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Background: While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM. Methods: We initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay. Results: The herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells – with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir. Conclusion: Our results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.

The incidence of zoonotic diseases, such as coronavirus disease 2019 and Ebola virus disease, is increasing worldwide. However, drug and vaccine development for zoonotic diseases has been hampered because the experiments involving live viruses are limited to high-containment laboratories. The Ebola virus minigenome system enables researchers to study the Ebola virus under BSL-2 conditions. Here, we found that the addition of the nucleocapsid protein of human coronaviruses, such as severe acute respiratory syndrome coronavirus 2, can increase the ratio of green fluorescent protein-positive cells by 1.5–2 folds in the Ebola virus minigenome system. Further analysis showed that the nucleocapsid protein acts as an activator of the Ebola virus minigenome system. Here, we developed an EBOV MiniG Plus system based on the Ebola virus minigenome system by adding the SARS-CoV-2 nucleocapsid protein. By evaluating the antiviral effect of remdesivir and rupintrivir, we demonstrated that compared to that of the traditional Ebola virus minigenome system, significant concentration-dependent activity was observed in the EBOV MiniG Plus system. Taken together, these results demonstrate the utility of adding nucleocapsid protein to the Ebola virus minigenome system to create a powerful platform for screening antiviral drugs against the Ebola virus.

Abstract In obese adults, nonalcoholic fatty liver disease (NAFLD) is accompanied by multiple metabolic dysfunctions. Although upregulated hepatic fatty acid synthesis has been identified as a crucial mediator of NAFLD development, the underlying mechanisms are yet to be elucidated. In this study, we reported upregulated expression of gene related to anergy in lymphocytes (GRAIL) in the livers of humans and mice with hepatic steatosis. Grail ablation markedly alleviated the high-fat diet-induced hepatic fat accumulation and expression of genes related to the lipid metabolism, in vitro and in vivo. Conversely, overexpression of GRAIL exacerbated lipid accumulation and enhanced the expression of lipid metabolic genes in mice and liver cells. Our results demonstrated that Grail regulated the lipid accumulation in hepatic steatosis via interaction with sirtuin 1. Thus, Grail poses as a significant molecular regulator in the development of NAFLD.

Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis.

Tuberculosis (TB) is a leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb). Although progress has been made in TB control, still about 10 million people worldwide develop TB annually and 1.5 million die of the disease. The rapid emergence of aggressive, drug-resistant strains and latent infections have caused TB to remain a global health challenge. TB treatments are lengthy and their side effects lead to poor patient compliance, which in turn has contributed to the drug resistance and exacerbated the TB epidemic. The relatively low output of newly approved antibiotics has spurred research interest toward alternative antibacterial molecules such as silver nanoparticles (AgNPs). In the present study, we use the natural biopolymer alginate to serve as a stabilizer and/or reductant to green synthesize AgNPs, which improves their biocompatibility and avoids the use of toxic chemicals. The average size of the alginate-capped AgNPs (ALG-AgNPs) was characterized as nanoscale, and the particles were round in shape. Drug susceptibility tests showed that these ALG-AgNPs are effective against both drug-resistant Mtb strains and dormant Mtb. A bacterial cell-wall permeability assay showed that the anti-mycobacterial action of ALG-AgNPs is mediated through an increase in cell-wall permeability. Notably, the anti-mycobacterial potential of ALG-AgNPs was effective in both zebrafish and mouse TB animal models in vivo. These results suggest that ALG-AgNPs could provide a new therapeutic option to overcome the difficulties of current TB treatments.

