A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.

Currently, SARS-CoV-2 has caused a global pandemic and threatened many lives. Although SARS-CoV-2 mainly causes respiratory diseases, growing data indicate that SARS-CoV-2 can also invade the central nervous system (CNS) and peripheral nervous system (PNS) causing multiple neurological diseases, such as encephalitis, encephalopathy, Guillain-Barré syndrome, meningitis, and skeletal muscular symptoms. Despite the increasing incidences of clinical neurological complications of SARS-CoV-2, the precise neuroinvasion mechanisms of SARS-CoV-2 have not been fully established. In this review, we primarily describe the clinical neurological complications associated with SARS-CoV-2 and discuss the potential mechanisms through which SARS-CoV-2 invades the brain based on the current evidence. Finally, we summarize the experimental models were used to study SARS-CoV-2 neuroinvasion. These data form the basis for studies on the significance of SARS-CoV-2 infection in the brain.

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

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Toll-like receptor (TLR) agonists as the potent stimulants of an innate immune system hold promises for applications in anticancer immunotherapy. However, most of them are limited in the clinical translation due to the uncontrolled systemic inflammatory response. In the current study, 1V209, a small molecule TLR7 agonist, was conjugated with cholesterol (1V209-Cho) and prepared into liposomes (1V209-Cho-Lip). 1V209-Cho-Lip exerted minimal toxic effects and enhanced the transportation ability in lymph nodes (LNs) compared with 1V209. 1V209-Cho-Lip treatment inhibited tumor progression in CT26 colorectal cancer, 4T1 breast cancer, and Pan02 pancreatic ductal cancer models through inducing effective DC activation and eliciting CD8 + T cell responses. Furthermore, 1V209-Cho-Lip induced tumor-specific memory immunity to inhibit cancer recurrence and metastasis. These results indicate that cholesterol conjugation with 1V209 is an effective approach to target lymph nodes and to reduce the adverse effects. This work provides a rational basis for the distribution optimization of TLR agonists for potential clinical use.

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell–cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.

The development of animal models for COVID-19 is essential for basic research and drug/vaccine screening. Previously reported COVID-19 animal models need to be established under a high biosafety level condition for the utilization of live SARS-CoV-2, which greatly limits its application in routine research. Here, we generate a mouse model of COVID-19 under a general laboratory condition that captures multiple characteristics of SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) observed in humans. Briefly, human ACE2-transgenic (hACE2) mice were intratracheally instilled with the formaldehyde-inactivated SARS-CoV-2, resulting in a rapid weight loss and detrimental changes in lung structure and function. The pulmonary pathologic changes were characterized by diffuse alveolar damage with pulmonary consolidation, hemorrhage, necrotic debris, and hyaline membrane formation. The production of fatal cytokines (IL-1β, TNF-α, and IL-6) and the infiltration of activated neutrophils, inflammatory monocyte-macrophages, and T cells in the lung were also determined, suggesting the activation of an adaptive immune response. Therapeutic strategies, such as dexamethasone or passive antibody therapy, could effectively ameliorate the disease progression in this model. Therefore, the established mouse model for SARS-CoV-2-induced ARDS in the current study may provide a robust tool for researchers in the standard open laboratory to investigate the pathological mechanisms or develop new therapeutic strategies for COVID-19 and ARDS.

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