Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus.

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

Colour Preference and Healing in Digital Roaming Landscape: A Case Study of Mental Subhealth Populations.

From the perspective of emotional preference, studies have been conducted about landscape healing for subhealth people in the National High-Tech Zone (the study area). Combined with a preliminary practice investigation, Unity 2019 was used to make a digital roaming landscape, and 91 subjects with a history of mental subhealth diseases were randomly invited to participate in the immersive experimentation of the Human-Machine Environment Synchronisation (ErgoLAB) platform. After the experiment, the subjects were invited to fill in a Likert scale as the control group. The interest preference, emotion fluctuation, and healing effect of landscape colour were verified. The results show that: (1) The variation trend of interest reflected the concentration of interest in landscape, and the order of degree of interest, ranging from high to low, went Green > Yellow > Blue > Red > Orange > Purple > Cyan. (2) The subjects' interest in landscape colour was correlated with the arousal of positive emotions. The correlation between interest in landscape colour and positive emotions, from high to low, went Blue > Green > Yellow, while the correlation between red, cyan, orange, and purple was not significant. (3) The mean skin conductance (SC) fluctuation variance of subjects was 5.594%, which confirmed that the healing effect of digital roaming landscape scenes was significant under the state of low arousal. According to the Likert scale data, subjects' scores of the healing effect of landscapes, from high to low, went as follows: Green > Yellow > Red > Blue > Cyan > Orange > Purple. The results provide a new method for demonstrating the logical relationship between the digital landscape interest experience-emotional awakening-healing effect and providing a theoretical method and construction scheme for landscape colour configuration in the implementation of landscape healing projects.

Real-Time Temperature Distribution Monitoring in Chinese Solar Greenhouse Using Virtual LAN

The internal air temperature of Chinese solar greenhouse (CSG) has the problem of uneven spatial and temporal distribution. To determine temperature distribution at different locations, we designed a greenhouse temperature real-time monitoring system based on virtual local area network (VLAN) and estimate, including interpolation estimation module, data acquisition, and transmission module. The temperature data were obtained from 24 sensors, and the Ordinary Kriging algorithm estimated the temperature distribution of the whole plane according to the data. The results showed that the real-time temperature distribution monitoring method established was fast and robust. In addition, data validity rate for VLAN technology deployed for data transmission was 2.64% higher than that of cellular network technology. The following results are obtained by interpolation estimation of temperature data using gaussian model. The average relative error (ARE) of estimate, mean absolute error (MAE), root mean square error (RMSE), and determination coefficient (R2) were −0.12 °C, 0.42 °C, 0.56 °C, and 0.9964, respectively. After simple optimization of the number of sensors, the following conclusions are drawn. When the number of sensors were decreased to 12~16, MAE, RMSE, and R2 were 0.40~0.60 °C, 0.60~0.80 °C, and >0.99, respectively. Furthermore, temperature distribution in the greenhouse varied in the east–west and north–south directions and had strong regularity. The calculation speed of estimate interpolation algorithm was 50~150 ms, and greenhouse Temperature Distribution Real-time Monitoring System (TDRMS) realized simultaneous acquisition, processing, and fast estimate.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

How to Effectively Identify Patients With Rifampin-Resistant Tuberculosis in China: Perspectives of Stakeholders Among Service Providers.

To evaluate China's current rifampin-resistant tuberculosis (RR-TB) screening strategy from stakeholders' perspectives, the perceptions, attitudes, and interests of 245 stakeholders from three eastern, central, and western China provinces on RR-TB screening strategies, were investigated through stakeholder survey and interview. The attitudes toward three RR-TB screening strategies were statistically different: inclination to choose who to screen (Z = 98.477; P < 0.001), funding for rapid diagnostic technology screening either by reimbursed health insurance or directly subsidized financial assistance (Z = 4.142, P < 0.001), and respondents' attitude during RR-TB screening implementation levels (Z = 2.380, P = 0.017). In conclusion, RR-TB screening scope could be expanded by applying rapid diagnostic technologies. Provinces with different economic status could adjust their screening policies accordingly.

Comprehensive and Integrative Analysis of Two Novel SARS-CoV-2 Entry Associated Proteases CTSB and CTSL in Healthy Individuals and Cancer Patients.

