PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale

Effects of self-efficacy on oral health behaviours and gingival health in university students aged 18- or 19-years-old

Aim Although self-efficacy is known to affect various health-related practises, few studies have clearly examined how self-efficacy correlates with oral health behaviors or the oral health condition. We examined the relationship between gingivitis, oral health behaviors and self-efficacy in university students. Material & Methods A total of 2,111 students (1,197 males, 914 females) aged 18 and 19 years were examined. The degree of gingivitis was expressed as the percentage of bleeding on probing (%BOP). Additional information was collected via a questionnaire regarding oral health behaviors (daily frequency of tooth-brushing, use of dental floss and regular check-up). Self-efficacy was assessed using the Self-Efficacy Scale for Self-care (SESS). Path analysis was used to test pathways from self-efficacy to oral health behaviors and %BOP. Results In the final structural model, self-efficacies were related to each other, and they affected oral health behaviors. Good oral health behaviors reduced dental plaque and calculus, and lower levels of dental plaque and calculus resulted in lower %BOP. Conclusion Higher self-efficacy correlated with better oral health behaviours and gingival health in university students. Improving self-efficacy may be beneficial for maintaining good gingival health in university students. To prevent gingivitis, the approach of enhancing self-efficacy in university students would be useful

Irradiation by a Combination of Different Peak-Wavelength Ultraviolet-Light Emitting Diodes Enhances the Inactivation of Influenza A Viruses

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock. This study aimed to determine the ideal irradiation by ultraviolet-light emitting diodes (UV-LEDs) for IAV disinfection. We irradiated the IAV H1N1 subtype with 4.8 mJ/cm2 UV using eight UV-LEDs [peak wavelengths (WL) = 365, 310, 300, 290, 280, 270, and 260 nm)] or a mercury low pressure (LP)-UV lamp (Peak WL = 254 nm). Inactivation was evaluated by the infection ratio of Madin–Darby canine kidney (MDCK) cells or chicken embryonated eggs. Irradiation by the 260 nm UV-LED showed the highest inactivation among all treatments. Because the irradiation-induced inactivation effects strongly correlated with damage to viral RNA, we calculated the correlation coefficient (RAE) between the irradiant spectrum and absorption of viral RNA. The RAE scores strongly correlated with the inactivation by the UV-LEDs and LP-UV lamp. To increase the RAE score, we combined three different peak WL UV-LEDs (hybrid UV-LED). The hybrid UV-LED (RAE = 86.3) significantly inactivated both H1N1 and H6N2 subtypes to a greater extent than 260 nm (RAE = 68.6) or 270 nm (RAE = 42.2) UV-LEDs. The RAE score is an important factor for increasing the virucidal effects of UV-LED irradiation

Radically Different Thioredoxin Domain Arrangement of ERp46, an Efficient Disulfide Bond Introducer of the Mammalian PDI Family

SummaryThe mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER

紫外線発光ダイオード照射は宿主細胞内でのウイルスRNAの複製と転写を抑制することでA型インフルエンザウイルスを不活化する

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock, especially poultry and pigs. This study aimed to investigate how to inactivate IAVs by using different ultraviolet-light-emitting diodes (UV-LEDs). We developed sterilization equipment with light-emitting diodes (LEDs) those peak wavelengths were 365 nm (UVA-LED), 310 nm (UVB-LED), and 280 nm (UVC-LED). These UV-LED irradiations decreased dose fluence-dependent plaque-forming units of IAV H1N1 subtype (A/Puerto Rico/8/1934) infected Madin-Darby canine kidney (MDCK) cells, but the inactivation efficiency of UVA-LED was significantly lower than UVB- and UVC-LED. UV-LED irradiations did not alter hemagglutination titer, but decreased accumulation of intracellular total viral RNA in infected MDCK cells was observed. Additionally, UV-LED irradiations suppressed the accumulation of intracellular mRNA (messenger RNA), vRNA (viral RNA), and cRNA (complementary RNA), as measured by strand-specific RT-PCR. These results suggest that UV-LEDs inhibit host cell replication and transcription of viral RNA. Both UVB- and UVC-LED irradiation decreased focus-forming unit (FFU) of H5N1 subtype (A/Crow/Kyoto/53/2004), a highly pathogenic avian IAV (HPAI), in infected MDCK cells, and the amount of FFU were lower than the H1N1 subtype. From these results, it appears that IAVs may have different sensitivity among the subtypes, and UVB- and UVC-LED may be suitable for HPAI virus inactivation

General characterization of Antarctic micrometeorites collected by the 39th Japanese Antarctic Research Expedition: Consortium studies of JARE AMMs (III)

