Expression and Purification of Recombinant SARS-CoV-2 Accessory Protein ORF7a and Functional Analysis of Its Role in Up-Regulating Cytokine Production

The severity of coronavirus disease 2019 is closely linked to dysregulated immune responses. The search for viral proteins associated with immune regulation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to reveal the pathogenicity of the virus. In this study, accessory proteins ORF7a (referred to as ORF7a-1 and ORF7a-2, respectively) from two SARS-related coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2, were produced through the denaturing and refolding of inclusion body proteins. The recombinant protein was incubated with alveolar epithelial cells, and the transcription and expression levels of major cytokines were determined by reverse transcription-quantitative PCR and enzyme-linked immunosorbent assay. SARS-related coronavirus ORF7a can up-regulate the transcription and expression of interleukin-6, C-C motif chemokine ligand 8, interferon α, and interferon β. The results also indicated that the two highly conserved ORF7a had certain differences in promoting the transcription and expression of cytokines. The study showed that ORF7a is a virus-encoded immune regulator by alveolar epithelial cells that plays an important role in the pathogenicity of SARS-related coronaviruses.

Speaking-Induced Charge-Laden Face Masks with Durable Protectiveness and Wearing Breathability.

Face masks, which serve as personal protection equipment, have become ubiquitous for combating the ongoing COVID-19. However, conventional electrostatic-based mask filters are disposable and short-term effective with high breathing resistance, causing respiratory ailments and massive consumption (129 billion monthly), intensifying global environmental pollution. In an effort to address these challenges, the introduction of a piezoelectric polymer was adopted to realize the charge-laden melt-blown via the melt-blowing method. The charge-laden melt-blown could be applied to manufacture face masks and to generate charges triggered by mechanical and acoustic energy originated from daily speaking. Through an efficient and scalable industrial melt-blown process, our charge-laden mask is capable of overcoming the inevitable electrostatic attenuation, even in a high-humidity atmosphere by long-wearing (prolonging from 4 to 72 h) and three-cycle common decontamination methods. Combined with outstanding protective properties (PM2.5 filtration efficiency >99.9%), breathability (differential pressure <17 Pa/cm2), and mechanical strength, the resultant charge-laden mask could enable the decreased replacement of masks, thereby lowering to 94.4% of output masks worldwide (∼122 billion monthly) without substituting the existing structure or assembling process.

Exploring the Influencing Factors of the Recreational Utilization and Evaluation of Urban Ecological Protection Green Belts for Urban Renewal: A Case Study in Shanghai.

With the continuous expansion of urban construction land, the green belts aiming for ecological protection have ensured a sustainable and effective function of regional ecosystem services. At the same time, these ecological green belts are expected to develop their compound service potentials with the development of cities. In order to meet the increasing demand of urban residents for the recreational utilization of urban green space, the primary function of the ecological green belts has transformed from being purely ecological to a combination of being ecological and recreational. Based on social media data, which has the characteristics of a large amount of accessible geographic information, this study used multiple regression models to analyze the recreational utilization intensity of ecological protection green belts with a case study in the green belt of Shanghai, China. The research results showed that the internal elements (total external area, water area, etc.) of the Shanghai green belt have positive correlations with its recreational utilization. The impact of external factors was inconclusive on the recreational utilization of the outer forest belt (the number of subway stations in accessibility factors was negatively correlated; the number of cultural facilities and the number of restaurants in the surrounding service facilities were positively related). Combined with the "Shanghai City Master Plan (2017-2035)", this study suggests potential zones for the recreational transformation of the Shanghai green belt, provides a theoretical and practical basis for improving the recreational utilization of an urban ecological protection green belt and contributes to the sustainable development of ecological protection green belts in high-density cities.

