Dual-Gene-Controlled Rolling Circle Amplification Strategy for SARS-CoV-2 Analysis.

The development of sensitive, accurate, and conveniently operated methods for the simultaneous assay of two nucleic acids is promising while still challenging. In this work, by using two genes (the N gene and RdRp gene) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we have designed an ingenious dual-gene-controlled rolling circle amplification (RCA) strategy to propose an accurate and sensitive electrochemical method. Specifically, the coexistence of the two target genes can trigger the RCA reaction to generate a number of repeated G-quadruplex (G4)-forming sequences. These sequences then switch into G4/hemin complexes with redox activity after the incubation of hemin, which can catalyze the TMB/H2O2 substrates to produce significantly enhanced current responses. Experimental results reveal that the proposed method exhibits satisfying feasibility and analytical performance, enabling the sensitive detection of SARS-CoV-2 in the range of 0.1-5000 pM, with the detection limit of 57 fM. Meanwhile, because only the simultaneous existence of the two target genes can effectively trigger the downstream amplification reaction, this method can effectively avoid false-positives and ensure specificity as well as accuracy. Furthermore, our method can distinguish the COVID-19 samples from healthy people, and the outcomes show a satisfying agreement with the results of RT-PCR, manifesting that our label-free dual-gene-controlled RCA strategy exhibits great possibility in clinical application.

A new PEDV strain CH/HLJJS/2022 can challenge current detection methods and vaccines.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) variant strains cause great economic losses to the global swine industry. However, vaccines do not provide sufficient protection against currently circulating strains due to viral mutations. This study traced the molecular characteristics of the most recent isolates in China and aimed to provide a basis for the prevention and treatment of PEDV. METHODS: We obtained samples from a Chinese diarrheal swine farm in 2022. Reverse transcription polymerase chain reaction and immunofluorescence were used to determine the etiology, and the full-length PEDV genome was sequenced. Nucleotide similarity was calculated using MEGA to construct a phylogenetic tree and DNASTAR. Mutant amino acids were aligned using DNAMAN and modeled by SWISS-MODEL, Phyre2 and FirstGlance in JMOL for protein tertiary structure simulation. Additionally, TMHMM was used for protein function prediction. RESULTS: A PEDV virulent strain CH/HLJJS/2022 was successfully isolated in China. A genome-wide based phylogenetic analysis suggests that it belongs to the GII subtype, and 96.1-98.9% homology existed in the whole genomes of other strains. For the first time, simultaneous mutations of four amino acids were found in the highly conserved membrane (M) and nucleocapsid (N) proteins, as well as eight amino acid mutations that differed from the vast majority of strains in the spike (S) protein. Three of the mutations alter the S-protein spatial structure. In addition, typing markers exist during strain evolution, but isolates are using the fusion of specific amino acids from multiple variant strains to add additional features, as also demonstrated by protein alignments and 3D models of numerous subtype strains. CONCLUSION: The newly isolated prevalent strain CH/HLJJS/2022 belonged to the GII subtype, and thirteen mutations different from other strains were found, including mutations in the highly conserved m and N proteins, and in the S1° and COE neutralizing epitopes of the S protein. PEDV is breaking through original cognitions and moving on a more complex path. Surveillance for PEDV now and in the future and improvements derived from mutant strain vaccines are highly warranted.

An electrochemical biosensor for SARS-CoV-2 detection via its papain-like cysteine protease and the protease inhibitor screening.

The persistent coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still infecting hundreds of thousands of people every day. Enriching the kits for SARS-CoV-2 detection and developing the drugs for patient treatments are still urgently needed for combating the spreading virus, especially after the emergence of various mutants. Herein, an electrochemical biosensor has been fabricated in this work for the detection of SARS-CoV-2 via its papain-like cysteine protease (PLpro) and the screening of protease inhibitor against SARS-CoV-2 by using our designed chimeric peptide-DNA (pDNA) nanoprobes. Utilizing this biosensor, the sensitive and specific detection of SARS-CoV-2 PLpro can be conducted in complex real environments including blood and saliva. Five positive and five negative patient throat swab samples have also been tested to verify the practical application capability of the biosensor. Moreover, we have obtained a detection limit of 27.18 fM and a linear detection range from 1 pg mL-1 to 10 µg mL-1 (I = 1.63 + 4.44 lgC). Meanwhile, rapid inhibitor screening against SARS-CoV-2 PLpro can be also obtained. Therefore, this electrochemical biosensor has the great potential for COVID-19 combating and drug development.

