A Real-World Retrospective Study on the Efficacy and Safety of Four Antiviral Drugs for Hospitalized COVID-19 Patients: Nirmatrelvir/Ritonavir, Simnotrelvir/Ritonavir, Molnupiravir and Azvudine.

Purpose: This retrospective study aims to compare the effectiveness and safety of four oral antiviral drugs including Simnotrelvir/Ritonavir, Nirmatrelvir/Ritonavir, Azvudine and Molnupiravir in hospitalized patients with Coronavirus Disease 2019 (COVID-19) in a real-world setting, providing evidence to guide clinical practice against COVID-19. Patients and Methods: Patients with mild or moderate COVID-19 hospitalized at Wuxi City's Second People's Hospital during December 2022 to June 2023 were included in this study. Patients were grouped by the antiviral drug received. The primary endpoint was the length of hospital stay. Patients were further divided into subgroups for stratified analysis, considering age, timing of medication, and drug mechanisms, to explore whether these factors could influence the treatment efficacy. Results: Of the enrolled 195 patients receiving any treatment, 42 received Nirmatrelvir/Ritonavir, 33 received Molnupiravir, 81 received Simnotrelvir/Ritonavir, and 39 received Azvudine. Patients in Nirmatrelvir/Ritonavir and Simnotrelvir/Ritonavir groups had significantly shorter hospital stays compared to those in Azvudine group (P < 0.05). No significant difference was observed in hospital stays between those initiating antiviral therapy within or more than five days after symptom onset (P = 0.1109). Among patients with comorbidities, the Nirmatrelvir/Ritonavir and Simnotrelvir/Ritonavir group showed shorter hospital stays than the Azvudine group (P < 0.05). No serious treatment-related adverse events were observed across the groups. Conclusion: In this retrospective study, Nirmatrelvir/Ritonavir and Simnotrelvir/Ritonavir exerts stronger potency on reducing duration of hospital stays in hospitalized patient with COVID-19, suggestive of a better choice for antiviral therapy. Patients who fail to take antiviral drugs in time after symptom onset would still benefit from these antiviral regimens. Additional well-designed clinical trials with large sample size are still needed to further confirm the effectiveness of these antivirals.

Global school plate waste estimates highlight the need for building a sustainable food education system.

Food waste reduction is essential for supporting the sustainability of food systems. Wasteful behaviours are difficult to change after they have been formed, highlighting the importance of early interventions. Here we present an assessment of school plate food waste from 29 countries, and examine the environmental implications, causes, and interventions. School plate waste ranged from 4% to 46% per capita per meal and was positively correlated with country income levels. On a global scale, this waste embodies ∼150 Mha of cropland and ∼770 MtCO2e of greenhouse gas emissions; hence, reducing school plate food waste offers potentially large environmental gains. We propose a comprehensive, multistakeholder framework centred around sustainable food education that cultivates food systems knowledge and skills, and an appreciation for nature and food labour to reduce the psychological distance between youth and their food waste. To effectively implement the framework requires the support and engagement of families, communities and the broader society beyond the confines of schools.

Identification of algicidal monoterpenoids from four chemotypes of Cinnamomum camphora and their algicidal mechanisms on Microcystis aeruginosa.

Cyanobacterial blooms cause serious environmental issues, and plant secondary metabolites are considered as new algaecide for controlling them. Cinnamomum camphora produces a wide spectrum of terpenoids and has 4 main chemotypes, including linalool, camphor, eucalyptol and borneol chemotype. To develop the new cyanobacterial algaecide by using suitable chemotype of Cinnamomum camphora and the main terpenoids, we analyzed the terpenoid composition in the 4 chemotype extracts, evaluated the algicidal effects of the extracts and their typical monoterpenoids on Microcystis aeruginosa, and investigated the algicidal mechanism of the stronger algicidal agents. Among the 4 chemotypes, eucalyptol and borneol chemotype extracts exhibited stronger algicidal effects. In the 4 chemotype extracts, monoterpenoids were the main compounds, of which linalool, camphor, eucalyptol and borneol were the typical components. Among the 4 typical monoterpenoids, eucalyptol and borneol showed stronger algicidal effects, which killed 78.8% and 100% M. aeruginosa cells, respectively, at 1.2 mM after 48 h. In 1.2 mM eucalyptol and borneol treatments, the reactive oxygen species levels markedly increased, and the caspase-3-like activity also raised. With prolonging the treatment time, M. aeruginosa cells gradually shrank and wrinkled, and the cell TUNEL fluorescence intensity and DNA degradation gradually enhanced, indicating that the lethal mechanism is causing apoptosis-like programmed cell death (PCD). Therefore, eucalyptol and borneol chemotype extracts and their typical monoterpenoids have the potential for developing as algaecides to control cyanobacteria through triggering apoptosis-like PCD.

Ferulic Acid Induces Autophagy and Apoptosis in Colon Cancer CT26 Cells via the MAPK Pathway.

Ferulic acid (FA) is a bioactive compound found in traditional Chinese herbal medicine; for example, it is present in Xinjiang Ferula, but also in strong-flavor Chinese baijiu. FA has been shown to play a crucial role in treating oxidative stress, skin whitening, and eye diseases. In this study, the potential role of FA as a means of inducing apoptosis and inhibiting colon cancer induced by the transplantation of CT26 cells was investigated. The results show that FA adjuvant treatment caused an upregulation in the expression of genes related to autophagy while simultaneously suppressing the expression of inflammatory response elements and improving the bodyweight, glutamic pyruvic transaminase (ALT), and glutamic oxaloacetic transaminase (AST) in vivo. Furthermore, FA inhibited the proliferation of CT26 cells and induced apoptosis, specifically by activating the phosphorylation of ERK and JNK to enhance the essential proteins BCL-2 and BAX in the apoptosis pathway. These results suggest that FA could be a promising auxiliary therapeutic agent for the treatment of colon cancer. Further research is needed to better understand the mechanisms underlying the beneficial effects of FA and its synergistic effects with other compounds.