Comprehensive engineering of a therapeutic neutralizing antibody targeting SARS-CoV-2 spike protein to neutralize escape variants.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

Low Humoral Immune Response and Ineffective Clearance of SARS-Cov-2 in a COVID-19 Patient With CLL During a 69-Day Follow-Up

Background: A recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), which began in Wuhan, China, with a high level of human-to-human transmission has been reported. There are limited data available on Coronavirus Disease 2019 (COVID-19) patients with hematological malignancies with more than 60 days of follow-up. This study describes the clinical characteristics, including multiple recurrences of COVID-19, in a patient with chronic lymphocytic leukemia (CLL) during 69 days of follow-up. Case Presentation: A 72-year-old female was admitted to hospital isolation after being infected with COVID-19 as part of a family cluster on January 30, 2020. Apart from SARS-Cov-2 virus infection, laboratory results revealed lymphocytosis of uncertain etiology and abnormal distribution of T lymphocytes. On blood smears, small blue lymphocytes with scant cytoplasm were observed, and the presence of high levels of circulating clonal B cells was also demonstrated by flow cytometry. The patient was diagnosed with COVID-19 and CLL. Among her family members, she had the highest viral loads and the fastest progression on lung injury and developed severe pneumonia. Serological results showed she had both 2019-nCoV-specific IgM and IgG antibodies;however, only IgG antibodies were detected in her husband's plasma. Results: A combination regimen of antiviral therapy and high-dose intravenous immunoglobulin (IVIG) in the early stage seemed to be effective for treating CLL and SARS-Cov-2 infection. Because of the low humoral immune response, the CLL patient could not effectively clear the SARS-Cov-2 infection and suffered from recurrence twice during the 69-day follow-up. Conclusion: In CLL, a neoplastic antigen-specific B-cell clone proliferates, and the progeny cells accumulate and outgrow other B cells, leading to immune deficiency. Considering the low humoral immune response and ineffective clearance of SARS-Cov-2 in CLL patients, the follow-up and home quarantine period should be extended. We need further studies to clarify suspending or continuing CLL therapy during COVID infection. For those patients who are prone to progression to severe disease, administering humoral immunity therapies can help to prevent disease progression and quickly meet the cure criteria.

Uncertain regression model with autoregressive time series errors.

Uncertain regression model is a powerful analytical tool for exploring the relationship between explanatory variables and response variables. It is assumed that the errors of regression equations are independent. However, in many cases, the error terms are highly positively autocorrelated. Assuming that the errors have an autoregressive structure, this paper first proposes an uncertain regression model with autoregressive time series errors. Then, the principle of least squares is used to estimate the unknown parameters in the model. Besides, this new methodology is used to analyze and predict the cumulative number of confirmed COVID-19 cases in China. Finally, this paper gives a comparative analysis of uncertain regression model, difference plus uncertain autoregressive model, and uncertain regression model with autoregressive time series errors. From the comparison, it is concluded that the uncertain regression model with autoregressive time series errors can improve the accuracy of predictions compared with the uncertain regression model.

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants.

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

Clinical characteristics and outcome of hemodialysis patients with COVID-19: a large cohort study in a single Chinese center.

BACKGROUND: Novel coronavirus disease (COVID-19) is spreading rapidly, which poses great challenges to patients on maintenance hemodialysis. Here we report the clinical features of 66 hemodialysis patients with laboratory confirmed COVID-19 infection. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Retrospective, single-center case series of the 66 hemodialysis patients with confirmed COVID-19 from 1 January to 5 March 2020; the final date of follow-up was 25 March 2020. RESULTS: The clinical data were collected from 66 hemodialysis patients with confirmed COVID-19. The incidence of COVID-19 in our center was 11.0% (66/602), of which 18 patients died. According to different prognosis, hemodialysis patients with COVID-19 were divided into the survival and death group. A higher incidence of fever and dyspnea was found in the death group compared with the survival group. Meanwhile, patients in the death group were often accompanied by higher white blood cell count, prolonged PT time, increased D-dimer (p < .05). More patients in the death group showed hepatocytes and cardiomyocytes damage. Furthermore, logistic regression analysis suggested that fever, dyspnea, and elevated D-dimer were independent risk factors for death in hemodialysis patients with COVID-19 (OR, 1.077; 95% CI, 1.014 to 1.439; p = .044; OR, 1.146; 95% CI, 1.026 to 1.875; p = .034, OR, 4.974; 95% CI, 3.315 to 6.263; p = .007, respectively). CONCLUSIONS: The potential risk factors of fever, dyspnea, and elevated D-dimer could help clinicians to identify hemodialysis patients with poor prognosis at an early stage of COVID-19 infection.

Clinical characteristics of deceased hemodialysis patients affected by COVID-19.