More than 200 million people have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 4 million deaths have been reported worldwide to date. Cathepsin B/cathepsin L (CTSB/L) are SARS-CoV-2 entry-associated proteases and facilitate SARS-CoV-2 to infect host cells. However, the expressions of CTSB/L in healthy individuals and cancer patients remain not fully elucidated yet. Here, we comprehensively profiled the expressions and distributions of CTSB/L in human normal tissues, cancer tissues, and cell lines. Moreover, we compared CTSB/L expressions between various cancers and matched normal tissues, and investigated their genetic alteration and prognostic values in pan-cancer. Finally, we also explored the correlation between CTSB/L expressions and immune infiltration. We found that CTSB was highly expressed in most tissues, and CTSL was highly expressed predominantly in the digestive, urinary, and respiratory systems, such as the lungs, liver and gallbladder, and kidney tissues in the translational level. Moreover, cancer patients may be more susceptible to SARS-CoV-2 infection. Our data suggested that CTSB/L are overexpressed in aerodigestive and genitourinary cancers when compared with that in matched normal tissues, and their expressions were closely related to the prognosis of some cancer types. Interestingly, CTSB/L expressions were significantly correlated with immune cell infiltration in manifold cancer tissues and their corresponding normal tissues. In conclusion, our study shows a comprehensive bioinformatic analysis of two important SARS-CoV-2 entry-related proteases, which could provide a potential indication on prevention of SARS-CoV-2 infection.

Dissemination and Refutation of Rumors During the COVID-19 Outbreak in China: Infodemiology Study.

BACKGROUND: During the outbreak of COVID-19, numerous rumors emerged on the internet in China and caused confusion among the public. However, the characteristics of these rumors in different phases of the epidemic have not been studied in depth, and the official responses to the rumors have not been systematically evaluated. OBJECTIVE: The aims of this study were to evaluate the rumor epidemic and official responses during the COVID-19 outbreak in China and to provide a scientific basis for effective information communication in future public health crises. METHODS: Data on internet rumors related to COVID-19 were collected via the Sina Weibo Official Account to Refute Rumors between January 20 and April 8, 2020, extracted, and analyzed. The data were divided into five periods according to the key events and disease epidemic. Different classifications of rumors were described and compared over the five periods. The trends of the epidemic and the focus of the public at different stages were plotted, and correlation analysis between the number of rumors and the number of COVID-19 cases was performed. The geographic distributions of the sources and refuters of the rumors were graphed, and analyses of the most frequently appearing words in the rumors were applied to reveal hotspots of the rumors. RESULTS: A total of 1943 rumors were retrieved. The median of the response interval between publication and debunking of the rumors was 1 day (IQR 1-2). Rumors in text format accounted for the majority of the 1943 rumors (n=1241, 63.9%); chat tools, particularly WeChat (n=1386, 71.3%), were the most common platform for initial publishing of the rumors (n=1412, 72.7%). In addition to text rumors, Weibo and web pages were more likely to be platforms for rumors released in multimedia formats or in a combination of formats, respectively. Local agencies played a large role in dispelling rumors among social media platforms (1537/1943, 79.1%). There were significant differences in the formats and origins of rumors over the five periods (P<.001). Hubei Province accounted for most of the country's confirmed rumors. Beijing and Wuhan City were the main centers for debunking of disinformation. The words most frequently included in the core messages of the rumors varied by period, indicating shifting in the public's concern. CONCLUSIONS: Chat tools, particularly WeChat, became the major sources of rumors during the COVID-19 outbreak in China, indicating a requirement to establish rumor monitoring and refuting mechanisms on these platforms. Moreover, targeted policy adjustments and timely release of official information are needed in different phases of the outbreak.

Differences between COVID-19 and suspected then confirmed SARS-CoV-2-negative pneumonia: A retrospective study from a single center.

Coronavirus disease 2019 (COVID-19) broke out in Wuhan, Hubei, China in December 2019. Tens thousands of people have been infected with the disease. Our aim was to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive patients from SARS-CoV-2-negative patients. We retrospectively compared the data of COVID-19 patients with those of suspected and confirmed SARS-CoV-2-negative patients (control patients). There were 78 COVID-19 patients and 26 control patients, whose median ages were significantly different (P = .001). The percentage of COVID-19 patients admitting exposure to Wuhan was obviously higher than that of control patients (X2 = 29.130; P < .001). Fever and cough appeared more frequently in COVID-19 patients than in the control patients. The routine blood workup parameters of COVID-19 patients did not change much and their mean counts were in the normal range. There were 38.5% of control patients had higher procalcitonin (PCT) levels than 0.5 ng/mL, which was significantly higher than that percentage of COVID-19 patients (X2 = 22.636; P < .05), and COVID-19 patients were also more likely to have decreased or normal urea and creatinine levels than control patients (X2 = 24.930, 8.480; P < .05).Younger age, exposure to Wuhan, fever, cough, and slight changes in routine blood workup parameters, urea and creatinine were important features discriminating COVID-19 from control patients. Slightly increased, but far less than 0.5 ng/mL, PCT levels also differentiated COVID-19 patients from control patients.