Photocrosslinkable Artificial Nucleic Acid Probe Based miRNA Biosensor

Molecular recognition elements like enzymes, antibodies, and nucleic acids, which are involved in specific binding, are important components in biosensing technologies. These biomolecular recognition elements are based on molecular interactions such as hydrogen bonding, van der Waals forces, and hydrophobic interactions. However, these interactions are often affected by the solution environment such as pH, temperature, and salt concentration, which are the rate-limiting factors for biosensing applications. In this study, we focused on molecular recognition using photocrosslinkable artificial nucleic acids. Photocrosslinkable artificial nucleic acids can form covalent bonds with target nucleic acids upon photoirradiation after hybridization. The covalent bonds formed are stronger than those in conventional molecular recognition and are not affected by the solution environment. Herein, we propose a biosensing system that combines molecular recognition by photocrosslinkable artificial nucleic acids, isothermal amplification by hybridization chain reaction, and electrochemical detection of miR-21 as the target molecule, which has recently attracted attention as a cancer biomarker. This technology eliminates non-specific binding and enables biosensing measurements with a suppressed background

A bienzyme electrochemical biosensor for the detection of collagen l-hydroxyproline

l-hydroxyproline (l-Hyp: trans-4-hydroxy-l-proline) is commonly found at high concentrations in connective tissue proteins such as collagen. It is a remarkably useful molecular marker because variation in the level of l-Hyp is associated with various diseases. Recently, the novel enzymes l-hydroxyproline epimerase and d-hydroxyproline dehydrogenase were isolated from bacteria. In this study, a novel electrochemical biosensor for l-Hyp was constructed using these two enzymes. l-hydroxyproline epimerase epimerized l-Hyp to d-hydroxyproline (d-Hyp: cis-4-hydroxy-d-proline), and d-Hyp was oxidized with the reaction catalyzed by d-hydroxyproline dehydrogenase and mediated by ferrocene. We found that the sensor could determine l-Hyp concentrations of 10–100 μM with high-selectivity

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Abstract Background Effects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts. Methods Under treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression. Results MTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high. Conclusions We present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA

Additional file 3: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Plasmid constructs of Per2/Bik promoter. D-box(+), plasmid constructs containing D-box. D-box(−), plasmid constructs without D-box. D-box motifs of Per2 promoter were mutated from 5′-TTATGTAA-3′ to 5′-CGCCAGGC-3′ (−372 to −365), and 5′-TTACGTAA-3′ to 5′-CAGCGTAA-3′ (−47 to −40). Human Bik promoter containing D-box (−780 to +176) and human Bik promoter without D-box (−260 to +323) constructed. (PDF 215 kb

Additional file 2: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Detailed information of patients with RA enrolled in this study. Joint samples obtained from 10 different patients to establish primary cultured synovial fibroblast cell lines. Age, sex, disease duration, CRP, DAS28-ESR, and treatment with MTX, PSL, and other DMARDs shown in the table. (PDF 252 kb

Additional file 5: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

The mRNA expression of circadian clock genes over time. mRNA expression of circadian clock genes measured at –4 h (before synchronization), 0 h (just before MTX stimulation), 24 h, 32 h, and 48 h. Controls and 10/100 nM of MTX showed almost the same expression rhythms, and MTX influenced their expression levels. (PDF 264 kb

Additional file 6: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Two different transcriptional pathways by which MTX induces apoptosis to synovial fibroblasts: PAR bZIP–Per2 transcriptional pathway and PAR bZIP–Bik transcriptional pathway. We propose MTX induces apoptosis in synovial cells through activated binding of PAR bZIP to D-box in two different genes, Per2 and Bik, and these dual pathways work independently but synergistically. (PDF 206 kb

Additional file 4: of Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

Cell viability of MTX-treated fibroblasts. Cell viability of RA synovial fibroblasts measured by WST-8 assay after 24 h of stimulation of MTX (1 pM to 1 μM). MTX (1 and 10 nM) significantly decreased cell viability as shown in Fig. 1, while 1–100 pM of MTX did not. (PDF 274 kb