BACKGROUND: The recent outbreak of Coronavirus Disease 2019 (COVID-19) is a public health emergency of international concern. In China, Wuhan, Hubei Province was the epicenter. The disease is more severe in patients with high comorbidities and dialysis patients fall into this category. METHODS: In this report, we reviewed the whole course of the epidemic emerged in the HD center of Wuhan NO.1 Hospital by 28 February 2020. We compared the differences on the epidemiological characteristics and clinical features between patients surviving from COVID-19 and patients who died. RESULT: In this hospital, at time of the present report, 627 patients were on chronic hemodialysis and the prevalence of affected cases was 11.8% (74/627).The median age of the COVID-19-positive patients was 63 years (range 31-88), with an almost even gender distribution (females accounted for 54.4%).The most frequent presenting symptom was cough (41.9%), followed by fatigue (24.2%), fever (17.2%) and dyspnea (14.8%); 22.4% of the cases were and asymptomatic. Fourteen of the 74 patients died (19%), as for presenting symptoms, cough was more frequent in patients who died (P < 0.05). Surviving patients had higher levels of phosphate and albumin, and lower levels of C-reactive protein (CRP). Chest CT scan was positive in all cases, including in asymptomatic ones, and revealed in about three fourths of the cases bilateral (76.2%) lesions; in each lung lesions were multiple in about half of the cases of the cases (52.3%). After diagnosis, median time to death was 7 days in the 14 patients who died, with a range between 4 and 18 days. CONCLUSION: This preliminary, single Center study identifies hemodialysis patients as a population at high risk of severe, and deadly COVID-19 infection. While classic baseline clinical conditions, including age, kidney disease and gender, are not significantly associated with survival in COVID-19 infected hemodialysis, our study also suggests a significant association between risk of and death, poor nutritional status and less than optimal metabolic balance.

Cross-cultural adaptation, validity, and reliability of the Chinese version of the Nurses' Perceptions of Disaster Core Competencies Scale (NPDCC).

BACKGROUND: In recent years, disasters occurred frequently all over the world, and the role of nurses in public health emergencies and disaster emergencies was highlighted under the background of the covid19 epidemic. However, there was a lack of education and evaluation. Our study aims to cross-cultural adapt the Nurses' Perceptions of Disaster Core Competencies Scale (NPDCC) and evaluate the reliability and validity of the Chinese version. METHODS: We translated the scale following the translation-integration-back translation-expert review procedure, adapted according to Chinese culture. We evaluated the reliability and validity of the scale, and a total sample of 911 nurse data from the Yangtze River Delta Regional Nursing Alliance Hospital was gathered. RESULTS: The Chinese version of NPDCC included 45 items, 5 factors (critical thinking skills, special diagnostic skills, general diagnostic skills, technical skills, and communication skills) were extracted from the analysis, which could explain the 68.289% of the total variance. The content validity index was 0.925. The Cronbach's α of the total NPDCC score was 0.978, and 0.884-0.945 for every factor. The split-half for the scale was 0.930, and every factor was 0.861-0.894. CONCLUSIONS: The Chinese version of NPDCC has excellent reliability and validity, and it is suitable to measure nurses' perceptions of disaster core competencies in China. The next step is to promote the application in a large scale.

Low Humoral Immune Response and Ineffective Clearance of SARS-Cov-2 in a COVID-19 Patient With CLL During a 69-Day Follow-Up.

Background: A recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), which began in Wuhan, China, with a high level of human-to-human transmission has been reported. There are limited data available on Coronavirus Disease 2019 (COVID-19) patients with hematological malignancies with more than 60 days of follow-up. This study describes the clinical characteristics, including multiple recurrences of COVID-19, in a patient with chronic lymphocytic leukemia (CLL) during 69 days of follow-up. Case Presentation: A 72-year-old female was admitted to hospital isolation after being infected with COVID-19 as part of a family cluster on January 30, 2020. Apart from SARS-Cov-2 virus infection, laboratory results revealed lymphocytosis of uncertain etiology and abnormal distribution of T lymphocytes. On blood smears, small blue lymphocytes with scant cytoplasm were observed, and the presence of high levels of circulating clonal B cells was also demonstrated by flow cytometry. The patient was diagnosed with COVID-19 and CLL. Among her family members, she had the highest viral loads and the fastest progression on lung injury and developed severe pneumonia. Serological results showed she had both 2019-nCoV-specific IgM and IgG antibodies; however, only IgG antibodies were detected in her husband's plasma. Results: A combination regimen of antiviral therapy and high-dose intravenous immunoglobulin (IVIG) in the early stage seemed to be effective for treating CLL and SARS-Cov-2 infection. Because of the low humoral immune response, the CLL patient could not effectively clear the SARS-Cov-2 infection and suffered from recurrence twice during the 69-day follow-up. Conclusion: In CLL, a neoplastic antigen-specific B-cell clone proliferates, and the progeny cells accumulate and outgrow other B cells, leading to immune deficiency. Considering the low humoral immune response and ineffective clearance of SARS-Cov-2 in CLL patients, the follow-up and home quarantine period should be extended. We need further studies to clarify suspending or continuing CLL therapy during COVID infection. For those patients who are prone to progression to severe disease, administering humoral immunity therapies can help to prevent disease progression and quickly meet the cure criteria.

Serum Cystatin C and Coronavirus Disease 2019: A Potential Inflammatory Biomarker in Predicting Critical Illness and Mortality for Adult Patients.

This study aimed at determining the relationship between baseline cystatin C levels and coronavirus disease 2019 (COVID-19) and investigating the potential prognostic value of serum cystatin C in adult patients with COVID-19. 481 patients with COVID-19 were consecutively included in this study from January 2, 2020, and followed up to April 15, 2020. All clinical and laboratory data of COVID-19 patients with definite outcomes were reviewed. For every measure, COVID-19 patients were grouped into quartiles according to the baseline levels of serum cystatin C. The highest cystatin C level was significantly related to more severe inflammatory conditions, worse organ dysfunction, and worse outcomes among patients with COVID-19 (P values < 0.05). In the adjusted logistic regression analyses, the highest cystatin C level and ln-transformed cystatin C levels were independently associated with the risks of developing critically ill COVID-19 and all-cause death either in overall patients or in patients without chronic kidney disease (P values < 0.05). As a potential inflammatory marker, increasing baseline levels of serum cystatin C might independently predict adverse outcomes for COVID-19 patients. Serum cystatin C could be routinely monitored during hospitalization, which showed clinical importance in prognosticating for adult patients with COVID-19.

Association of procalcitonin levels with the progression and prognosis of hospitalized patients with COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) was first announced in Wuhan, and has rapidly evolved into a pandemic. However, the risk factors associated with the severity and mortality of COVID-19 are yet to be described in detail. Methods: We retrospectively reviewed the information of 1525 cases from the Leishenshan Hospital in Wuhan. Univariate and multivariate Cox regression analyses were generated to explore the relationship between procalcitonin (PCT) level and the progression and prognosis of COVID-19. Univariate and multivariate logistic regression analyses were performed to explore the relationship between disease severity in hospitalized patients and their PCT levels. Survival curves and the cumulative hazard function for COVID-19 progression were conducted in the two groups. To further detect the relationship between the computed tomography score and survival days, curve-fitting analyses were performed. Results: Patients in the elevated PCT group had a higher incidence of severe and critical severity conditions (P < 0.001), death, and higher computed tomography (CT) scores. There was an association between elevated PCT levels and mortality in the univariate ((hazard ratio [1], 3.377; 95% confidence interval [2], 1.012-10.344; P = 0.033) and multivariate Cox regression analysis (HR, 4.933; 95% CI, 1.170-20.788; P = 0.030). Similarly, patients with elevated PCT were more likely to have critically severe disease conditions in the univariate (odds ratio [2], 7.247; 95% CI, 3.559-14.757; P < 0.001) and multivariate logistic regression analysis (OR, 10.679; 95% CI, 4.562-25.000; P < 0.001). Kaplan-Meier curves showed poorer prognosis for patients with elevated PCT (P = 0.024). The CT score 1 for patients with elevated PCT peaked at day 40 following the onset of symptoms then decreased gradually, while their total CT score was relatively stable. Conclusion: PCT level was shown as an independent risk factor of in-hospital mortality among COVID-19 patients. Compared with inpatients with normal PCT levels, inpatients with elevated PCT levels had a higher risk for overall mortality and critically severe disease. These findings may provide guidance for improving the prognosis of patients with critically severe COVID-19.