SARS-CoV-2 variant with the spike protein mutation F306L in the southern border provinces of Thailand.

The southernmost part of Thailand is a unique and culturally diverse region that has been greatly affected by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak during the coronavirus disease-2019 pandemic. To gain insights into this situation, we analyzed 1942 whole-genome sequences of SARS-CoV-2 obtained from the five southernmost provinces of Thailand between April 2021 and March 2022, together with those publicly available in the Global Initiative on Sharing All Influenza Data database. Our analysis revealed evidence for transboundary transmissions of the virus in and out of the five southernmost provinces during the study period, from both domestic and international sources. The most prevalent viral variant in our sequence dataset was the Delta B.1.617.2.85 variant, also known as the Delta AY.85 variant, with many samples carrying a non-synonymous mutation F306L in their spike protein. Protein-protein docking and binding interface analyses suggested that the mutation may enhance the binding between the spike protein and host cell receptor protein angiotensin-converting enzyme 2, and we found that the mutation was significantly associated with an increased fatality rate. This mutation has also been observed in other SARS-CoV-2 variants, suggesting that it is of particular interest and should be monitored.

Surface cysteine to serine substitutions in IL-18 reduce aggregation and enhance activity.

Background: Interleukin-18 (IL-18) is prone to form multimers resulting in inactive aggregates, making this cytokine unstable for clinical use. Therefore, mutations have been introduced into recombinant IL-18 to overcome this issue. Methods: To prevent the formation of disulfide bonds between the IL-18 molecules, multiple mutations targeting surface cysteines (C38, C68, C76, and C127) were introduced into our previously modified human IL-18 double mutant E6K+T63A (IL-18 DM) by direct gene synthesis. The open reading frames of IL-18 wild-type (WT), IL-18 DM, and IL-18 multiple mutant E6K+T63A+C38S+C68S+C76S+C127S (IL-18 DM1234) were inserted in the pET28a expression vector and transformed into Escherichia coli Rosetta2 (DE3) pLysS cells for protein production. The inclusion bodies of WT and mutated IL-18 were extracted by sonication and refolded by stepwise dialysis using 8 M urea as the starting concentration. The refolded IL-18 proteins were tested for aggregation using the ProteoStat protein aggregation assay. Their activity was also investigated by treating NK-92MI cells with each IL-18 at concentrations of 75, 150, and 300 ng/ml with 0.5 ng/ml of human IL-12 and interferon-gamma (IFN-γ) levels in the supernatant were evaluated using ELISA. The structure of modified IL-18 was visualized using molecular dynamics (MD) simulations. Results: IL-18 DM1234 exhibited the lowest aggregation signal, approximately 1.79- and 1.63-fold less than that of the WT and IL-18 DM proteins. Additionally, the IFN-γ inducing activity of IL-18 DM1234 was about 10 and 2.8 times higher than that of the WT and IL-18 DM, respectively. MD simulations revealed that binding site I of IL-18 DM1234 was altered mainly due to surface cysteine replacement with serine (C-to-S substitution). This is the first report showing that C-to-S substitutions in IL-18 improved its activity and stability, suggesting the use of this modified IL-18 for medical purposes in the future.

Low Piperine Fractional Piper nigrum Extract Enhanced the Antitumor Immunity via Regulating the Th1/Th2/Treg Cell Subsets on NMU-Induced Tumorigenesis Rats.

Cancer is one of the major causes of death worldwide. In addition to standard regimens, tumor suppression ability has been demonstrated in many types of natural products, including Piper nigrum, or black pepper. In previous reports, we demonstrated the antitumor effect of low piperine fractional Piper nigrum extract in vitro and in vivo. However, the effects of low piperine fractional P. nigrum extract in the aspect of antitumor immunity has not yet been investigated. In this study, tumor-bearing rats were fed with 100 mg/kg BW or 200 mg/kg BW of low piperine fractional P. nigrum extract 3 times per week for 4 weeks. Tumor burden and hematological data were then evaluated. Immunological data was investigated using a cytokine array and flow cytometry. The results showed that both doses of low piperine fractional P. nigrum extract significantly suppressed tumor progression in N-nitrosomethylurea-induced mammary tumor rats. There were no significant changes observed in the total white blood cells, red blood cells, and hemoglobin. Low piperine fractional P. nigrum extract suppressed some cytokine and chemokine levels including CXCL7, sICAM-1, and L-selectin 0.2- to 0.6-fold. Interestingly, 200 mg/kg BW of low piperine fractional P. nigrum extract significantly promoted type 1 T helper cell, and suppressed neutrophil, basophil, type 2 T helper cell, and regulatory T cell compared to the control group. In summary, these results indicate that low piperine fractional P. nigrum extract had a high efficacy in supporting antitumor activity at immunological levels via regulating Th1/Th2/Treg cells.

The attenuation effect of low piperine Piper nigrum extract on doxorubicin-induced toxicity of blood chemical and immunological properties in mammary tumour rats.

CONTEXT: Many natural extracts have been shown to minimize the toxicity of doxorubicin (Dox). Low piperine Piper nigrum L. (Piperaceae) extract (PFPE) is a natural extract containing many types of antioxidants that may reduce Dox toxicities. OBJECTIVE: To evaluate the effect of PFPE in attenuating the side effects of Dox. MATERIALS AND METHODS: Tumour-bearing Sprague Dawley rats were divided into five groups including normal, vehicle, 100 mg/kg BW of PFPE plus 2 mg/kg BW of Dox (P100 + Dox), 100 mg/kg BW of PFPE plus 2 mg/kg BW of Dox (P200 + Dox) and Dox. Rats were treated with Dox and/or PFPE three times/week for 4 weeks. Tumour burden, blood parameters, weight of internal organs and immunological data were investigated. RESULTS: The addition of 200 mg/kg PFPE significantly restored the levels of AST from 174.60 ± 45.67 U/L in the Dox group near to normal levels at 109.80 ± 4.99 U/L. The combination of PFPE and Dox also decreased the levels of CXCL7, TIMP-1, sICAM-1 and l-selectin about 1.4-1.6-fold compared to Dox group. Feeding rats with 200 mg/kg BW of PFPE combination with Dox slightly increased Th1 from 161.67 ± 14.28 cells in Dox group to 200.75 ± 5.8 cells meanwhile suppressed Treg from 3088 ± 78 cells in Dox to 2561 ± 71 cells. DISCUSSION AND CONCLUSIONS: This study showed that PFPE ameliorated Dox toxicity in many aspects indicating the role of antioxidant and other substances in the extract on toxicity attenuation. This suggested the using of PFPE may be valuable for Dox treated patients.