Integration of transcriptomics and proteomics to elucidate inhibitory effect and mechanism of rosmarinic acid from Perilla frutescens (L.) Britt. in treating Trichophyton mentagrophytes.

BACKGROUND: Dermatophyte caused by Trichophyton mentagrophytes is a global disease with a growing prevalence that is difficult to cure. Perilla frutescens (L.) Britt. is an edible and medicinal plant. Ancient books of Traditional Chinese Medicine and modern pharmacological studies have shown that it has potential anti-fungi activity. This is the first study to explore the inhibitory effects of compounds from P. frutescens on Trichophyton mentagrophytes and its mechanism of action coupled with the antifungal activity in vitro from network pharmacology, transcriptomics and proteomics. METHODS: Five most potential inhibitory compounds against fungi in P. frutescens was screened with network pharmacology. The antifungal activity of the candidates was detected by a broth microdilution method. Through in vitro antifungal assays screening the compound with efficacy, transcriptomics and proteomics were performed to investigate the pharmacological mechanisms of the effective compound against Trichophyton mentagrophytes. Furthermore, the real-time polymerase chain reaction (PCR) was applied to verify the expression of genes. RESULTS: The top five potential antifungal compounds in P. frutescens screened by network pharmacology are: progesterone, luteolin, apigenin, ursolic acid and rosmarinic acid. In vitro antifungal assays showed that rosmarinic acid had a favorable inhibitory effect on fungi. The transcriptomic findings exhibited that the differentially expressed genes of fungus after rosmarinic acid intervention were mainly enriched in the carbon metabolism pathway, while the proteomic findings suggested that rosmarinic acid could inhibit the average growth of Trichophyton mentagrophytes by interfering with the expression of enolase in the glycolysis pathway. Comparison of real-time PCR and transcriptomics results showed that the trends of gene expression in glycolytic, carbon metabolism and glutathione metabolic pathways were identical. The binding modes and interactions between rosmarinic acid and enolase were preliminary explored by molecular docking analysis. CONCLUSION: The key findings of the present study manifested that rosmarinic acid, a medicinal compound extracted from P. frutescens, had pharmacological activity in inhibiting the growth of Trichophyton mentagrophytes by affecting its enolase expression to reduce metabolism. Rosmarinic acid is expected to be an efficacious product for prevention and treatment of dermatophytes.

Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display.

Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.

High efficiency CHO cell display-based antibody maturation.

Previously, we developed a CHO cell display-based antibody maturation procedure in which an antibody (or other protein) gene of interest was induced to mutate by activation-induced cytidine deaminase (AID) and then form a library by simply proliferating the CHO cells in culture. In this study, we further improved the efficiency of this maturation system by reengineering AID, and optimizing the nucleic acid sequence of the target antibody gene and AID gene as well as the protocol for AID gene transfection. These changes have increased both the mutation rate and the number of mutation type of antibody genes by more than 10 fold, and greatly improved the maturation efficiency of antibody/other proteins.

An Efficient Exogenous Gene Insertion Site in CHO Cells with High Transcription Level to Enhance AID-Induced Mutation.

Antibodies have been extensively used for the purpose of scientific research, clinical diagnosis, and therapy. Combination of in vitro somatic hypermutation and mammalian cell surface display has been an efficient technology for antibody or other proteins optimization, in which the efficiency of activation-induced cytidine deaminase (AID) mutations in genes is one of the most important key factors. Gene transcriptional level has been found to be positively proportional to AID-induced mutation frequency. Thus, construction of the cell clone bearing a gene of interest (GOI) with high transcription level can increase AID-induced mutations. In this study, a retargetable gene cassette is inserted onto predetermined chromosome site (ywhae gene site) which is among the genes with the highest as well as stable transcription, and is found that one subsite is suitable to be retargeted for efficient protein display in Chinese hamster ovary (CHO) cells. The resultant cell clone (T31) has higher and more stable transcription/expression than CHO-puro clone which was previously established through the strategy of random insertion followed by a high-throughput selection. It also possesses a significantly higher mutation frequency to GOI than CHO-puro cells; thus, it is a better clone for the in vitro improvement of antibody affinity, and probably other properties.

Activation-Induced Cytidine Deaminase Aided In Vitro Antibody Evolution.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) by converting deoxycytidines (dC) to deoxyuracils (dU) which then can induce other mutations, and plays a central role in introducing diversification of the antibody repertoire in B cells. Ectopic expression of AID in bacteria and non-B cells can also lead to frequent mutations in highly expressed genes. Taking advantage of this feature of AID, in recent years, systems coupling in vitro somatic hypermutation and mammalian cell surface display have been developed, with unique benefits in antibody discovery and optimization in vitro. Here, we provide a protocol for AID mediated in vitro protein evolution. A CHO cell clone bearing a single gene expression cassette has been constructed. The gene of an interested protein for in vitro evolution can be easily inserted into the cassette by dual recombinase-mediated cassette exchange (RMCE) and constantly expressed at high levels. Here, we matured an anti-TNFα antibody as an example. Firstly, we obtained a CHO cell clone highly displaying the antibody by dual RMCE. Then, the plasmid expressing AID is transfected into the CHO cells. After a few rounds of cell sorting-cell proliferation, mutant antibodies with improved features can be generated. This protocol can be applied for improving protein features based on displaying levels on cell surface and protein-protein interaction, and thus is able to enhance affinity, specificity, and stability besides others.