(Background) The coronavirus disease 2019 (COVID-19) that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries high infectivity and mortality. Efficient intervention strategies are urgently needed. Avian immunoglobulin Y (IgY) showed efficacy against viral infection whereas the in vivo efficacy remains unclear. (Methods) We immunized laying hens with S1, S1 receptor-binding domain (S1-RBD), or S2 subunits of the SARS-CoV-2 spike (S) protein. After immunization, IgYs were collected and extracted from the egg yolks. The neutralization potential of IgYs was examined by the plaque reduction neutralization test (PRNT). The bioutility of IgYs was examined in Syrian hamsters in vivo. (Results) IgYs exhibited typical banding patterns in SDS-PAGE and Western blot and were immunoreactive against S1, S1-RBD, and S2 subunits. The plaque reduction neutralization test (PRNT) showed that all purified IgYs potently neutralized different SARS-CoV-2 strains in vitro. In Syrian hamsters, the combination of IgYs for S1-RBD and S2 subunits administered before or after SARS-CoV-2 infection effectively restored body weight loss and reduced intrapulmonary lesions and the amount of immunoreactive N protein-positive cells, which were caused by SARS-CoV-2 infection. (Conclusions) Collectively, IgYs specific for S protein subunits effectively neutralized SARS-CoV-2 in vitro and in vivo and may serve as prophylactic or therapeutic antibodies in the prevention or treatment of COVID-19.

The flexible, anti-fouling, and bionic surface-enhanced Raman scattering (SERS) biochip, which has a Nepenthes peristome-like structure, was fabricated by photolithography, replicated technology, and thermal evaporation. The pattern of the bionic Nepenthes peristome-like structure was fabricated by two layers of photolithography with SU-8 photoresist. The bionic structure was then replicated by polydimethylsiloxane (PDMS) and grafting the zwitterion polymers (2-methacryloyloxyethyl phosphorylcholine, MPC) by atmospheric plasma polymerization (PDMS-PMPC). The phospholipid monomer of MPC immobilization plays an important role; it can not only improve hydrophilicity, anti-fouling and anti-bacterial properties, and biocompatibility, but it also allows for self-driving and unidirectional water delivery. Ag nanofilms (5 nm) were deposited on a PDMS (PDMS-Ag) substrate by thermal evaporation for SERS detection. Characterizations of the bionic SERS chips were measured by a scanning electron microscope (SEM), optical microscope (OM), X-ray photoelectron spectrometer (XPS), Fourier-transform infrared spectroscopy (FTIR), and contact angle (CA) testing. The results show that the superior anti-fouling capability of proteins and bacteria (E. coli) was found on the PDMS-PMPC substrate. Furthermore, the one-way liquid transfer capability of the bionic SERS chip was successfully demonstrated, which provides for the ability to separate samples during the flow channel, and which was detected by Raman spectroscopy. The SERS intensity (adenine, 10−4 M) of PDMS-Ag with a bionic structure is ~4 times higher than PDMS-Ag without a bionic structure, due to the multi-reflection of the 3D bionic structure. The high-sensitivity bionic SERS substrate, with its self-driving water capability, has potential for biomolecule separation and detection.

On analyzing the results of cell-based assays, we have previously shown that perilla (Perilla frutescens) leaf extract (PLE), a food supplement and orally deliverable traditional Chinese medicine approved by the Taiwan Food and Drug Administration, effectively inhibits SARS-CoV-2 by directly targeting virions. PLE was also found to modulate virusinduced cytokine expression levels. In this study, we explored the anti-SARS-CoV-2 activity of PLE in a hamster model by examining viral loads and virus-induced immunopathology in lung tissues. Experimental animals were intranasally challenged with different SARS-CoV-2 doses. Jugular blood samples and lung tissue specimens were obtained in the acute disease stage (3-4 post-infection days). As expected, SARS-CoV-2 induced lung inflammation and hemorrhagic effusions in the alveoli and perivascular areas; additionally, it increased the expression of several immune markers of lung injury -- including lung Ki67-positive cells, Iba-1-positive macrophages, and myeloperoxidase-positive neutrophils. Virus-induced lung alterations were significantly attenuated by orally administered PLE. In addition, pretreatment of hamsters with PLE significantly reduced viral loads and immune marker expression. A purified active fraction of PLE was found to confer higher antiviral protection. Notably, PLE prevented SARS-CoV-2-induced increase in serum markers of liver and kidney function as well as the decrease in serum high-density lipoprotein and total cholesterol levels in a dose-dependent fashion. Differently from lung pathology, monitoring of serum biomarkers in Syrian hamsters may allow a more humane assessment of the novel drugs with potential anti-SARS-CoV-2 activity. Our results expand prior research by confirming that PLE may exert an in vivo therapeutic activity against SARS-CoV-2 by attenuating viral loads and lung tissue inflammation, which may pave the way for future clinical applications. [ABSTRACT FROM AUTHOR]