Distal Protein-Protein Interactions Contribute to SARS-CoV-2 Main Protease Substrate Binding and Nirmatrelvir Resistance (preprint)

SARS-CoV-2 main protease, Mpro, is responsible for the processing of the viral polyproteins into individual proteins, including the protease itself. Mpro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g. E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g. P252L, T21I, L50F), which restore the fitness of viral replication. Although the mechanism of resistance for the active site mutations is apparent, the role of the non-active site mutations in fitness rescue remains elusive. In this study, we use the model system of a Mpro triple mutant (L50F/E166A/L167F) that confers not only nirmatrelvir drug resistance but also a similar fitness of replication compared to the wild-type both in vitro and in vivo. By comparing peptide and full-length Mpro protein as substrates, we demonstrate that the binding of Mpro substrate involves more than residues in the active site. In particular, L50F and other non-active site mutations can enhance the Mpro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of Mpro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying Mpro and substrate interactions, and offers important insights into Mpro function, resistance development, and inhibitor design.

Restrained memory CD8+ T cell responses favors viral persistence and elevated IgG responses in patients with severe Long COVID. (preprint)

During the COVID-19 pandemic it was widely described that certain individuals infected by SARS-CoV-2 experience persistent disease signs and symptoms, Long COVID, which in some cases is very severe with life changing consequences. To maximize our chances of identifying the underpinnings of this illness, we have focused on 121 of the most severe cases from >1000 patients screened in specialized clinics in Sweden and Belgium. We restricted this study to subjects with objective measures of organ damage or dysfunction, >3 months following a verified, but mild-to-moderate SARS-CoV-2 infection. By performing systems-level immunological testing and comparisons to controls fully convalescent following a similar mild/moderate COVID-19 episode, we identify elevated serological responses to SARS-CoV-2 in severe Long COVID suggestive of chronic antigen stimulation. Persistent viral reservoirs have been proposed in Long COVID and using multiple orthogonal methods for detection of SARS-CoV-2 RNA and protein in plasma we identify a subset of patients with detectable antigens, but with minimal overlap across assays, and no correlation to symptoms or immune measurements. Elevated serologic responses to SARS-CoV-2 on the other hand were inversely correlated with clonally expanded memory CD8+ T cells, indicating that restrained clonal expansion enables viral persistence, chronic antigen exposure and elevated IgG responses, even if antigen-detection in blood is not universally possible.

Investigation and analysis of current situation of rehabilitation therapists in Hunan province after the COVID-19 epidemic: a cross-sectional study (preprint)

Background This study aims to comprehend the prevailing circumstances of rehabilitation therapists in the aftermath of the COVID-19 outbreak and to analysis the variables that affect the job satisfaction of these therapists who volunteered to take part in this study. In total, 769 rehabilitation were enrolled in the study.Method Based on the experts' discussions and revisions at the Xiangya Boai Rehabilitation Hospital, this questionnaire was subsequently distributed to rehabilitation therapists via WeChat. rehabilitation were sampled using a cross-sectional approach on Wenjuanxing through the WeChat APP.Results Based on an analysis of data distribution and composition ratio, it is evident that there have been significant transformations in multiple data dimensions between pre-pandemic therapy and the current era. Comparative analyses revealed statistically significant differences in age (H = 32.90, P < 0.001), years of working experience (H = 32.14, P < 0.001), job title (H = 29.93, P < 0.001), and monthly salary level (H = 114.81, P < 0.001). Further analyses using multiple factor regression indicated that monthly income significantly influenced the outcome measure. Participants with a monthly income below 3,000 yuan had a significantly higher Odds Ratio (OR) of 17.9 (OR = 17.95, 95%CI 8.49–38.29, P < 0.001) for experiencing job dissatisfaction compared to those with a monthly income exceeding 10,000 yuan. Similarly, therapists earning between 3,000 and 5,000 yuan monthly had an OR of 5.3 (OR = 5.32, 95%CI 2.91–9.83, P < 0.001) for job dissatisfaction, while those in the 5,001–7,000 yuan income range had an OR of 2.46 (OR = 2.46, 95%CI 1.36–4.49, P < 0.05).Conclusion After the epidemic, the distribution of age, gender, hospital type, and job title among rehabilitation therapists has changed. The job classification of therapists has become more refined, and the monthly income level has become an important factor affecting job satisfaction. The forefront of this academic endeavor lies in carefully investigate the existing landscape that defines rehabilitation therapists in Hunan, which will provide a deeper insight into whole China's community of therapists.

Design of SARS-CoV-2 papain-like protease inhibitor with antiviral efficacy in a mouse model (preprint)

The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PLpro) is a promising but challenging drug target. In this study, we designed and synthesized 85 noncovalent PLpro inhibitors that bind to the newly discovered Val70Ub site and the known BL2 groove pocket. Potent compounds inhibited PLpro with inhibitory constant Ki values from 13.2 to 88.2 nM. The co-crystal structures of PLpro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC50 from 0.44 to 2.02 microM. Oral treatment with Jun12682 significantly improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting PLpro inhibitors are promising oral SARS-CoV-2 antiviral candidates.

SARS-CoV-2 NSP13 suppresses the Hippo pathway downstream effector YAP (preprint)

The Hippo pathway plays critical roles in tissue development, regeneration, and immune homeostasis. The widespread pandemic of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has resulted in a global healthcare crisis and strained health resources. How SARS-CoV-2 affects Hippo signaling in host cells has remained poorly understood. Here, we report that SARS-CoV-2 infection in patient lung cells and cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) suppressed YAP target gene expression, as evidenced by RNA sequencing data. Furthermore, in a screening of nonstructural proteins from SARS-CoV-2, nonstructural protein 13 (NSP13) significantly inhibited YAP transcriptional activity independent of the YAP upstream suppressor kinase Lats1/2. Consistent with this, NSP13 suppressed active YAP (YAP5SA) in vivo, whereby NSP13 expression reverted the phenotype of YAP5SA transgenic mice. From a mechanistic standpoint, NSP13 helicase activity was shown to be required for its suppression of YAP. Furthermore, through the interaction of NSP13 with TEAD4, which is the most common YAP-interacting transcription factor in the nucleus, NSP13 recruited endogenous YAP suppressors such as CCT3 and TTF2 to inactivate the YAP/TEAD4 complex. These findings reveal the function and mechanism of the SARS-CoV-2 helicase NSP13 in host cells and partially explain the toxic effect of SARS-CoV-2 in particular host tissue with high YAP activity.

CD4+ T cell senescence is associated with reduced reactogenicity in severe/critical COVID-19 (preprint)

Background: Aging is a critical risk factor for unfavorable clinical outcomes among COVID-19 patients and may affect vaccine efficacy. However, whether the senescence of T cells impact the progression to severe COVID-19 in the elderly individuals remains unclear. Methods: By using flow cytometry, we analyzed the frequency of senescent T cells (Tsens) in the peripheral blood from 100 elderly patients hospitalized for COVID-19 and compared the difference between mild/moderate and severe/critical illness. We also assessed correlations between the percentage of Tsens and the quantity and quality of spike-specific antibodies by ELISA, neutralizing antibody test kit and Elispot assay respectively, cytokine production profile of COVID-19 reactive T cells as well as plasma soluble factors by cytometric bead array (CBA). Results: We found a significant elevated level of CD4+ Tsens in severe/critical disease compared to mild/moderate illness and patients with a higher level of CD4+ Tsens (>19.78%) showed a decreased survival rate as compared to those with a lower level (<19.78%), especially in the breakthrough infection. The percentage of CD4+ Tsens was negatively correlated with spike-specific antibody titers, neutralization ability and COVID-19 reactive IL-2+ CD4+ T cells. Additionally, IL-2 producing T cells and plasma levels of IL-2 were positively correlated with antibody levels. Conclusion: Our data illustrated that the percentage of CD4+ Tsens in the peripheral blood could act as an efficient biomarker for the capacity of spike-specific antibody production and the prognosis of severe COVID-19, especially in the breakthrough infection. Therefore, restoration of the immune response of CD4+ Tsens is one of the key factors to prevent severe illness and improve vaccine efficacy in older adults.

First close insight into global daily gapless 1 km PM2.5 pollution, driving factors, and health impact (preprint)

Fine particulate matter (PM2.5) is the largest environmental risk factor impacting human health. While PM2.5 has been measured widely across the world, there has been no high-resolution and gapless global PM2.5 data on a daily scale. We generate a global daily PM2.5 concentration at 1 km resolution using satellite gap-filled aerosol products and machine learning. Daily PM2.5 retrievals agreed well with ground measurements, with sample-, space-, and time-based cross-validated correlations of 0.93, 0.89, and 0.88, respectively. This enables us to unprecedentedly monitor the day-to-day variations of PM2.5, exposure risk, and mortality burden around the globe. More than 96% of the days exceeded the World Health Organization (WHO) recommended daily air quality guidelines (AQG) level (15 μg m-3) in 2020, and 99% of populated areas were exposed to PM2.5 risk at least one day; in particular, the proportions are 91% and 64% similarly in 7 and 30 days, respectively. The annual population-weighted mean PM2.5 concentration was 27.6 μg m-3 (~5.5 times higher than the WHO annual AQG level of 5 μg m-3), resulting in estimated premature deaths of ~4.2 million people and accounting for ~6.6% of total global deaths. Substantial differences are noted in many parts of the world between 2019 and 2020 associated with widespread episodes of wildfires or the COVID-19 shutdowns. The overall air quality in 2020 was significantly better than in 2019 in more than 70% of major cities. The global population-weighted mean PM2.5 decreased by ~5.1%, and the associated number of premature deaths dropped by 56,700.

Safety and Efficacy of Oral administrated Cepharathine in Non-hospitalized, asymptomatic or mild COVID-19 patients: A Double-blind, Randomized, Placebo-controlled Trial (preprint)

Cepharanthine (CEP) is a natural remedy that potently inhibits SARS-CoV-2 activity both in vitro and in vivo. We conducted a proof-of-concept, double-blind, randomized, placebo-controlled trial among adults with asymptomatic or mild coronavirus disease 2019 (COVID-19). Patients were stratified randomly to de novo infection or viral rebound, and assigned in a 1:1:1 ratio to receive 60 mg/day or 120 mg/day of CEP or placebo. Primary outcome the time from randomization to negative nasopharyngeal swab, and safety were evaluated. A total of 262 de novo infected and 124 viral rebound patients underwent randomization. In the 188 de novo patients included in modified intention-to-treat (mITT) population, when compared with placebo, 60 mg/day CEP slightly shortened the time to negative (difference=-0.77 days, hazard ratio (HR)=1.40, 95% CI 0.97 to 2.01, p=0.072), and 120 mg/day CEP did not show the trend. Among de novo patients in the per-protocol set (PPS), 60 mg/day CEP significantly shortened the time to negative (difference=-0.87 days, HR=1.56, 95% CI 1.03 to 2.37, p=0.035). Among viral rebound patients in the mITT population, neither 120 mg/day nor 60 mg/day CEP significantly shortened the time to negative compared to placebo. Adverse events were not different among the three groups, and no serious adverse events occurred. Treatment of asymptomatic or mild Covid-19 with 120 mg/day or 60 mg/day CEP did not shorten the time to negative compared with placebo, without evident safety concerns. Among de novo infected patients with good compliance, 60 mg/day CEP significantly shortened the time to negative compared with placebo ( NCT05398705 ).

1:4 case-control study on Coronavirus disease 2019

Objective: To analyze the risk factors of COVID-19 infection in Danzhou, Hainan province.

Practices, Knowledge, and Attitudes of Chinese University Students Toward Traditional Chinese Herbal Medicine for the Control of COVID-19.