From November 1998 to January 1999,the 39th Japanese Antarctic Research Expedition (JARE-39) undertook Japanese first large-scale collection of Antarctic micrometeorites (AMMs), with sizes larger than 10μm, at the Meteorite Ice Field around the Yamato Mountains in Antarctica (at three different locations, for a total of 24 collection sites). The number of collected AMMs larger than 40μm is estimated to be about 5000. Here we present the general characterization (i.e., micro-morphology and surface chemical composition using SEM/EDS) of ∿810 AMMs chosen from 5 of the 24 sites. Additionally, the mineral composition of 61 out of 810 AMMs was determined by Synchrotron X-ray radiation. Preliminary results on mineralogical and chemical compositions show similarities with that of previous studies, even though a pronounced alteration of some AMMs is noticed. A correlation is found between the Mg/Si ratio at the sample\u27s surfaces of unmelted AMMs and the age of snow/ice in which the AMMs are embedded

ブラジル・南パンタナールの伝統的な農場経営とその課題 : バイアボニータ農場の事例

ブラジル南パンタナールのバイアボニータ農場では，天然草地の放牧地に依存した，肥育用の仔ウシ生産を目的とする仔取り繁殖経営が中心に営まれている。ここでは，季節的な河川の水位変化に起因して発現する多様な天然草地の状況に合わせて牛群を管理したり，火入れや伐採・巻き枯らしにより草地の森林化を抑制したりするなど，農場間の相互扶助システムとともに，パンタナールで培われてきた牧畜経営のワイズユース（wise use）が現在も継承されている。しかし，農場規模が小さいうえに，労働力不足，ウシの受胎率の低さ，生産性や品質の低さといった諸課題に直面して，伝統的な牧畜経営の維持・発展は困難な状況にある。こうした現状を打開して経営の安定化を実現するためには，繁殖・出荷カレンダーを作成してウシの繁殖時期を特定化し管理作業の効率化を図ることや，質の高い種牡ウシや牝ウシを導入して，より厳格な牧区規制の下で両者の頭数管理や繁殖を効果的に実施することなどが必要である。The people of Pantanal (pantaneiros) have conducted the extensive calf production that depends excessively on natural grassland in the Pantanal of Brazil. According to the case of Fazenda Baía Bonita in the Nhecolândia of south Pantanal, this study examines the problems of traditional cattle grazing and appropriate measures for the future management by focusing on the relationships between natural environment and social surroundings. The wise use of wetlands fostered for centuries still remains in the ranch, e.g., management of cattle herds corresponding with seasonal water level, methods for preventing grassland from plant succession to semi-deciduous forest by means of intentional burn, tree girdling and cleaning, and mutual aid customs between neighboring ranches. However, the management of extensive calf production confronts difficulties at present because of the scarcity of labor, small-scale grassland of the ranch, low conception rate of heifers and cows, and low productivity and quality of calves. In order to resolve current situation and to achieve stable management of calf production, it needs to create a calendar of breeding and shipping that especially specifies the season of breeding to promote the efficiency of administrative work. It is also necessary to introduce stud bull and cows of high quality, and to practice appropriate controls on their numbers and breeding under strict regulations of grazing areas

Establishment of an Epicutaneously Sensitized Murine Model of Shellfish Allergy and Evaluation of Skin Condition by Raman Microscopy

Background: Shellfish allergy is one of the most common food allergies. Recent studies have shown that sensitization to allergens via the skin is involved in the development of food allergies. In this study, a mouse model of shrimp allergy was generated by epicutaneous sensitization and used to identify skin conditions associated with susceptibility to sensitization. Methods: Four-week-old female BALB/c mice were sensitized by repeated application of 0.1 mg of tropomyosin to tape-stripped skin on days 0, 7, and 15, followed by a challenge on days 28 and 35. Results: Epicutaneously sensitized mice exhibited higher serum levels of tropomyosin-specific IgE on day 15 than control mice. After the oral challenge, model mice had higher anaphylaxis scores and lower rectal temperature. After three tape-strip treatments for sensitization, the skin was analyzed by Raman microscopy. The sensitized mice exhibited lower relative intensities of Raman bands at 399, 915, and 1073 cm−1 than control mice, which could be helpful noninvasive markers in screening for potential sensitization via the skin. Conclusions: An epicutaneous sensitization shellfish allergy model was generated. This model will be useful in studies to elucidate the pathogenesis of skin sensitization. Raman microscopy may also be valuable for capturing subtle skin changes leading to sensitization

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution