C. elegansにおける酸素濃度依存的な低温馴化に関わる神経回路

温度は生物が常に感知する環境情報であり、生物は温度変化に馴化や適応することで生存・繁栄してきた。本研究では、動物の温度馴化の基本原理の理解のために、線虫Caenorhabditis elegansの低温馴化を指標に解析をおこなった。低温馴化に関わる新規分子としてカリウムチャネルKQT-2を同定した。KQT-2は、ヒトにおいて心臓病やてんかん等に関わるKCNQ型カリウムチャネルのホモログであり、そのノックアウト線虫であるkqt-2変異体では野生株よりも低温に早く馴化する異常が見られた。興味深いことに、直径3.5 cmの寒天培地で飼育した場合よりも、直径6 cmの寒天培地で飼育した際にkqt-2変異体の低温馴化異常が強くなった。GFPレポーターを用いた解析から、kqt-2遺伝子は頭部のASKとADL感覚ニューロンと腸で発現しており、kqt-2変異体のADL感覚ニューロンで特異的にkqt-2遺伝子を発現させることによってkqt-2変異体の低温馴化異常が回復した。これまでに、ADLは温度受容ニューロンであることが知られていたため、カルシウムイメージング法を用いてkqt-2変異体のADLの温度応答性を測定したところ、kqt-2変異体では異常が見られた。神経回路上において、ADLは酸素受容ニューロンURXの下流に位置することが報告されている。そこで、低温馴化およびADLの温度応答性における酸素情報の関連性を調べるために、ADLで機能しているカリウムチャネルKQT-2の変異体にURXで機能する酸素受容体GCY-35の機能欠損変異を導入し、gcy-35; kqt-2二重変異体の低温馴化およびADLの温度応答性を測定した。その結果、kqt-2変異体のこれらの異常はgcy-35変異によって抑圧された。つまり、URX酸素受容ニューロンの下流でADL温度受容ニューロンが働き、酸素濃度依存的な温度情報伝達がおこなわれている可能性が考えられた。kqt-2変異体の低温馴化異常に酸素濃度が関わる可能性を考え、kqt-2変異体を飼育する際の酸素濃度を変化させた。その結果、酸素濃度の低下に応じてkqt-2変異体の低温馴化の低下が見られた。さらに、直径6 cmと直径3.5 cmの各々の寒天培地上の酸素濃度を測定したところ、直径6 cmの寒天培地の方が直径3.5 cmの寒天培地よりも約5 %高い酸素濃度を示した。以上の結果から、ADL感覚ニューロンにおいてカリウムチャネルKQT-2は、URX酸素受容ニューロンからの酸素情報依存的に温度情報伝達を調節し、低温馴化を変化させていることが示唆された。この神経回路は、温度と酸素という質的に異なる複数の感覚情報の統合の解析モデルとなりえると考えられる。甲南大学令和元年度(2019年度

Electronic transport in DNA

We study the electronic properties of DNA by way of a tight-binding model applied to four particular DNA sequences. The charge transfer properties are presented in terms of localization lengths (crudely speaking, the length over which electrons travel). Various types of disorder, including random potentials, are employed to account for different real environments. We have performed calculations on poly(dG)-poly(dC), telomeric-DNA, random-ATGC DNA, and l-DNA. We find that random and l-DNA have localization lengths allowing for electron motion among a few dozen basepairs only. A novel enhancement of localization lengths is observed at particular energies for an increasing binary backbone disorder. We comment on the possible biological relevance of sequence-dependent charge transfer in DNA

Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs

Direct site-specific biotinylation of RNA molecules was achieved by specific transcription mediated by unnatural base pairs. Unnatural base pairs between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y), or 2-amino-6-(2-thiazolyl)purine (denoted as v) and y specifically function in T7 transcription. Using these unnatural base pairs, the substrate of biotinylated-y (Bio-yTP) was selectively incorporated into RNA, opposite s or v in the DNA templates, by T7 RNA polymerase. This method was applied to the immobilization of an RNA aptamer on sensor chips, and the aptamer accurately recognized its target protein. This direct site-specific biotinylation will provide a tool for RNA-based biotechnologies

Ultrasonographic Changes of the Knee Joint Reflect Symptoms of Early Knee Osteoarthritis in General Population; The Nagahama Study

Objective: Radiographic changes in knee osteoarthritis (OA) are not always associated with symptoms, especially in its early stages. Ultrasonography (US) can detect early changes in the knee joint, but the changes that reflect symptoms have not been fully elucidated. This study aimed to identify US-detectable changes in the knee that are often associated with knee symptoms and demonstrate the feasibility of early diagnosis in symptomatic knee OA using US. Design: In this cross-sectional community-based study, 1, 667 participants aged ≥60 years (1, 103 women [66%]) were included. All participants concurrently underwent US and radiography of the knee and completed the Knee Society Knee Scoring System (KSS) questionnaire. Simple and multiple regression analyses were used to examine the associations between US findings and KSS symptom subscales. Results: Among all participants, medial meniscus protrusion and medial osteophytes, age, and body mass index showed significant associations with KSS symptom scores. Among 894 participants with Kellgren-Lawrence (KL) grade ≤1, medial osteophytes and age were significantly associated with KSS symptom score. US measures were more related to KSS symptoms than KL grades. Conclusions: Among the knee US-detectable changes, medial osteophytes were strongly associated with knee symptoms. Osteophytes are reliable predictors of symptomatic early knee OA, even in participants with few radiographic OA changes

Ab-initio study of model guanine assemblies: The role of pi-pi coupling and band transport

Several assemblies of guanine molecules are investigated by means of first-principle calculations. Such structures include stacked and hydrogen-bonded dimers, as well as vertical columns and planar ribbons, respectively, obtained by periodically replicating the dimers. Our results are in good agreement with experimental data for isolated molecules, isolated dimers, and periodic ribbons. For stacked dimers and columns, the stability is affected by the relative charge distribution of the pi orbitals in adjacent guanine molecules. pi-pi coupling in some stacked columns induces dispersive energy bands, while no dispersion is identified in the planar ribbons along the connections of hydrogen bonds. The implications for different materials comprised of guanine aggregates are discussed. The bandstructure of dispersive configurations may justify a contribution of band transport (Bloch type) in the conduction mechanism of deoxyguanosine fibres, while in DNA-like configurations band transport should be negligible.Comment: 21 pages, 6 figures, 3 tables, to be published in Phys. Rev.

Environmentally Responsive Core/Shell Particles via Electrohydrodynamic Co-Jetting of Fully Miscible Polymer Solutions

Herein it is demonstrated that electrohydrodynamic co-jetting is not limited to Janus-type particles, but can also be used for the preparation of core/shell particles. Using side-by-side flow of miscible polymer solutions, electrohydrodynamic co-jetting offers an elegant and scalable route towards preparation of core/shell particles with otherwise difficult-to-prepare particle architectures, including particles with hydrophilic shell and core. Throughout this study, electrohydrodynamic co-jetting of aqueous solutions consisting of a mixture of PAAm-co-AA and PAA is used, and a range of different types of particles with distinct compartments are observed. Transition from Janus particles to core/shell particles appears to be caused by changes in the relative conductivity of the two jetting solutions. After crosslinking, the core/shell particles are stable in aqueous solution and exhibit reproducible swelling behavior while maintaining the original core/shell geometry. In addition, the pH-responsiveness of the particles is demonstrated by repeatedly switching the environmental pH between 1.3 and 12. Moreover, the core/shell particles show surprising uptake selectivity. For instance, a 450% increase in uptake of 6-carboxyfluorescein over rhodamine B base is found.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61234/1/1756_ftp.pd

Dipping-Induced Azimuthal Helix Orientation in Langmuir-Blodgett Monolayers of α-Helical Amphiphilic Diblock Copolypeptides

The azimuthal helix orientation of the rigid-rod amphiphilic diblock copolypeptides (PLGA-b-PMLGSLGs) of poly(α-L-glutamic acid) (PLGA) and poly(γ-methyl-L-glutamate-ran-γ-stearyl-L-glutamate) with 30 mol % of stearyl substituents (PMLGSLG) in Langmuir-Blodgett (LB) monolayers was investigated using polarized transmission Fourier transform infrared spectroscopy. The relative position of dipping with respect to the previous transfer position can be used to manipulate the azimuthal orientation of the helices parallel to or tilted by an angle of 45° with respect to the dipping direction in the transferred films. The study of the azimuthal order for the LB monolayers of PLGA-b-PMLGSLGs of various block lengths revealed that the observed effect arises mainly from the deformation of the PMLGSLG top brush layer, induced by the flow orientation around the transfer region. In those cases where the PMLGSLG block is tilted by a sufficiently large angle with respect to the surface normal, high azimuthal order parameters of 0.5-0.75 were obtained.

Effect of probe characteristics on the subtractive hybridization efficiency of human genomic DNA

<p>Abstract</p> <p>Background</p> <p>The detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics.</p> <p>Findings</p> <p>In this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA.</p> <p>Conclusions</p> <p>The characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase.</p

Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging

A single-molecule detection setup based on total internal reflection fluorescence (TIRF) microscopy has been used to investigate association and dissociation kinetics of unlabeled 30mer DNA strands. Single-molecule sensitivity was accomplished by letting unlabeled DNA target strands mediate the binding of DNA-modified and fluorescently labeled liposomes to a DNA-modified surface. The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (Kd < 1 nM, koff < 0.01 s−1) to be collected under equilibrium conditions at low pM concentrations. The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1–100 pM and 23–46°C, respectively. This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis