in serum samples of 84 CHIKV-infected patients, with qRT-PCR as the reference standard, and compared it with 93 healthy and 94 DENV-1-infected controls. The assay achieved a sensitivity of 94.05% (95% CI: 87.3–97.3) and a specificity of 98.4% (95% CI: 95.3–99.5). This E1 antigen-based EIA enables rapid and accurate diagnosis of CHIKV infection to facilitate the control of outbreaks of this emerging arbovirus.

Neutralising monoclonal antibody (mAb) is an important weapon in our arsenal for combating respiratory viral infections. However, the effectiveness of neutralising mAb has been impeded by the rapid emergence of mutant variants. Early administration of broad-spectrum mAb with improved delivery efficiency can potentially enhance efficacy and patient outcomes. WKS13 is a humanised mAb which was previously demonstrated to exhibit broad-spectrum activity against SARS-CoV-2 variants. In this study, a dual targeting formulation strategy was designed to deliver WKS13 to both the nasal cavity and lower airways, the two critical sites of infection caused by SARS-CoV-2. Dry powders of WKS13 were first prepared by spray drying, with cyclodextrin used as stabiliser excipient. Two-fluid nozzle (TFN) was used to produce particles below 5 μm for lung deposition (C-TFN formulation) and ultrasonic nozzle (USN) was used to produce particles above 10 μm for nasal deposition (C-USN formulation). Gel electrophoresis and size exclusion chromatography studies showed that the structural integrity of mAb was successfully preserved with no sign of aggregation after spray drying. To achieve dual targeting property, C-TFN and C-USN were mixed at various ratios. The aerosolisation property of the mixed formulations dispersed from a nasal powder device was examined using a Next Generation Impactor (NGI) coupled with a glass expansion chamber. When the ratio of C-TFN in the mixed formulation increased, the fraction of particles deposited in the lung increased proportionally while the fraction of particles deposited in the nasal cavity decreased correspondingly. A customisable aerosol deposition profile could therefore be achieved by manipulating the mixing ratio between C-TFN and C-USN. Dual administration of C-TFN and C-USN powders to the lung and nasal cavity of hamsters, respectively, was effective in offering prophylactic protection against SARS-CoV-2 Delta variant. Viral loads in both the lung tissues and nasal wash were significantly reduced, and the efficacy was comparable to systemic administration of unformulated WKS13. Overall, dual targeting powder formulation of neutralising mAb is a promising approach for prophylaxis of respiratory viral infections. The ease and non-invasive administration of dual targeting nasal powder may facilitate the widespread distribution of neutralising mAb during the early stage of unpredictable outbreaks.

Introduction: Therapeutic monoclonal antibodies (mAbs) against the SARSCoV-2 spike protein have been shown to improve the outcome of severe COVID-19 patients in clinical trials. However, novel variants with spike protein mutations can render many currently available mAbs ineffective.Methods: We produced mAbs by using hybridoma cells that generated from mice immunized with spike protein trimer and receptor binding domain (RBD). The panel of mAbs were screened for binding and neutralizing activity against different SARSCoV-2 variants. The in vivo effectiveness of WKS13 was evaluated in a hamster model.Results: Out of 960 clones, we identified 18 mAbs that could bind spike protein. Ten of the mAbs could attach to RBD, among which five had neutralizing activity against the ancestral strain and could block the binding between the spike protein and human ACE2. One of these mAbs, WKS13, had broad neutralizing activity against all Variants of Concern (VOCs), including the Omicron variant. Both murine or humanized versions of WKS13 could reduce the lung viral load in hamsters infected with the Delta variant.Conclusions: Our data showed that broad-spectrum high potency mAbs can be produced from immunized mice, which can be used in humans after humanization of the Fc region. Our method represents a versatile and rapid strategy for generating therapeutic mAbs for upcoming novel variants.

Although γδ-T cell-based tumor immunotherapy using phosphoantigens to boost γδ-T cell immunity has shown success in some cancer patients, the clinical application is limited due to the rapid exhaustion of Vγ9Vδ2-T cells caused by repetitive stimulation from phosphoantigens and the profoundly immunosuppressive tumor microenvironment (TME). In this study, using a cell culture medium containing human and viral interleukin-10 (hIL-10 and vIL-10) secreted from EBV-transformed lymphoblastoid B cell lines (EBV-LCL) to mimic the immunosuppressive TEM, we found that the antitumor activity of Vγ9Vδ2-T cells was highly suppressed by endogenous hIL-10 and vIL-10 within the TME. CD137 costimulation could provide an anti-exhaustion signal to mitigate the suppressive effects of IL-10 in TME by suppressing IL-10R1 expression on Vγ9Vδ2-T cells. CD137 costimulation also improved the compromised antitumor activity of Vγ9Vδ2-T cells in TME with high levels of IL-10 in Rag2-/- γc-/- mice. In humanized mice, CD137 costimulation boosted the therapeutic effects of aminobisphosphonate pamidronate against EBV-induced lymphoma. Our study offers a novel approach to overcoming the obstacle of the hIL-10 and vIL-10-mediated immunosuppressive microenvironment by costimulating CD137 and enhancing the efficacy of γδ-T cell-based tumor therapy.

Numerous serological detection kits are being rapidly developed and approved for screening and diagnosing suspected coronavirus disease 2019 (COVID-19) cases. However, cross-reactivity between pre-existing antibodies against other coronaviruses and the captured antigens in these kits can affect detection accuracy, emphasizing the necessity for identifying highly specific antigen fragments for antibody detection. Thus, we performed a conservation and specificity analysis of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein. We also integrated various B-cell epitope prediction methods to obtain possible dominant epitope regions for the N protein, analyzed the differences in serological antibody levels for different epitopes using ELISA, and identified N protein epitopes for IgG and IgM with high-specificity. The SARS-CoV-2 N protein showed low mutation rates and shared the highest amino acid similarity with SARS-CoV; however, it differed substantially from other coronaviruses. Tests targeting the SARS-CoV-2 N protein produce strong positive results in patients recovering from SARS-CoV. The N18-39 and N183-197 epitopes for IgG and IgM detection, respectively, can effectively overcome cross-reactivity, and even exhibit good specificity between SARS-CoV-2 and SARS-CoV. The antibody levels detected with these were consistent with those detected using the complete N protein. These findings provide a basis for serological diagnosis and determining the kinetics of SARS-CoV-2 antibody detection in patients.

Objective To prepare neutralizing monoclonal antibodies (mAbs) against envelope protein domain III of the four dengue virus serotypes (DENV-EDIII) and identify its specificity. Methods BALB/c mice were immunized with recombinant EDIII protein (rDENV-EDIII) of the four DENV serotypes. Hybridoma cells secreting DENV-EDIII antibodies were screened by indirect ELISA. The specificity of positive hybridoma cells were further tested by indirect immunofluorescence assay (IFA). The neutralizing activities of DENV-EDIII mAbs in vitro were determined by the enzyme-linked immunospot microneutralization test (ELISPOT-MNT). Results 6 mAbs specific for the EDIII of the four DENV serotypes and 11 mAbs specific for only one serotype of DENV-EDIII protein were obtained, of which one monoclonal antibody had a balanced strong neutralizing activity against the four DENV serotypes in vitro, and its 50% inhibitory concentrations (IC50) for the four DENV serotypes was 0.05 μg/mL, 1.89 μg/mL, 0.02 μg/mL, 3.91 μg/mL, respectively. Conclusion A specific monoclonal antibody against DENV-EDIII is successfully screened and obtained to neutralize the four DENV serotypes.

通过对12根CFRP筋珊瑚混凝土梁进行抗弯试验,研究配筋率、珊瑚混凝土强度对CFRP筋珊瑚混凝土梁开裂性能的影响,对裂缝的分布与裂缝宽度的发展规律、卸载后裂缝闭合情况、正常使用极限状态最大裂缝宽度限值对应荷载水平进行了分析.研究结果表明,提高CFRP筋配筋率和提高珊瑚混凝土强度均能有效控制CFRP筋珊瑚混凝土梁的最大裂缝宽度;梁试件卸载后裂缝出现闭合,配筋率越高,裂缝闭合越显著;CFRP筋珊瑚混凝土梁在达到正常使用极限状态时相对其极限承载力状态处于较低的荷载水平,因此其设计主要由正常使用极限状态控制.通过对试验数据的分析,对现有规范GB 50608中的短期最大裂缝宽度计算公式进行了修正,采用修正公式得到的最大裂缝宽度计算值与试验值吻合良好.

为了研究CFRP筋珊瑚混凝土梁变形性能,以珊瑚混凝土强度等级(C20、C30)和CFRP筋配筋率(0.31％、0.47％、0.73％、1.06％、1.37％、1.54％)作为变量,设计了12根CFRP筋珊瑚混凝土梁,通过对CFRP筋珊瑚混凝土梁进行四点弯曲试验,取得了CFRP筋珊瑚混凝土梁的挠度数据并对其进行分析.试验结果表明:梁试件的荷载挠度曲线呈双线性的特征,卸荷后其形变恢复明显,且配筋率越高,形变恢复能力越强.梁试件在达到正常使用极限状态挠度限值时的荷载往往处于较低的荷载水平.在《纤维增强复合材料建设工程应用技术规范》(GB 50608—2010)的基础上,通过对试验数据回归分析,修正了受拉纵筋应变不均匀系数ψ,得出了适用于CFRP筋珊瑚混凝土梁的短期抗弯刚度计算公式,计算结果与试验值吻合较好.

SummaryBACKGROUNDNucleic acid test and antibody assay have been employed in the diagnosis for SARS-CoV-2 infection, but the use of viral antigen for diagnosis has not been successfully developed. Theoretically, viral antigen is the specific marker of the virus and precedes antibody appearance within the infected population. There is a clear need of detection of viral antigen for rapid and early diagnosis.METHODSWe included a cohort of 239 participants with suspected SARS-CoV-2 infection from 7 centers for the study. We measured nucleocapsid protein in nasopharyngeal swab samples in parallel with the nucleic acid test. Nucleic acid test was taken as the reference standard, and statistical evaluation was taken in blind. We detected nucleocapsid protein in 20 urine samples in another center, employing nasopharyngeal swab nucleic acid test as reference standard.RESULTSWe developed a fluorescence immunochromatographic assay for detecting nucleocapsid protein of SARS-CoV-2 in nasopharyngeal swab sample and urine within 10 minutes. 100% of nucleocapsid protein positive and negative participants accord with nucleic acid test for same samples. Further, earliest participant after 3 days of fever can be identified by the method. In an additional preliminary study, we detected nucleocapsid protein in urine in 73.6% of diagnosed COVID-19 patients.CONCLUSIONSThose findings indicate that nucleocapsid protein assay is an accurate, rapid, early and simple method for diagnosis of COVID-19. Appearance of nucleocapsid protein in urine coincides our finding of the SARS-CoV-2 invading kidney and might be of diagnostic value.

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we demonstrated that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids. Ciliated cells, alveolar type 2 (AT2) cells and rare club cells were virus target cells. Electron microscopy captured typical replication, assembly and release ultrastructures and revealed the presence of viruses within lamellar bodies in AT2 cells. Virus infection induced more severe cell death in alveolar organoids than in airway organoids. Additionally, RNA-seq revealed early cell response to SARS-CoV-2 infection and an unexpected downregulation of ACE2 mRNA. Further, compared to the transmembrane protease, serine 2 (TMPRSS2) inhibitor camostat, the nucleotide analog prodrug Remdesivir potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model for SARS-CoV-2 infection and drug discovery.### Competing Interest StatementThe authors have declared no competing interest.

Coronavirus Disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a crude case fatality rate of about 0.5–10 % depending on locality. A few clinically approved drugs, such as remdesivir, chloroquine, hydroxychloroquine, nafamostat, camostat, and ivermectin, exhibited anti-SARS-CoV-2 activity in vitro and/or in a small number of patients. However, their clinical use may be limited by anti-SARS-CoV-2 50 % maximal effective concentrations (EC50) that exceeded their achievable peak serum concentrations (Cmax), side effects, and/or availability. To find more immediately available COVID-19 antivirals, we established a two-tier drug screening system that combines SARS-CoV-2 enzyme-linked immunosorbent assay and cell viability assay, and applied it to screen a library consisting 1528 FDA-approved drugs. Cetilistat (anti-pancreatic lipase), diiodohydroxyquinoline (anti-parasitic), abiraterone acetate (synthetic androstane steroid), and bexarotene (antineoplastic retinoid) exhibited potent in vitro anti-SARS-CoV-2 activity (EC50 1.13–2.01 μM). Bexarotene demonstrated the highest Cmax:EC50 ratio (1.69) which was higher than those of chloroquine, hydroxychloroquine, and ivermectin. These results demonstrated the efficacy of the two-tier screening system and identified potential COVID-19 treatments which can achieve effective levels if given by inhalation or systemically depending on their pharmacokinetics.

Aimed at a new kind of overheated ablated fault on the pole shoe surface of a large tubular hydro-generator, based on the real site characteristics of the fault with combination of a physical field analysis, this paper reveals the occurrence mechanism of such faults from operational conditions, generator structure, material characteristics, and so on. On the basis of the results, the fault treatment and the generator improvement strategy are proposed. In addition, the treatment method has been applied to a faulty generator, and it successfully eliminating the fault. The research is of great significance for restraining the loss and heat of poles in large tubular hydro-generators as well as improving the safety and reliability of the operation of generators and power grids.

Influenza epidemics and pandemics are constant threats to global public health. Although strategies including vaccines and antiviral drugs have achieved great advances in controlling influenza virus infection, the efficacy of these strategies is limited by the highly frequent mutations in the viral genome and the emergence of drug-resistant strains. Our previous study indicated that boosting the immunity of human Vγ9Vδ2-T cells with the phosphoantigen pamidronate could be a therapeutic strategy to treat seasonal and avian influenza virus infections. However, one notable drawback of γδ-T cell-based immunotherapy is the rapid exhaustion of proliferation and effector responses due to repeated treatments with phosphoantigens. Here, we found that the expression of CD137 was inducible in Vγ9Vδ2-T cells following antigenic stimulation. CD137 + Vγ9Vδ2-T cells displayed more potent antiviral activity against influenza virus than their CD137 − counterparts in vitro and in Rag2 -/- γc -/- mice. We further demonstrated that CD137 costimulation was essential for Vγ9Vδ2-T cell activation, proliferation, survival and effector functions. In humanized mice reconstituted with human peripheral blood mononuclear cells, CD137 costimulation with a recombinant human CD137L protein boosted the therapeutic effects of pamidronate against influenza virus. Our study provides a novel strategy of targeting CD137 to improve the efficacy of Vγ9Vδ2-T cell-based immunotherapy.

Background The current coronavirus disease (COVID-19) pandemic has created a pressing need to diagnose and screen a large number of close contacts of confirmed and suspected cases. Numerous nucleic acid detection kits are being rapidly developed and approved for viral etiological diagnosis; however, these are limited by the number of false negatives produced in clinical practice. Therefore, there is an urgent need to establish serological detection methods to serve as supplementary diagnostics. Methods We (1) performed a conservation and specificity analysis of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein, which is the main target of serological diagnosis; (2) integrated various B-cell epitope prediction methods to obtain possible dominant epitope regions for the N protein; (3) applied ELISA to analyze differences in the serological antibody levels for different epitopes; and (4) identified N protein epitopes for IgG and IgM with high specificity. Results SARS-CoV-2 strains showed low mutation rates for the N protein, and the construction of a phylogeny was a good characterization of its molecular evolutionary lineage in relation to other coronaviruses. SARS-CoV-2 showed the closest genetic relationship with SARS-CoV, which showed multiple consecutive long conserved regions at the amino acid level, but differed substantially from other coronaviruses. Tests targeting the SARS-CoV-2 N protein produced strong positive results in SARS-CoV patients in recovery. Of the five epitope dominant regions, using N18-39 and N183-197 for IgG and IgM detection, respectively, can effectively overcome the limitations of cross-reactivity. Conclusions The patients infected with both SARS viruses may exhibit cross-reactivity when using the N protein for antibody detection. However, there are regions of the N protein that can be used for antibody detection and some of these regions showed good specificity even between SARS-CoV-2 and SARS-CoV, and the antibody levels detected were consistent with those detected by the complete N protein. These findings provide a basis for serological diagnosis of SARS-CoV-2 patients, and research ideas for developing vaccines.

ObjectivesRapid, reliable and easy-to-implement diagnostics that can be adapted in early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis are critical to combat the epidemic. SARS-CoV-2 nucleocapsid protein (NP) is an ideal target for viral antigen-based detection. A rapid and convenient method was developed based on fluorescence immunochromatographic (FIC) assay to detect the SARS-CoV-2 NP antigen. However, the accuracy of this diagnostic method needs to be examined.MethodsThis prospective study was carried out between 10 and 15 February 2020 in seven hospitals in Wuhan and one hospital in Chongqing, China. Participants with clinically suspected SARS-CoV-2 infection were enrolled. NP antigen testing by FIC assay and nucleic acid (NA) testing by real-time reverse transcriptase PCR (RT-PCR) were performed simultaneously in a blinded manner with the same nasopharyngeal swab sample. The diagnostic accuracy of NP antigen testing was calculated by taking NA testing of RT-PCR as the reference standard, in which samples with a cycle threshold (Ct) value of ≤40 were interpreted as positive for SARS-CoV-2.ResultsA total of 253 participants were enrolled; two participants were excluded from the analyses because of invalid NP testing results. Of 251 participants (99.2%) included in the diagnostic accuracy analysis, 201 (80.1%) had a Ct value of ≤40. With Ct value 40 as the cutoff of NA testing, the sensitivity, specificity and percentage agreement of the FIC assay was 75.6% (95% confidence interval, 69.0–81.3), 100% (95% confidence interval, 91.1–100) and 80.5% (95% confidence interval, 75.1–84.9) respectively.ConclusionsWith RT-PCR assay as the reference standard, NP antigen testing by FIC assay shows high specificity and relatively high sensitivity in SARS-CoV-2 diagnosis in the early phase of infection.

Acetaminophen (APAP) toxicity is the leading cause of drug-induced liver failure, which is closely related to mitochondrial dysfunction and oxidative damage. Studies in clinical trials and in animal models have shown that omega-3 polyunsaturated fatty acids (n-3 PUFAs) affect the progression of various types of liver damage. Interestingly, the sex-dependent effect of n-3 PUFAs on human health has also been well documented. However, it is unknown whether supplementation of n-3 PUFAs modulates the pathogenesis of APAP-induced liver failure with sex-specificity. Our results showed that both endogenous and exogenous n-3 PUFAs significantly aggravated the APAP-induced liver injury in male mice, whereas the opposite effects were observed in females. In vivo and in vitro studies demonstrated that estrogen contributes to the gender difference in the regulation of n-3 PUFAs on APAP overdose. We found that n-3 PUFA-mediated regulation of hepatic oxidative stress response and autophagy upon APAP challenge is distinct between male and female mice. Moreover, we provided evidence that β-catenin signaling activation is responsible for the sex-dependent regulation of APAP hepatotoxicity by n-3 PUFAs. Together, these findings indicated that supplementation with n-3 PUFAs displays sex-differential effect on APAP hepatotoxicity and could have profound significance in the clinical management for drug-induced liver injury.

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived selforganized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.

To obtain more precise and rational calculation details of the loss and heat of a damper winding in a tubular hydro-generator, this study develops a three-dimensional, finite-element, electromagnetic-temperature field close-coupling model of the damper winding. On the basis of multi-physical field coupled theory, the model fully considers the temperature effects of the damper winding resistivities and heat conductivities, and the eddy current loss in the end region of the damper winding. The model was verified by direct tests of the damper winding temperature. Unlike the conventional weak-coupling model, the proposed close-coupling model fully captures the interaction between the electromagnetic and temperature fields. Therefore, of the model more accurately and reasonably calculates the loss and heat of the damper winding than the conventional model. The proposed calculation model can properly assess the loss and heat of damper windings in large hydro-generators, which is helpful for improving the design standards of hydro-generators.

1 引言 在各类变压器间接试验检测手段发展迅速的今天,吊罩检查在掌握变压器内部状态、监督变压器设计制造工艺质量、保障安装及运检质量方面,仍然具有难以替代的直接性、具体性、精准性与可靠性.特别是对于大多数中小变压器制造商生产的电力主变而言,采取吊罩检查措施,能够最大限度地发现其设计制造工艺缺陷,使得用户能够更好地做到防患于未然,进而能够为变压器运维检修水平的持续提升,提供更为直接的指导和参考.

钢筋连接技术是影响预制装配式结构发展的重要因素之一,对比约束浆锚连接和套筒灌浆连接等传统连接方式,提出一种新型钢筋连接方式,即钢筋灌胶搭接连接.该方式具有可靠的受力性能,且制作成本低、加工简单,有利于推广使用.进行9组18个钢筋灌胶连接件拉拔试验,研究其在不同钢筋搭接长度、套筒尺寸和钢筋直径影响下的破坏过程和破坏形态,研究结果表明钢筋灌胶搭接连接件受力性能可靠.搭接长度对连接件破坏过程和破坏形态的影响最大,当搭接长度≥12d时连接件表现为钢筋断裂破坏.此外,3个应用该连接件的装配式偏压柱拉拔试验结果表明柱破坏截面均出现在后浇段以外的部分,验证了连接件的可靠性.