Background: The application of traditional Chinese herbal medicine has been officially recommended and strongly promoted in China as an important complement to conventional prevention and treatment for COVID-19. Capturing the practices, knowledge and attitudes of young adult population toward using Chinese herbs for COVID-19 is important for understanding the future of Chinese herbal medicine over the coming decades. Methods: This cross-sectional questionnaire-based study was conducted from May to June, 2022, among 313 student volunteers in Wuhan University of Science and Technology, a provincial comprehensive university in China. Results: Results showed that only 18% of students had used Chinese herbs to prevent COVID-19. The main information sources were social media, the students' family members, relatives, friends, etc. as well as the healthcare professionals. However, most students only sometimes paid close attention to related reports and news articles in social media. Clear pharmacological and toxicological properties of herbs were considered by 43% students as the most important factor to promote their own application of Chinese herbs to fight COVID-19. The mean knowledge score was 1.64 out of 5. Students' overall attitudes toward application of Chinese herbal medicine to fight COVID-19 were very positive. Conclusion: These data suggests most university young adults had a positive attitude but lack of practices and knowledge towards traditional Chinese herbal medicine for COVID-19 control.

Rituximab successful in COVID-19 vaccine-induced refractory pemphigus vulgaris (preprint)

Coronavirus disease 2019 (COVID-19) is the most prevalent disease in society today, and vaccination is a powerful weapon against COVID-19. However, there have been many recent reports of vaccine-induced skin side effects. Autoimmune bullous reactions caused by the COVID-19 vaccine are rare. We identified a case of refractory PV associated with COVID-19 vaccination that improved after 2 RTX injections.

Carbon Emission Measurement of Urban Green Passenger Transport: A Case Study of Qingdao

Urban passenger transport is one of the most significant sources of fossil energy consumption and greenhouse gas emission, especially in developing countries. The rapid growth of urban transport makes it a critical target for carbon reduction. This paper establishes a method for calculating carbon emission from urban passenger transport including ground buses, private cars, cruising taxis, online-hailing taxis, and rail transit. The scope of the study is determined according to the transportation mode and energy type, and the carbon emission factor of each energy source is also determined according to the local energy structure, etc. Taking into consideration the development trend of new energy vehicles, a combination of “top-down” and “bottom-up” approaches is used to estimate the carbon dioxide emission of each transportation mode. The results reveal that carbon emission from Qingdao’s passenger transport in 2020 was 8.15 million tons, of which 84.31% came from private cars, while the share of private cars of total travel was only 45.66%. Ground buses are the most efficient mode of transport. Fossil fuels emit more greenhouse gases than other clean energy sources. The emission intensity of hydrogen fuel cell buses is better than that of other fuel type vehicles. Battery electric buses have the largest sensitivity coefficient, therefore the carbon emission reduction potentially achieved by developing battery electric buses is most significant.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations (preprint)

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations (preprint)

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir (preprint)

The SARS-CoV-2 main protease (Mpro) is a cysteine protease and a validated antiviral drug target. Paxlovid is an FDA-approved oral COVID-19 antiviral that contains the Mpro inhibitor nirmatrelvir and the metabolic booster ritonavir. The emergence of SARS-CoV-2 variants mutations in the Mpro raised the alarm of potential drug resistance. In this study, we aim to discover Mpro drug resistant mutants from naturally observed polymorphisms. Through analyzing the SARS-CoV-2 sequences deposited in Global initiative on Sharing Avian influenza Data (GISAID) database, we identified 66 prevalent Mpro mutations located at the nirmatrelvir binding site. The Mpro mutant proteins were expressed and characterized for enzymatic activity and nirmatrelvir inhibition. While the majority of the Mpro mutants had reduced enzymatic activity (kcat/Km >10-fold decrease), 11 mutants including S144M/F/A/G/Y, M165T, E166Q, H172Q/F, and Q192T/S/V showed comparable enzymatic activity as the wild-type (kcat/Km <10-fold change) and resistance to nirmatrelvir (Ki > 10-fold increase). We further demonstrate that the enzymatic activity and inhibitor resistance of these single mutations can be enhanced by additional substitutions in a double mutant. X-ray crystal structures were determined for six of the single mutants with and/or without GC-376/nirmatrelvir. The structures illustrate how mutations can reduce ligand binding by impacting the conformational stability of the active site. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance.

