FRET probe for detecting two mutations in one EGFR mRNA

Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET

Field Induced Transitions in Rare Earth Intermetallic Compounds RX and RX_2 (R=Er, Ho, Dy, Tb and Gd and X=Ag and Au)(Magnetism)

Rare earth intermetallic compounds RX and RX_2 ( R=Gd, Tb, Dy, Ho and Er : X=Ag and Au) are antiferromagnetic compounds with the CsCl-type crystal structure and the MoSi_2-type one, respectively. Magnetization process is investigated for these compounds under static magnetic fields up to 270 kOe and pulsed ones up to 300 kOe. The observed field induced transitions are reviewed together with their magnetic phase diagrams

Improved photodynamic activities of liposome‐incorporated [60]fullerene derivatives bearing a polar group

[60]Fullerene (C60) derivatives were incorporated into liposomes using a fullerene exchange method involving the transfer of the fullerene from the cavity of two γ‐cyclodextrin molecules to a liposome. A lipid‐membrane‐incorporated C60 derivative bearing a polar group showed much higher photodynamic activity than the analogous system incorporating pristine C60.This work was supported by JSPS KAKENHI Grant‐in‐Aid for Scientific Research (B) (Grant No. JP16H04133) and Grant‐in‐Aid for Challenging Exploratory Research (Grant No. JP16K13982)

Molecular cloning of doublesex genes of four cladocera (water flea) species

BACKGROUND: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. RESULTS: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5’ upstream regulatory elements are preserved in D. magna and D. pulex. CONCLUSIONS: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans

Porphyrin‐uptake in liposomes and living cells using an exchange method with cyclodextrin

The water‐solubilisation of porphyrin derivatives is very important for biological applications. Although liposomal drug carriers for porphyrin derivatives have shown significant promise in the field of medicinal chemistry (e.g., as sensitisers for photodynamic therapy), it is currently not possible to prepare lipid‐membrane‐incorporated tetraphenylporphyrin (TPP) with a high concentration of TPP using conventional methods. In this study, we have succeeded in preparing lipid-membrane‐incorporated TPP and zinc(II) tetraphenylporphyrin (ZnTPP) from the corresponding TPP or ZnTPP•cyclodextrin complex using the exchange method in lipid‐membranes composed of liposomes. Furthermore, the exchange method allowed for the incorporation of TPP or ZnTPP into the plasma membranes of HeLa cells. However, it was not possible to prepare lipid‐membrane‐incorporated porphyrin derivatives with polar and hydrophilic groups in the meso positions using this exchange reaction.Electronic supplementary information (ESI) available: Experimental procedures, 1H NMR spectra, DLS measurements, cryo-TEM images, phase contrast and fluorescence images. See DOI: 10.1039/c5ra24985This work was supported by JSPS KAKENHI a Grant‐in‐Aid for Scientific Research (B) (Grant No. 25288037) and a Grant‐in‐Aid for Young Scientists (A) (Grant No. 24681028)

HLA-DRB1 and DQB1 alleles in Japanese type 1 autoimmune hepatitis: The predisposing role of the DR4/DR8 heterozygous genotype

ObjectiveAutoimmune hepatitis (AIH) is a chronic progressive liver disease. AIH is composed predominantly of type 1 in Japanese populations. The genetic and environmental factors are associated with the pathogenesis of AIH. HLA-DRB1*03:01 and *04:01 are associated with type 1 AIH in European and *04:05 in Japanese populations. Here, we conducted an HLA association study in order to find HLA alleles or haplotypes predisposing or protective for Japanese AIH.MethodsHLA-DRB1 and DQB1 genotyping of 360 type 1 AIH patients and 1026 healthy controls was performed.ResultsThe predisposing association of DRB1*04:01 (P = 0.0006, corrected P [Pc] = 0.0193, odds ratio [OR] 2.97, 95% confidence interval [CI] 1.62–5.43), DRB1*04:05 (P = 1.89×10−21, Pc = 5.86×10−20, OR 3.41, 95% CI 2.65–4.38), and DQB1*04:01 (P = 4.66×10−18, Pc = 6.99×10−17, OR 3.89, 95% CI 2.84–5.33) and the protective association of DRB1*13:02 (P = 0.0003, Pc = 0.0080, OR 0.48, 95% CI 0.32–0.72) with Japanese type 1 AIH were observed. An association of the DR4/DR8 heterozygous genotype with Japanese AIH was identified for the first time (P = 3.12×10−9, OR 3.52, 95% CI 2.34–5.29). Susceptible diplotypes were DRB1*04:05-DQB1*04:01/DRB1*08:02-DQB1*03:02 (P = 0.0004, OR 24.77, 95% CI 1.45–424.31) and DRB1*04:05-DQB1*04:01/DRB1*08:03-DQB1*06:01 (P = 1.18×10−6, OR 10.64, 95% CI 3.19–35.46). Serum levels of Immunoglobulin G and Immunoglobulin M, International Autoimmune Hepatitis Group score, positive rate of anti-smooth muscle antibodies, and the rate of definite AIH were higher in AIH patients with DRB1*04:05 than without.ConclusionsThe important roles of specific combinations of DRB1 and DQB1 alleles or haplotypes in the pathogenesis of type 1 AIH were suggested. The association of DR4/DR8 heterozygous genotype suggested the pathologic importance of trans-complementing DQα-β heterodimer molecules encoded by DQA1 allele of one haplotype and the DQB1 allele of the other haplotype, as it was proposed in the HLA association studies of Type 1 diabetes

Independent association of HLA-DPB1*02:01 with rheumatoid arthritis in Japanese populations

ObjectiveRheumatoid arthritis (RA) is a chronic autoimmune disease characterized with joint destructions; environmental and genetic factors were thought to be involved in the etiology of RA. The production of anti-citrullinated peptide antibodies (ACPA) is specifically associated with RA. DRB1 is associated with the susceptibility of RA, especially ACPA-positive RA [ACPA(+)RA]. However, a few studies reported on the independent associations of DPB1 alleles with RA susceptibility. Thus, we investigated the independent association of DPB1 alleles with RA in Japanese populations.MethodsAssociation analyses of DPB1 were conducted by logistic regression analysis in 1667 RA patients and 413 controls.ResultsIn unconditioned analysis, DPB1*04:02 was nominally associated with the susceptibility of ACPA(+)RA (P = 0.0021, corrected P (Pc) = 0.0275, odds ratio [OR] 1.52, 95% confidence interval [CI] 1.16–1.99). A significant association of DPB1*02:01 with the susceptibility of ACPA(+)RA was observed, when conditioned on DRB1 (Padjusted = 0.0003, Pcadjusted = 0.0040, ORadjusted 1.47, 95%CI 1.19–1.81). DPB1*05:01 was tended to be associated with the protection against ACPA(+)RA, when conditioned on DRB1 (Padjusted = 0.0091, Pcadjusted = 0.1184, ORadjusted 0.78, 95%CI 0.65–0.94). When conditioned on DRB1, the association of DPB1*04:02 with ACPA(+)RA was disappeared. No association of DPB1 alleles with ACPA-negative RA was detected.ConclusionThe independent association of DPB1*02:01 with Japanese ACPA(+)RA was identified

Transport of trace metals (Mn, Fe, Ni, Zn and Cd) in the western Arctic Ocean (Chukchi Sea and Canada Basin) in late summer 2012

