Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna

It is well known that most daphnid species have several attractive life history characteristics such as cyclical parthenogenesis, environmental sex determination, and predator-induced defense formation. Recent advances in high-throughput omics technologies make it easy to obtain a huge number of potential candidate factors involved in environmental stimuli-triggered phenotypic alterations. Furthermore, our group has developed a microinjection system to introduce foreign materials such as nucleotides and chemicals into the early-stage (one-cell stage) egg of Daphnia pulex and Daphnia magna. Consequently, we established a microinjection-based RNAi system that allows arbitrary gene functions to be investigated. However, this microinjection system does not seem to have pervaded in the daphnid research community due to its low throughput and high level of skills required. In this chapter, we review the microinjection method and its RNAi system in water fleas, D. pulex and D. magna, providing some technical tips and making challenging proposals for the development of novel high-throughput RNAi methods. Finally, we provide an overview of recently developed gene functional analysis methods such as overexpression and genome-editing systems

Na-catalyzed rapid synthesis and characterization of intercalated graphite CaC6

In this study, we conducted experiments on CaC6 for elucidating the Na-catalyzed formation mechanism and achieving rapid mass synthesis of graphite intercalation compounds (GICs). Rapidly synthesized CaC6 was characterized by analysis of its crystal structure and physical properties. We found that the formation of the reaction intermediate Na-GIC (NaCx, x = 64) requires a larger amount of Na than is intercalated between the graphite interlayers. The requirement for excess Na may provide insights into the mechanism of Na-catalyzed GIC formation. A Na-to-C molar mixing ratio of 1.5-2.0:6 was suitable for the efficient formation of CaC6 under heat treatment at 250{\deg}C for 2 h, and the catalytic Na remaining in the sample was demonstrably reduced to a Na:Ca ratio of approximately 3:97. The upper critical field Hc2 was enhanced approximately three times compared to those of previous reports. Based on X-ray diffraction and experimental parameter analysis, we concluded that the enhancement of Hc2 was attributed to the disordered stacking sequence in CaC6, possibly because of the rapid and low-temperature formation. Physical properties derived from specific heat measurements were comparable to those of high-quality CaC6, which is slowly synthesized using the molten Li-Ca alloy method. This study provides new avenues for future research and exploration in the rapid mass synthesis of GICs as practical materials, for applications such as battery electrodes and superconducting wires.Comment: 17 pages, 6 figures, 1 tabl

Efficacy and safety of second-line chemotherapy for patients with advanced non-small cell lung cancer complicated by interstitial lung disease

Background: Treatment of non-small cell lung cancer (NSCLC) patients with interstitial lung disease (ILD) is limited because of the risk of its acute exacerbation (AE). Furthermore, the efficacy and safety of second-line chemotherapy for these patients is unclear. Methods: To investigate the efficacy and safety of second-line chemotherapy for NSCLC patients with ILD, we retrospectively reviewed patients who were treated at our institute between April 2010 and December 2018. Results: Thirty-five patients received two or more regimens. Thirty-four patients were male and the median age at the initiation of second-line chemotherapy was 70 years. Almost all patients had a smoking history. Fourteen patients had adenocarcinoma and 15 had squamous cell carcinoma histology. Stages III and IV were observed in 20 and 11 patients, respectively. With respect to the type of ILD, 12 patients had usual interstitial pneumonia (UIP). The overall response rate and disease control rate were 11.4 and 68.6%, respectively. The median progression-free and median overall survival were 4.1 and 6.4 months, respectively. The AE of ILD was observed in eight patients, five of whom died. UIP and low percentage vital capacity were detected as significant risk factors for the AE of ILD. Conclusion: Second-line chemotherapy among patients with NSCLC complicated by ILD showed a certain effectiveness, but some patients experienced the AE of ILD, which may lead to death. The risk of the AE of ILD must be considered especially for patients with UIP and low percentage VC

Antibacterial Effects of Disulfiram in Helicobacter pylori

Background: Helicobacter pylori infection poses a risk of the occurrence of gastrointestinal diseases, such as gastric cancer. Its incidence rate is significantly reduced by eradication, and thereby, eradication therapy is generally performed. Disulfiram is an oral prescription drug mainly used for the treatment of alcohol dependence. In recent years, reports have been made on its anticancer and antibacterial effects, and thus, it has recently become an interesting subject. This study aimed to examine the antibacterial activity of disulfiram, investigate the presence or absence of its antibacterial activity on H. pylori, and determine whether it could be a new bactericidal drug against drug-resistant H. pylori. Materials and Methods: Drug-sensitive strains of H. pylori and amoxicillin-resistant, clarithromycin-resistant, and metronidazole-resistant strains were used, and a growth inhibition test of H. pylori using disulfiram was performed. Furthermore, the expression of urease, vacuolating cytotoxin A (VacA), and CagA, the virulence proteins of H. pylori, was quantitatively analyzed using the Western blotting method. In addition, for H. pylori used in this study, the 16SrDNA sequence, a ribosomal gene involved in protein production, was analyzed to examine the presence or absence of gene mutation. Results: Disulfiram suppressed the growth of 7 out of 12 H. pylori strains at 1 mu g/mL, and no correlation was observed between their susceptibility/resistance to current eradication antimicrobial drugs and disulfiram resistance. Disulfiram reduced the expression levels of urease, VacA, and CagA proteins. H. pylori, which showed resistance to disulfiram, tended to have fewer gene deletions/insertions in the 16S rDNA sequence; however, no specific mutation was detected. Conclusion: Disulfiram has a bactericidal effect on H. pylori at low concentrations, suggesting that it can be used as a supplement for current H. pylori eradication drugs

Melanin pigments in the melanocytic nevus regress spontaneously after inactivation by high hydrostatic pressure

We report a novel treatment for giant congenital melanocytic nevi (GCMN) that involves the reuse of resected nevus tissue after high hydrostatic pressurization (HHP). However, the remaining melanin pigments in the inactivated nevus tissue pose a problem; therefore, we performed a long-term observation of the color change of inactivated nevus tissue after HHP. Pressurized nevus specimens (200 MPa group, n = 9) and non-pressurized nevus tissues (control group, n = 9) were subcutaneously implanted into nude mice (BALB/c-nu) and then harvested 3, 6, and 12 months later. Color changes of the nevus specimens were evaluated. In the 200 MPa group, the specimen color gradually regressed and turned white, and brightness values were significantly higher in the 200 MPa group than in the control group after 6 months. This indicated that melanin pigments in the pressurized nevus tissue had spontaneously degraded and regressed. Therefore, it is not necessary to remove melanin pigments in HHP-treated nevus tissue

Correlation between immune-related adverse events and therapeutic effects of nivolumab in patients with malignant pleural mesothelioma

Background: Nivolumab is used for the treatment of malignant pleural mesothelioma (MPM). However, immune-related adverse events (irAEs) occur in patients treated with nivolumab. Several studies have reported the correlation between irAEs and therapeutic effects of immune checkpoint inhibitor, but none have reported the correlation in MPM. Here we report a retrospective study which shows the correlation between irAEs and therapeutic effects of nivolumab in patients with MPM. Methods: This study included patients treated with nivolumab at Tokushima University Hospital from February 2009 to September 2021. We retrospectively reviewed the medical records to evaluate the several clinical factors, such as the presence or absence of irAEs, their severities, progression-free survival (PFS), overall survival (OS) or objective response to the treatment. Results: Eleven patients received treatment with nivolumab. Objective response rate was 18.2% and the disease control rate was 90.9%. Median PFS was 6.8 months (95% confidence interval, 1.3 to 11.9 months) and median OS was 15.2 months (95% confidence interval, 8.9 to 21.5 months). IrAEs occurred in eight patients (72.7%), and grade ≥ 2 irAEs occurred in six patients (54.5%). PFS and OS were significantly longer in the grade ≥ 2 irAEs group than in grade < 2 irAEs group (median PFS 13.6 vs. 3.8 months, p = 0.0093; median OS not reached vs. 8.6 months, p = 0.0108). Conclusions: This is the first study to report the correlation between irAEs and therapeutic effects in patients with MPM. Because the presence of irAEs may be associated with a favorable clinical outcome, early detection and appropriate management of irAEs will increase the therapeutic benefits to patients