Specific Pupylation as IDEntity Reporter (SPIDER) for the identification of Protein-Biomolecule interactions (preprint)

Protein-biomolecule interactions play pivotal roles in almost all biological processes, the identification of the interacting protein is essential. By combining a substrate-based proximity labelling activity from the pupylation pathway of Mycobacterium tuberculosis , and the streptavidin (SA)-biotin system, we developed S pecific P upylation as IDE ntity R eporter (SPIDER) for identifying protein-biomolecular interactions. As a proof of principle, SPIDER was successfully applied for global identification of interacting proteins, including substrates for enzyme (CobB), the readers of m 6 A, the protein interactome of mRNA, and the target proteins of drug (lenalidomide). In addition, by SPIDER, we identified SARS-CoV-2 Omicron variant specific receptors on cell membrane and performed in-depth analysis for one candidate, Protein-g. These potential receptors could explain the differences between the Omicron variant and the Prototype strain, and further serve as target for combating the Omicron variant. Overall, we provide a robust technology which is applicable for a wide-range of protein-biomolecular interaction studies.

Interaction Context-Aware Network Behavior Anomaly Detection for Discovering Unknown Attacks

Network behavior anomaly detection is an effective approach to discover unknown attacks, where generating high-efficacy network behavior representation is one of the most crucial parts. Nowadays, complicated network environments and advancing attack techniques make it more challenging. Existing methods cannot yield satisfied representations that express the semantics of network behaviors comprehensively. To tackle this problem, we propose XNBAD, a novel unsupervised network behavior anomaly detection framework, in this work. It integrates the timely high-order host states under the dynamic interaction context with the conversation patterns between hosts for behavior representation. High-order states can better summarize latent interaction patterns, but they are hard to be obtained directly. Therefore, XNBAD utilizes a graph neural network (GNN) to automatically generate high-order features from series of extracted base ones. We evaluated the detection performance of XNBAD in a publicly available benchmark dataset ISCX-2012. To report detailed and precise experimental results, we carefully refined the dataset before evaluation. The results show that XNBAD discovered various attack behaviors more effectively, and it significantly outperformed the existing representative methods by at least 3.8% relative improvement in terms of the overall weighted AUC.

Invalidation of dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) as SARS-CoV-2 main protease inhibitors and the discovery of PGG as a papain-like protease inhibitor (preprint)

The COVID-19 pandemic spurred a broad interest in antiviral drug discovery. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are attractive antiviral drug targets given their vital roles in viral replication and modulation of host immune response. Structurally disparate compounds were reported as Mpro and PLpro inhibitors from either drug repurposing or rational design. Two polyphenols dieckol and 1,2,3,4,6-pentagalloylglucose (PGG) were recently reported as SARS-CoV-2 main protease (Mpro) inhibitors. With our continuous interest in studying the mechanism of inhibition and resistance of Mpro inhibitors, we report herein our independent validation/invalidation of these two natural products. Our FRET-based enzymatic assay showed that neither dieckol nor PGG inhibited SARS-CoV-2 Mpro (IC50 > 20 µM), which is in contrary to previous reports. Serendipitously, PGG was found to inhibit the SARS-CoV-2 papain-like protease (PLpro) with an IC50 of 3.90 µM. The binding of PGG to PLpro was further confirmed in the thermal shift assay. However, PGG was cytotoxic in 293T-ACE2 cells (CC50 = 7.7 µM), so its intracellular PLpro inhibitory activity could not be quantified by the cell-based Flip-GFP PLpro assay. In addition, we also invalidated ebselen, disulfiram, carmofur, PX12, and tideglusib as SARS-CoV-2 PLpro inhibitors using the Flip-GFP assay. Overall, our results call for stringent hit validation, and the serendipitous discovery of PGG as a putative PLpro inhibitor might worth further pursuing.