Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium.

KEY MESSAGE: This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3-4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

Treatment rationale and protocol design: an investigator-initiated phase II study of combination treatment of nivolumab and TM5614, a PAI-1 inhibitor for previously treated patients with non-small cell lung cancer.

Background: There is no established standard 3rd line treatment for patients with advanced non-small cell lung cancer (NSCLC). Although cytotoxic chemotherapeutic agents that are not used as 1st or 2nd line treatment are administrated as 3rd line treatment, their anti-tumor efficacy is insufficient. Anti-programmed death ligand-1 (PD-L1)/programmed death-1 (PD1) treatment is more effective and less toxic than chemotherapy in anti-PD-L1/PD-1 treatment-naïve patients with NSCLC. Therefore, anti-PD-L1/PD-1 therapy is considered an appropriate 3rd line treatment. However, the anti-tumor efficacy is limited in patients previously treated with anti-PD-L1/PD-1 antibody. Today, new drugs are needed to increase the efficacy of anti-PD-L1/PD-1 antibodies. Methods: This open-label, single-arm, investigator-initiated phase II study is designed to evaluate combination treatment of nivolumab and TM5614, a plasminogen activator inhibitor (PAI-1) inhibitor as 3rd or more line treatment in NSCLC patients who underwent standard treatment. The primary endpoint is the objective response rate and the secondary endpoints are progression-free survival (PFS), overall survival (OS), duration of response (DOR) and safety. Recruitment began in September 2023 and is expected to continue for approximately three years. Discussion: Currently, there is no standard 3rd line treatment for advanced NSCLC, and we hope that the findings of this study will facilitate more effective treatments in this setting. Ethics and dissemination: the study protocol conformed to the ethical principles outlined in the Declaration of Helsinki. All patients will provide written informed consent prior to enrollment. Results will be published in a peer-reviewed publication. Trial Registration: This study is registered to Japan Registry of Clinical Trials with number: jRCT2061230039 (19/July/2023).