Distributions of trace metals (Mn, Fe, Ni, Zn and Cd) in the western Arctic Ocean (Chukchi Sea and Canada Basin) in September 2012 were investigated to elucidate the mechanisms behind the transport of these metals from the Chukchi Shelf to the Canada Basin. Filtered (<0.22 μm) and unfiltered seawater samples were analyzed to determine dissolved (D) and total dissolvable (TD) trace metal concentrations, respectively. We identified maxima in vertical profiles for the concentrations of D-Fe and TD-Fe, as well as for the other four analyzed trace metals, which occurred in the halocline and/or near-bottom waters. Concentration profiles of all trace metals except for Cd also tended to show peaks near the surface, which suggest that the inflow of low-salinity Pacific-origin water from the Bering Strait, as well as local fresh water inputs such as river water and melting sea-ice, influenced trace metal concentrations. The distribution patterns and concentration ranges were generally similar between the D and TD fractions for Ni, Zn and Cd, which indicate that Ni, Zn and Cd were present mainly in their dissolved forms, whereas the concentrations of TD-Fe and TD-Mn were generally higher than those of D-Fe and D-Mn, respectively. These results are consistent with the results of previous studies of this region. For both Fe and Mn, labile particulate (LP) concentrations (the difference between the TD and D fractions, which is acid-leachable fraction in the particles during storage at pH 1.5?1.6) were highest in the near-bottom waters of the Chukchi Shelf region. The relationships between the distance from the shelf break and the concentrations of trace metals revealed that Fe and Mn concentrations in halocline waters tended to decrease logarithmically with distance, whereas changes in the concentrations of Ni, Zn, Cd and phosphate with distance were small. These results suggest that the distributions of Fe and Mn were controlled mainly by input from shelf sediment and removal through scavenging processes. Based on the phase distributions of Fe and Mn, which were calculated as ratios between the LP and D fractions, different behaviors between Fe and Mn were expressed during lateral transportation. The concentration of TD-Fe declined rapidly via removal of LP-Fe from the water column, whereas the concentration of TD-Mn declined more slowly through the transformation of D-Mn into LP-Mn. In contrast, the concentrations of D-Cd, D-Zn and D-Ni were more strongly correlated with phosphate levels, which suggest that, like phosphate, the distributions of Cd, Zn and Ni were generally controlled by the internal biogeochemical cycles of the ocean interior. Based on the findings of studies that have previously evaluated the concentration maxima of Ni, Zn and Cd within the halocline layer in the Canada Basin near the Canadian Arctic Archipelago, the elevated Ni, Zn and Cd concentrations in the halocline layer may extend across the Canada Basin from the Chukchi Sea shelf-break area. The determination coefficients for correlations with phosphate concentration varied between the concentrations of Ni, Zn and Cd, which suggest that the sources of these trace metals, such as sediments and sea-ice melting, affected their patterns of distributions differently. Our findings reveal the importance and impact of the halocline layer for the transport of trace metals in the western Arctic Ocean during the late summer. The existence of rich and various sources likely sustained the high concentrations of trace metals and their unique profiles in this region

Relationship between milking management practices and milk somatic cell counts on local dairy farms

推奨されている搾乳衛生が体細胞数に及ぼす影響を知るために、北海道十勝地方の1373戸の酪農家を対象に調査を行った。調査方法は、客観的に評価できる項目を獣医師による聞き取り調査で、また客観的に評価困難な項目については乳検検定時に検定員により調査した。統計学的分析はt検定、一元配置分散分析、多変量解析数量化I類を用いて行った。体細胞数に最も影響度の高かった搾乳衛生は、牛体清潔度、乾乳期治療の有無、ポストディッピングの実施時期、過搾乳の有無、ディッパーの種類、搾乳手袋装着の有無、乳房炎軟膏の注入の仕方であった。また、この7つの作業を影響度の高い作業から順に加えて同時実施していくと、平均バルク乳体細胞数（log（X/10^3））は2.21から2.14へ減少し、同時実施しない作業を増やしていくと2.31から2.45へ増加した。同様に影響度の高い作業から順に同時実施していくと、平均体細胞リニアスコアは2.65から2.36に減少し、同時実施しない作業を増やしていくと2.96から3.76に増加した。以上のことから、現在推奨されている搾乳衛生は、体細胞数を低減する上で重要であることが確認できた。また、体細胞数に相関が高い作業の同時実施により体細胞数の低減効果が高いことが明らかになったことから、これらの作業を含めた推奨作業の同時実施が重要であり、特に相関の高かった作業の確実な実施を指導する必要があると考えられた