An extracellular matrix biosensing mimetic for evaluating cathepsin as a host target for COVID-19.

To combat the new virus currently ravaging the whole world, every possible anti-virus strategy should be explored. As the main strategy of targeting the virus itself is being frustrated by the rapid mutation of the virus, people are seeking an alternative "host targeting" strategy: neutralizing proteins in the human body that cooperate with the virus. The cathepsin family is such a group of promising host targets, the main biological function of which is to digest the extracellular matrix (ECM) to clear a path for virus spreading. To evaluate the potential of cathepsin as a host target, we have constructed a biosensing interface mimicking the ECM, which can detect cathepsin from 3.3 pM to 33 nM with the limit of detection of 1 pM. Based on our quantitative analysis enabled by this biosensing interface, it is clear that patients with background diseases such as chronic inflammation and tumor, tend to have higher cathepsin activity, confirming the potential of cathepsin to serve as a host target for combating COVID-19 virus.

Safety and immunogenicity of COVID-19 vaccination in patients with hepatocellular carcinoma (CHESS-NMCID 2101): A multicenter prospective study.

Data on safety and immunogenicity of coronavirus disease 2019 (COVID-19) vaccinations in hepatocellular carcinoma (HCC) patients are limited. In this multicenter prospective study, HCC patients received two doses of inactivated whole-virion COVID-19 vaccines. The safety and neutralizing antibody were monitored. Totally, 74 patients were enrolled from 10 centers in China, and 37 (50.0%), 25 (33.8%), and 12 (16.2%) received the CoronaVac, BBIBP-CorV, and WIBP-CorV, respectively. The vaccines were well tolerated, where pain at the injection site (6.8% [5/74]) and anorexia (2.7% [2/74]) were the most frequent local and systemic adverse events. The median level of neutralizing antibody was 13.5 (interquartile range [IQR]: 6.9-23.2) AU/ml at 45 (IQR: 19-72) days after the second dose of vaccinations, and 60.8% (45/74) of patients had positive neutralizing antibody. Additionally, lower γ-glutamyl transpeptidase level was related to positive neutralizing antibody (odds ratio = 1.022 [1.003-1.049], p = 0.049). In conclusion, this study found that inactivated COVID-19 vaccinations are safe and the immunogenicity is acceptable or hyporesponsive in patients with HCC. Given that the potential benefits may outweigh the risks and the continuing emergences of novel severe acute respiratory syndrome coronavirus 2 variants, we suggest HCC patients to be vaccinated against COVID-19. Future validation studies are warranted.

Target-triggered cascade signal amplification for sensitive electrochemical detection of SARS-CoV-2 with clinical application.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the outbreak of the 2019 coronavirus (COVID-19) disease, which greatly challenges the global economy and health. Simple and sensitive diagnosis of COVID-19 at the early stage is important to prevent the spread of pandemics. Herein, we have proposed a target-triggered cascade signal amplification in this work for sensitive analysis of SARS-CoV-2 RNA. Specifically, the presence of SARS-CoV-2 RNA can trigger the catalytic hairpin assembly to generate plenty of DNA duplexes with free 3'-OH termini, which can be recognized and catalyzed by the terminal deoxynucleotidyl transferase (TdT) to generate long strand DNA. The prolonged DNA can absorb substantial Ru(NH3)63+ molecules via electrostatic interaction and produce an enhanced current response. The incorporation of catalytic hairpin assembly and TdT-mediated polymerization effectively lowers the detection limit to 45 fM, with a wide linear range from 0.1 pM to 3000 pM. Moreover, the proposed strategy possesses excellent selectivity to distinguish target RNA with single-base mismatched, three-base mismatched, and random sequences. Notably, the proposed electrochemical biosensor can be applied to analyze targets in complex circumstances containing 10% saliva, which implies its high stability and anti-interference. Moreover, the proposed strategy has been successfully applied to SARS CoV-2 RNA detection in clinical samples and may have the potential to be cultivated as an effective tool for COVID-19 diagnosis.

Safety and immunogenicity of SARS-CoV-2 vaccines in Chinese patients with cirrhosis: a prospective multicenter study.

BACKGROUND: Data on safety and immunogenicity of coronavirus disease 2019 (COVID-19) vaccination in patients with compensated (C-cirrhosis) and decompensated cirrhosis (D-cirrhosis) are limited. METHODS: In this prospective multicenter study, adult participants with C-cirrhosis and D-cirrhosis were enrolled and received two doses of inactivated whole-virion COVID-19 vaccines. Adverse events were recorded within 14 days after any dose of vaccination, and serum samples of enrolled patients were collected and tested for SARS-CoV-2 neutralizing antibodies at least 14 days after the second dose. Risk factors for negative neutralizing antibody were analyzed. RESULTS: In total, 553 patients were enrolled from 15 centers in China, including 388 and 165 patients with C-cirrhosis and D-cirrhosis. The vaccines were well tolerated, most adverse reactions were mild and transient, and injection site pain (23/388 [5.9%] vs 9/165 [5.5%]) and fatigue (5/388 [1.3%] vs 3/165 [1.8%]) were the most frequently local and systemic adverse events in both the C-cirrhosis and D-cirrhosis groups. Overall, 4.4% (16/363) and 0.3% (1/363) of patients were reported Grades 2 and 3 alanine aminotransferase (ALT) elevations (defined as ALT > 2 upper limit of normal [ULN] but ≤ 5 ULN, and ALT > 5 ULN, respectively). The positive rates of COVID-19 neutralizing antibodies were 71.6% (278/388) and 66.1% (109/165) in C-cirrhosis and D-cirrhosis groups. Notably, Child-Pugh score of B and C levels was an independent risk factor of negative neutralizing antibody. CONCLUSIONS: Inactivated COVID-19 vaccinations are safe with acceptable immunogenicity in cirrhotic patients, and Child-Pugh score of B and C levels is associated with hyporesponsive to COVID-19 vaccination.

Aptamer-Functionalized Nanochannels for One-Step Detection of SARS-CoV-2 in Samples from COVID-19 Patients.

With the outbreak of COVID-19, which is fast transmitting and highly contagious, the development of rapid, highly specific, and sensitive detection kits has become a research hotspot. The existing assay methods for SARS-CoV-2 are mainly based on enzymatic reactions, which require expensive reagents, hindering popular use, especially in resource-constrained areas. Herein, we propose an aptamer-based method for the assay of SARS-CoV-2 via binding of the spike protein using functionalized biomimetic nanochannels. To get the analogous effect of human ACE2, a receptor for the spike protein, the aptamer to bind to the spike S1 protein has been first screened by a SELEX technique and then immobilized on the previously prepared nanochannels. In the presence of SARS-CoV-2, the changes in steric hindrance and charge density on the surface of the nanochannels will affect the ion transport, along with a rapid electrochemical response. Our method has been successfully applied to detect the viral particles in clinical pharyngeal swab specimens in one step without sample treatment. We expect this rapid, reagent-free, and sensitive assay method to be developed as a useful tool for diagnosing COVID-19.

Electrochemical detection of ACE2 as a biomarker for diagnosis of COVID-19 and potential male infertility.

Knowing how heavily the body is burdened by SARS-CoV-2 infection is all important to avoid tragic outcomes. This demands fast and convenient assays with minimum requirement for instruments and reagents. Therefore, a short synthetic peptide is developed to perform direct serum assay, using portable hand-held potentiostat, in a reagent-less manner. The target is angiotensin converting enzyme 2 (ACE2), a protein secreted by the body into the blood to restrict viral invasion. Specifically, under electrochemical potential scanning, the peptide can covalently capture ACE2 from the serum, and then form a covalent gel-like 2D protein network with the serum proteins, in an ACE2-specific fashion. This formation of a covalent biosensing complex enables sensitive detection in serum samples of SARS-CoV-2 infected patients. The detected serum level of ACE2 can not only serve as an index of viral load, but may also hint at the associated risk of potential male infertility. These results may point to field application of this simple design in the clinical practice in treating COVID-19 in the near future.

"Covalent biosensing" enables a one-step, reagent-less, low-cost and highly robust assay of SARS-CoV-2.

We have established a new protocol for detecting severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) using a peptidomimetic to covalently detect a viral marker protease.

Visual naked-eye detection of SARS-CoV-2 RNA based on covalent organic framework capsules.

The ongoing outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted that new diagnosis technologies are crucial for controlling the spread of the disease. Especially in the resources-limit region, conveniently operated detection methods such as "naked-eye" detection are urgently required that no instrument is needed. Herein, we have designed a novel and facile strategy to fabricate covalent organic framework (COF) capsules, which can be utilized to establish a new colorimetric assay for naked-eye detection of SARS-CoV-2 RNA. Specifically, we employ the digestible ZIF-90 as the sacrificial template to prepare the hollow COF capsules for horseradish peroxidase (HRP) encapsulation. The fabricated COF capsules can provide an appropriate microenvironment for the enzyme molecules, which may improve the conformational freedom of enzymes, enhance the mass transfer, and endow the enzyme with high environmental resistance. With such design, the proposed assay exhibits outstanding analytical performance for the detection of SARS-CoV-2 RNA in the linear range from 5 pM to 50 nM with a detection limit of 0.28 pM which can go parallel to qTR-PCR analysis. Our method also possesses excellent selectivity and reproducibility. Moreover, this method can also be served to analyze the clinical samples, and can successfully differentiate COVID-19 patients from healthy people, suggesting the promising potential in clinical diagnosis.

Thiol-sensitive probe enables dynamic electrochemical assembly of serum protein for detecting SARS-Cov-2 marker protease in clinical samples.

The poor situational awareness about the spreading of the virus especially in the underdeveloped regions calls for novel virus assays of low cost and simple operation. Currently, such assays are exclusively restricted to nucleic acid detection. In this investigation, a virus protein serum assay has been proposed in a one-step and reagent-less route. Specifically, in this assay, the main protease of the virus is targeted by a short probe mimicking its substrate. While the probe-protein interaction brings them together, a fluorescent thiol targeting molecule reacts with the free thiol groups on the target protein near the probe, generating a fluorescence signal proportional to the concentration of the target. This induces an electroactive 2D peptide nano-network on the sensing surface only in the presence of the target protein. The sensitivity of the method is enhanced through potential electrochemical scanning during incubation with serum samples. The successful detection of the virus marker protein in the serum of the infected patients encourages further development of incorporation of this method into clinical practice.

Sustainable innovation in the context of organizational cultural diversity: The role of cultural intelligence and knowledge sharing.

With the in-depth development of globalization, individuals are increasingly embedded in a culturally diverse environment. Effective communication and management ability (Cultural Intelligence) of employees in this type of diverse and heterogeneous environment impacts behavior and performance, affecting the sustainable innovation ability of organizations. Researchers have not yet fully assessed the impact of individuals' cross-cultural management ability on sustainable innovation. Using Cultural Intelligence Theory and Trait Activation Theory, this paper discusses the influence of individual cultural intelligence on sustainable innovation behavior. The results showed that employees' cultural intelligence positively affected their sustainable innovation behavior. Employee knowledge sharing plays an mediating role between intelligence and behavior. Differences in organizational culture have a negative moderating effect on the impact of employees' cultural intelligence on knowledge sharing and sustainable innovation behaviors. The research results provide theoretical guidance for managing organizational cultural diversity and advancing cultural intelligence and sustainable innovation behaviors among employees.

An electrochemical biosensor for sensitive analysis of the SARS-CoV-2 RNA.

The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is continuously worsening globally, herein we have proposed an electrochemical biosensor for the sensitive monitoring of SARS-CoV-2 RNA. The presence of target RNA firstly triggers the catalytic hairpin assembly circuit and then initiates terminal deoxynucleotidyl transferase-mediated DNA polymerization. Consequently, a large number of long single-stranded DNA products can be produced, and these negatively charged DNA products will bind a massive of positively charged electroactive molecular of Ru(NH3)63+ due to the electrostatic adsorption. Therefore, significantly amplified electrochemical signals can be generated for sensitive analysis of SARS-CoV-2 RNA in the range of 0.1-1000 pM with the detection limit as low as 26 fM. Besides the excellent distinguishing ability for SARS-CoV-2 RNA against single-base mismatched RNA, the proposed biosensor can also be successfully applied to complex matrices, as well as clinical patient samples with high stability, which shows great prospects of clinical application.