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Additional file 3. Association between IC50 values for chloroquine and polymorphisms at position 184 in pfmdr1 in the parasites with the pfcrt K76T mutation.

Additional file 2. Allele prevalence in pfcrt and pfmdr1.

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Additional file 5. No growth defect by the expression of PfRab5b-YFP-DD or PfRab5bQ94L-YFP-DD seen in the growth curve. Growth curve of parasites expressing PfRab5b-YFP-DD (a) or PfRab5bQ94L-YFP-DD (b). Parasitemia (%) was measured after treatment with (green and yellow) or without (blue and red) 0.5 μM Shld1 in the presence of 5 nM WR99210-HCl. (c) Immunoblot using anti-GFP antibody to probe PfRab5-YFP-DD showed that PfRab5-YFP-DD was highly expressed after the incubation with 0.5 μM of Shld1 for 24 h. Tpx1 is a loading control. (d) Quantification of intensity of PfRab5-YFP-DD in Additional Figure 5c. (e) Immunoblot of PfRab5-YFP-DD before or after washout of Shld1 for 2 h. (f) Quantification of intensity of PfRab5-YFP-DD in Additional Figure 5e.

Additional file 1. Structures of constructs used in this study. Plasmids used for the analysis of P. berghei (a) or P. falciparum (b). For coexpression of PbRab5b-mAG and RFP, a fusion fragment comprising the promoter region of the gene encoding the P. falciparum chloroquine resistance transporter (CRT) promoter [1], TagRFP amplified from the pTagRFP-C plasmid (Evrogen), and PbDT were PCR-amplified using overlapping oligonucleotides, and the In Fusion HD cloning kit was used to insert the DNA fragment into the XhoI site of the PbRab5b-mAG plasmid (PbRab5b-mAG+RFP). For single crossover transfection of constitutive active PbRab5bQ91L mutant, an upstream sequence encompassing nucleotide positions at â 1,500 bp to â 500 bp of PbRab5b was inserted into HindIII site of the plasmid for double crossover (PbRab5bQ91L-mAG for single crossover). Construction of PbRab5b-mAG, PbRab5b Chimeric, GOI-YFP-DD, RFP, GOI-RFP was described in the Materials section of the main manuscript.Reference[1] van Dooren GG, Marti M, Tonkin CJ, Stimmler LM, Cowman AF, McFadden GI. (2005) Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle of Plasmodium falciparum. Mol Microbiol; 57:405-19.

Gastrointestinal symptoms, such as abdominal pain and diarrhea, are frequently observed in patients with Plasmodium falciparum malaria. However, the correlation between malaria intestinal pathology and intestinal microbiota has not been investigated. In the present study, infection of C57BL/6 mice with P. berghei ANKA (PbA) caused intestinal pathological changes, such as detachment of epithelia in the small intestines and increased intestinal permeability, which correlated with development with experimental cerebral malaria (ECM). Notably, an apparent dysbiosis occurred, characterized by a reduction of Firmicutes and an increase in Proteobacteria. Furthermore, some genera of microbiota correlated with parasite growth and/or ECM development. By contrast, BALB/c mice are resistant to ECM and exhibit milder intestinal pathology and dysbiosis. These results indicate that the severity of cerebral and intestinal pathology coincides with the degree of alteration in microbiota. This is the first report demonstrating that malaria affects intestinal microbiota and causes dysbiosis.

Additional file 6. N-terminal myristoyl and palmitoyl modification of PfRab5b is essential for its transport to the TVN. (a) Subcellular localization of wild-type and mutated PfRab5b-YFP-DD (green) after 24-h Shld1 stabilization (left, Shld1) and after following 2-h Shld1 washout (right, washout). The TR-ceramide red fluorescent dye (red) labels the parasites and iRBC membranes. Washing out of Shld1 resulted in the loss of GFP signals from G2A and C3A mutants. (b) Immunoblotting after proteinase K (ProK) treatment of iRBCs permeabilized with streptolysin O. Anti-PfEXP2 antibody is used as a control which is not processed with proteinase K.

Additional file 4. Peripheral localization of PbRab5b-mAG in trophozoite stage of parasites. (a) Fluorescence image of PbRab5b-mAG in trophozoite-stage parasites. Transgenic parasites expressing PbRab5b-mAG under the regulation of PbRab5b promoter were fixed with PFA, and faint cytosolic fluorescence of mAG were detected (green). Nuclei were stained with DAPI (blue). In control wild-type parasites, mAG fluorescence signal was not detected. Bar, 10 μm. (b) Magnified images of trophozoites expressing both PbRab5b-mAG and TagRFP. Trophozoite stage of parasites expressing PbRab5b-mAG (green) and cytosolic TagRFP (red) were fixed and the mAG and TagRFP fluorescence signals were obtained. Histograms of the green and red intensities along the white arrow are shown in the right graph. Black arrowheads indicate regions where stronger PbRab5b-mAG signal were detected compared to the TagRFP signal. Bar, 5 μm. (c) Peripheral localization of PbRab5bQ91L-mAG expressed under the regulation of PbRab5b promoter in trophozoite-stage parasites. A constitutively active PbRab5bQ91L-mAG mutant, in which Gln at aa 91 was replaced with Leu [1,2] was integrated into the upstream of PbRab5b genomic locus by single crossover method. Fluorescence of PbRab5bQ91L-mAG mutant protein was accumulated at the periphery of the parasite (green). Nuclei were stained with DAPI (blue). Bar, 5 μm.References[1] Li G, Barbieri MA, Colombo MI, Stahl PD (1994) Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis. J Biol Chem 269: 14631-14635.[2] Stenmark H, Parton RG, Steele-Mortimer O, Lutcke A, Gruenberg J, et al. (1994) Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J 13: 1287-1296.

Additional file 3. Multiple-alignment of amino acids sequences of Plasmodium Rab5b and Toxoplasma Rab5b. The GTP-binding box (red boxes) and the effector domain (light green box) are shown. Amino acids in the blue box indicate N-terminal myristoyl and palmitoyl modification sites. Toxoplasma gondii Rab5b possesses atypical insertion sequences at amino acid positions 165-182, which are not present in Plasmodium Rab5b.

資料番号: PAIS08015000

This paper intends to consider the elderly migration to retirement homes for the elderly (Yuryo-Rojin-Homu in Japanese) as a case study in Onjuku in Chiba prefecture. To achive this purpose, the attributes of 286 residents and their previous residences are analyzed based on the directory of residents. As the resultes, the attributes of residents are mostly uniform. Most of them moved independently at ages of 60s from central area of the Tokyo Metropolitan Area (Tokyo, Yokohama, Kawaswaki), and their moves were done by themselves or with spouses. As to their social-economic statuses, their previous occupations and educational status are very high. The intensions of their movements are not to enjoy their life after the retirement at the facility. For residents, most significant function of the private home is the nursing care that is secured until they die, namely "lifelong care". Their movements are not the "amenity move". It is obvious that the private home residents move to receive the "lifelong care".

Recently Projection Pursuit Learning (PPL) emerged in the Neural Network (NN) literatures in recent years which used smoothing functions as activation functions in hidden units of NN. As this network uses smoothing functions as activation functions for hidden units, adaptability for various problems is expected to be excellent than other networks....

In speech recognition under noisy environments, it is necessary to construct a system which reduces noise and enhances speech. It is effective to imitate the human auditory system which has an excellent mechanism to analyze the spectrum for speech enhancement....

We are producing a transgenic mosquito, a flying syringe, to deliver a vaccine protein to human beings via the saliva the mosquito deposits in the skin while biting. The mosquito produces a vaccine protein in the salivary gland (SG) and deposits the protein into the host's skin when it takes the host's blood. We chose circumsporozoite protein (CSP), currently the most promising malaria vaccine candidate, to be expressed in the SG of Anopheles stephensi. To transform the mosquitoes, plasmid containing the CSP gene under the promoter of female SG-specific gene, as well as the green fluorescent protein (GFP) gene under the promoter of 3xP3 as a selection marker in the eyes, was injected into more than 400 eggs. As a result, five strains of GFP-expressing mosquitoes were established, and successful CSP expression in the SG was confirmed in one strain. The estimated amount of CSP in the SG of the strain was 40 ng per mosquito. We allowed the CSP-expressing mosquitoes to feed on mice to induce the production of anti-CSP antibody. However, the mice did not develop anti-CSP antibody even after transgenic mosquitoes had bitten them several times. We consider that CSP in the SG was not secreted properly into the saliva. Further techniques and trials are required in order to realize vaccine-delivering mosquitoes.

2000, 【要旨】

Repolarization abnormalities after radiofrequency ablation in patients with manifest Wolff-Parkinson-White syndrome (WPW) have been attributed to cardiac memory of pre-existing changes in repolarization properties. We compared spatial ventricular gradient (VG) from vectorcardiograms with QRST values of 12-lead ECG in 41 patients with WPW (group A, manifest WPW due to left-sided accessory pathway (n = 20); group B, manifest WPW due to right-sided accessory pathway (n = 12); group C, concealed WPW (n = 9)) before and after ablation. Group N (n = 607) served as control. In groups A and B, the abnormalities of spatial VG and QRST values of 12-lead ECG that existed before and 1 day after ablation significantly decreased 1 week after ablation. In group C, spatial VG and QRST values of 12-lead ECG showed no significant changes. The diagnostic ability of spatial VG is almost equivalent to that of the QRST value of ECG in detecting repolarization abnormalities in patients with WPW before and after ablation. We propose a new concept of a "remodeling gradient" directing from the preexcited area to the opposite side of the ventricle as a result of preexcitation-induced electrical remodeling.

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