Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena

Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations

A role for the elongator complex in zygotic paternal genome demethylation

The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs 1. One of the molecular events that takes place during this transition is the demethylation of the paternal genome 2,3. Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified 4. To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through siRNA-mediated knockdown in zygotes, we identified Elp3/KAT9, a component of the elongator complex 5, to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulfite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of mRNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation indicating that the SAM radical domain is involved in the demethylation process. Thus, our study not only establishes a critical role for the elongator in zygotic paternal genome demethylation, but also suggests that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain

Retrospective comparison of clinical and angiographic outcomes after primary stenting using sirolimus-eluting and bare-metal stents in nonrandomized consecutive 568 patients with first ST-segment elevated myocardial infarctions

SummaryBackground and purposeThe long-term safety and efficacy of primary stenting using drug-eluting stents (DES) in patients with ST-segment elevation myocardial infarction (STEMI) are not fully understood in Japan. Therefore, we retrospectively examined the midterm clinical and angiographic outcomes in STEMI patients after primary stenting using sirolimus-eluting stents (SES) in a clinical setting through a historical comparison with those of bare-metal stents (BMS).Methods and resultsThe study design was a retrospective, nonrandomized, and single-center study. The clinical outcomes for 568 consecutive patients who presented within 12h of their first STEMI and who were treated with BMS (n=198; 184 STEMIs from June 2003 to August 2004 and 14 STEMIs from September 2004 to May 2007) or SES (n=370; from August 2004 to May 2007) at our medical center in Japan were retrospectively investigated in February 2010. The incidence of post-discharge events (comprising cardiac death and nonfatal recurrent MI) after SES placement (3.9%) was not significantly different from that after BMS placement (6.7%). SES was not related to the risk of post-discharge events (mean follow-up for SES, 1327±415 days; BMS, 1818±681 days) (hazard ratio of 0.369 at 95% CI, 0.119–1.147, p=0.085). The incidence of definite stent thromboses after SES placement (0.54%) was not significantly higher than that after BMS placement (0%). The incidence of binary in-stent restenosis (% diameter stenosis of more than 50% at secondary angiography) after SES placement (8.3%) was significantly lower than that after BMS placement (25.7%; p<0.001).ConclusionsFrom the present historical comparison of SES and BMS, we conclude that primary stenting using SES in a clinical setting has favorable clinical and angiographic outcomes in Japanese STEMI patients

Administration route-dependent induction of antitumor immunity by interferon-alpha gene transfer.

Type I interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis, and immunomodulation. We have demonstrated that intratumoral injection of an IFN-α-expressing adenovirus effectively induces cell death of cancer cells and elicits a systemic tumor-specific immunity in several animal models. On the other hand, reports demonstrated that an elevation of IFN in the serum following an intramuscular delivery of a vector is able to activate antitumor immunity. In this study, we compared the intratumoral and systemic routes of IFN gene transfer with regard to the effect and safety of the treatment. Intratumoral injection of an IFN-α adenovirus effectively activated tumor-responsive lymphocytes and caused tumor suppression not only in the gene-transduced tumors but also in distant tumors, which was more effective than the intravenous administration of the same vector. The expression of co-stimulatory molecules on CD11c+ cells isolated from regional lymph nodes was enhanced by IFN gene transfer into the tumors. Systemic toxicity such as an elevation of hepatic enzymes was much lower in mice treated by intratumoral gene transfer than in those treated by systemic gene transfer. Our data suggest that the intratumoral route of the IFN vector is superior to intravenous administration, due to the effective induction of antitumor immunity and the lower toxicity. © 2010 Japanese Cancer Association

Association of variations in HLA class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma

Lung adenocarcinoma driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). Here we investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive lung adenocarcinoma. In addition, two additional loci, HLA class II at 6p21.32 (rs2179920; P =5.1 × 10(-17), per-allele OR=1.36) and 6p21.1 (FOXP4) (rs2495239; P=3.9 × 10(-9), per-allele OR=1.19) were newly identified as loci associated with EGFR mutation-positive lung adenocarcinoma. This study indicates that multiple genetic factors underlie the risk of lung adenocarcinomas with EGFR mutations

モウナイケイヘイソクノメンエキオウトウニタイスルエイキョウ

京都大学0048新制・論文博士医学博士乙第3369号論医博第696号新制||医||228(附属図書館)5234UT51-52-L186(主査)教授 濱島 義博, 教授 花岡 正男, 教授 小川 和朗学位規則第5条第2項該当Kyoto UniversityDA