Common variants at the promoter region of the APOM confer a risk of rheumatoid arthritis

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 × 10-7). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 × 10-5). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 × 10-10). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.     

Generation of core-shell microcapsules with three-dimensional focusing device for efficient formation of cell spheroid

We present a microfluidic device generating three-dimensional (3D) coaxial flow by the addition of a simple hillock to produce an alginate core-shell microcapsule for the efficient formation of a cell spheroid. A hillock tapered at downstream of the two-dimensional focusing channel enables outside flow to enclose the core flow. The aqueous solution in the core flow was focused and surrounded by 1.8% alginate solution to be solidified as a shell. The double-layered coaxial flow (aqueous phase) was broken up into a droplet by the shear flow of oleic acid (oil phase) containing calcium chloride for the polymerization of the alginate shell. The droplet generated from the laminar coaxial flow maintained a double-layer structure and gelation of the alginate solution made a core-shell microcapsule. The shell-thickness of the microcapsule was adjusted from 8-21 μm by the variation of two aqueous flow rates. The inner shape of the shell was almost spherical when the ratio of the water-glycol mixture in the core flow exceeded 20%. The microcapsule was used to form a spheroid of embryonic carcinoma cells (embryoid body; EB) by injecting a cell suspension into the core flow. The cells inside the microcapsule aggregated into an EB within 2 days and the EB formation rate was more than 80% with strong compaction. The microcapsule formed single spherical EBs without small satellite clusters or a bumpy shape as observed in solid microbeads. The microfluidic chip for encapsulation of cells could generate a number of EBs with high rate of EB formation when compared with the conventional hanging drop method. The core-shell microcapsule generated by 3D focusing in the microchannel was effective in forming large number of spherical cell clusters and the encapsulation of cells in the microcapsule is expected to be useful in the transplantation of islet cells or cancer stem cell enrichment.     

Stable aluminium fluoride chelates with triazacyclononane derivatives proved by X-ray crystallography and 18F-labeling study

A single crystal structure of an aluminium-fluoride complex of a model compound (NODA-benzyl) was studied to understand the co-ordination chemistry. Series of ligands with an extra carboxylic acid linker for biomolecule conjugation were studied for improved (18)F-labeling applications.     

Pyrazole analogues of porphyrins and oxophlorins

A series of porphyrin analogues with pyrazole rings replacing one of the usual pyrrole subunits have been synthesized. This was accomplished by reacting 1-phenyl, 1-methyl and 1-ethyl pyrazole-1,3-dicarbaldehydes with a tripyrrane in the presence of TFA, followed by an oxidation step. The initially formed phlorin product was sufficiently stable for the N-phenyl system to be isolated and characterized, although the related N-alkyl phlorin analogues were less stable. Attempts to dehydrogenate the intermediary phlorins with DDQ resulted in decomposition, but the N-alkylphlorins could be oxidized with 0.2% aqueous ferric chloride solutions. Although the phenyl-substituted phlorin could not be oxidized under these conditions, it did afford the pyrazoloporphyrin upon treatment with silver acetate under acidic conditions. Oxidations with silver acetate also afforded oxophlorin analogues where the oxo-linkage was selectively formed at the 5-position. The pyrazole-containing porphyrin analogues are cross-conjugated and exhibit only a small degree of diatropic character. The internal CH resonances were observed between 5.27 and 5.87 ppm, while the external meso-protons fell into a range of 6.84-7.88 ppm. The borderline overall aromatic character was attributed to dipolar resonance contributors. Protonation considerably increased the diatropicity and the diprotonated dications formed from these porphyrin analogues gave the internal CH resonance at upfield values of 2.65-3.20 ppm. The aromatic character was enhanced by the presence of an electron-donating alkyl substituent on the nitrogen compared to the phenyl-substituted species. The pyrazoloporphyrins reacted with nickel(II) acetate in DMF, or palladium(II) acetate in acetonitrile, to give the corresponding organometallic derivatives. The metal complexes showed increased diatropic character but protonation afforded nonaromatic cations. The oxophlorin analogues were also nonaromatic in the free base and protonated forms. This work extends our understanding of carbaporphyrinoid systems and provides the first detailed studies on pyrazole-containing porphyrin analogues.     

A general approach to triphenylenes and azatriphenylenes: total synthesis of dehydrotylophorine and tylophorine

A convergent and flexible synthesis of substituted triphenylenes, azatriphenylenes, and the cytotoxic alkaloids dehydrotylophorine and tylophorine has been developed.     

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.     

Hysteretic sorption of light gases by a porous metal-organic framework containing tris(para-carboxylated) triphenylphosphine oxide

The porous metal-organic framework (MOF) PCM-4, based on tris(para-carboxylated) triphenylphosphine oxide, contains atypical, polar organic substituents; the material exhibits a hysteretic sorption of Ar, N2 and O2, and demonstrates the advantage of ligands of this type.     

A MELAS syndrome family harboring two mutations in mitochondrial genome

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.