Sequence specific visual detection of LAMP reactions by addition of cationic polymers

BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device

Distribution of ferritin and hemosiderin in the liver, spleen and bone marrow of normal, phlebotomized and iron overloaded rats.

The distribution of ferritin has been studied in many tissues, but has not yet been established on the cellular level. We investigated the cellular distribution of ferritin in the liver, spleen and bone marrow using the immunoperoxidase method, and compared it with that of hemosiderin. We also examined changes in the distribution of these proteins after phlebotomy and iron overload. In normal rats, ferritin was seen in centrilobular hepatocytes, Kupffer cells, macrophages in the red and white pulp of the spleen and central macrophages in bone marrow. Hemosiderin was observed almost exclusively in the red pulp and partly in tangible body macrophages of the white pulp. After phlebotomy, neither ferritin nor hemosiderin were detectable in these cells except for ferritin-positive cells in the white pulp, which showed little change after either phlebotomy or iron overload. In iron overloaded rats, both ferritin and hemosiderin increased in hepatocytes and reticulo-endothelial (RE) cells. Ferritin-positive cells in the liver were mainly located in the periportal area. These results indicated that hepatocytes and RE cells except for those in the white pulp may play an important role in iron storage, and that ferritin-positive cells in the white pulp may have a function other than iron reserve. They also suggested that the zonal distribution of ferritin-positive hepatocytes may be due to microcirculation in the hepatic lobules.</p

A case-control study of endometrial cancer especially with reference to lifestyle and other factors of Japanese women

Japanese women\u27s anthropometries and their lifestyles have been gradually changing since 1945. Therefore, we conducted a case-control study in order to explore the relationship of anthropometries, physical activity and other risk factors of endometrial cancer (EC) in Japanese women. This study of EC was conducted between January 2003 and March 2008 in Sapporo, Japan. Informed written consent was obtained from 191 patients and 419 population-based control subjects. The items surveyed were body height and weight, physical activity, past history, family history, menstruation, post-menopausal status, reproductive history, and so on. An adjusted odds ratio (OR) and its 95% confidence interval (95%CI) were estimated with the multivariate unconditional logistic regression model. The number of pregnancies (Ptrend=0.004) and number of live-births (Ptrend<0.001) were significantly associated with EC risk. Oral contraceptive use was not significantly associated with reduced EC risk when age, area, BMI and number of live-births were adjusted. Adult obesity significantly increased the risk of EC. In addition, the amount of maximum weight gained since the age of 20 years old was strongly related to an increase of EC risk (Ptrend<0.001). When, age, area, BMI, and the number of live-births were adjusted, the duration of physical activity at a certain time was marginally associated with reduced risk of EC (Ptrend=0.050). Light-moderate alcohol consumption (0.1~59.9g per opportunity) was significantly related to the decreased risk of EC even after age, area, BMI and number of live-births were adjusted (P<0.05). We conclude that further study is necessary to clarify the relationship between physical activity and light-moderate alcohol consumption with the reduced risk of EC

Mediastinal and Hilar Lymph Node Metastases from Renal Cell Carcinoma with Concomitant Lung Carcinoma: A Rare Case with Unique Diagnostic Challenges

A 75-year-old man presented to our hospital 1 year after partial renal resection for clear cell carcinoma. A right lower lobe lung nodule noted at the time of surgery had increased to 3.0 cm in diameter and was confirmed as squamous cell lung carcinoma by bronchoscopic cytology. Computed tomography had also revealed paratracheal lymph node swelling. He underwent right lower lobectomy with lymph node dissection by video-assisted thoracic surgery. Pathological examination confirmed squamous cell carcinoma of the lung but diagnosed the right hilar and mediastinal lymph node metastases as clear cell carcinoma

Genomic organization and promoter function of the mouse uncoupling protein 2 (UCP2) gene1The nucleotide sequences reported in this paper will appear in the DDBJ, and GenBank/EMBL Data Bank with accession number AB012159.1

AbstractWe cloned and characterized the mouse uncoupling protein 2 (UCP2) gene and its promoter region. The gene spans approximately 6.3 kb and contains eight exons and seven introns. Two short exons are located in the 5′ untranslated region, and each of the remaining exons encodes one of the transmembrane domains. 3′-RACE analysis showed that a polyadenylation signal 257 bp downstream from the stop codon was functional. Primer extension analysis indicated a single transcriptional start site 369 bp upstream from the translational start site. The promoter region lacks both TATA and CAAT boxes but is GC-rich. A construct containing 1250 bp of the promoter region showed significant activity in all 6 cell lines examined, and the region between −160 and −678 bp exhibited strong positive regulatory activity. These features of the UCP2 gene are different from those of the UCP1 gene and may contribute to its ubiquitous expression

Impact of Workplace on the Risk of Severe COVID-19

Indiscriminate regional lockdowns aim to prevent the coronavirus disease 2019 (COVID-19) infection by restricting the movement of people; however, this comes with psychological, social, and economic costs. Measures are needed that complement lockdowns and reduce adverse effects. Epidemiological studies, to date, have identified high-risk populations, but not workplaces appropriate for closure. This study was conducted to provide evidence-based measures that used exact and reliable follow-up data of the PCR-positive COVID-19 cases to complement lockdowns. The data are not subjected to selection or follow-up biases, since the Japanese government, by law, must register and follow all the PCR-positive cases until either recovery or death. Direct customer exposure may affect the quantity of viral inoculum received, which, in turn, may affect the risk of the severity of disease at infection. Therefore, the professions of the cases were grouped according to their frequency of direct customer exposure (FDCE) based on subjective observations, which resulted in five workplaces; hospital, school, food service, outdoor service, and indoor office being identified. Analyzing the follow-up data, we obtained precise estimates for the risk of severe disease, defined as intensive care unit (ICU) hospitalization or death, for the workplaces adjusted for age, sex, family status, and comorbidity. Major findings are as follows: hospital and school are the lowest risk, food and outdoor services are, despite higher FDCE, safer than indoor office. Unemployed and unclear are the highest risk, despite low FDCE. These results suggest the following workplace-specific measures complementing the lockdown: school should not be closed and indiscriminate closing of food and outdoor service industries should be avoided, since it would be more effective to reinforce their efforts to promote adherence to public health guidelines among students and customers. These actions would also reduce the adverse effects of the lockdown. This study is the first to address the causality between the workplaces and severe disease. We introduce FDCE and adherence to public health guidelines (APHGs) to associate the workplace characteristics with the risk of COVID-19 severity, which provided the basis for the measures complementing lockdowns

Ethanolamine Is a New Anti-Prion Compound

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco’s modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound

Specific Egg Yolk Immunoglobulin as a New Preventive Approach for Shiga-Toxin-Mediated Diseases

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection