Improving the Degree of Pubic Arch Post-Yoga Exercise During Pregnancy.

Yoga exercises undertaken during the maternity period may reduce unpleasant symptoms during pregnancy and reduce delivery pain. Flexibility of the hip joint improves the degree of pubic arch created in an open-leg posture and widens the pelvic outlet region, shortening the time required for delivery and facilitating easy delivery. The degree of pubic arch is related to the flexibility of the hip joint. Although many studies have shown the effect of yoga exercise during pregnancy, the effect on the degree of the pubic arch has not been elucidated. To elucidate whether the degree of the pubic arch is improved by yoga exercise during pregnancy, this study enrolled 177 pregnant subjects. The degree of pubic arch pre- and post-yoga exercise was measured using instruments in an open-leg posture (seated with femurs abducted). The mean pubic arch pre- and post-yoga was 122.61 and 127.93 degrees, respectively. The degree of pubic arch post-yoga sessions was significantly increased compared with pre-yoga sessions (p < 0.01). This study showed that yoga exercises during the pregnancy period appear to improve the degree of pubic arch.

Development of a Novel and Rapid Fully Automated Genetic Testing System.

We have developed a rapid genetic testing system integrating nucleic acid extraction, purification, amplification, and detection in a single cartridge. The system performs real-time polymerase chain reaction (PCR) after nucleic acid purification in a fully automated manner. RNase P, a housekeeping gene, was purified from human nasal epithelial cells using silica-coated magnetic beads and subjected to real-time PCR using a novel droplet-real-time-PCR machine. The process was completed within 13 min. This system will be widely applicable for research and diagnostic uses.

A Rapid and Automated Device for Purifying Nucleic Acids.

We have developed a rapid, automated nucleic acid purification device in a single cartridge containing silica-coated magnetic beads. We succeeded in extracting the matrix protein gene of influenza A virus from pharyngeal swab samples within 3 min. The device will be widely applicable to detect a specific gene from the various samples for clinical diagnosis and genetic research.

A novel automated device for rapid nucleic acid extraction utilizing a zigzag motion of magnetic silica beads.

We report a novel automated device for nucleic acid extraction, which consists of a mechanical control system and a disposable cassette. The cassette is composed of a bottle, a capillary tube, and a chamber. After sample injection in the bottle, the sample is lysed, and nucleic acids are adsorbed on the surface of magnetic silica beads. These magnetic beads are transported and are vibrated through the washing reagents in the capillary tube under the control of the mechanical control system, and thus, the nucleic acid is purified without centrifugation. The purified nucleic acid is automatically extracted in 3 min for the polymerase chain reaction (PCR). The nucleic acid extraction is dependent on the transport speed and the vibration frequency of the magnetic beads, and optimizing these two parameters provided better PCR efficiency than the conventional manual procedure. There was no difference between the detection limits of our novel device and that of the conventional manual procedure. We have already developed the droplet-PCR machine, which can amplify and detect specific nucleic acids rapidly and automatically. Connecting the droplet-PCR machine to our novel automated extraction device enables PCR analysis within 15 min, and this system can be made available as a point-of-care testing in clinics as well as general hospitals.

Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays.

BACKGROUND: Immunochromatographic antigen tests have been widely used for detection of influenza virus; however its low sensitivity restricts the use of clinical materials other than nasopharyngeal swabs. Saliva is obtained non-invasively and has utility for diagnosis of influenza. Polymerase chain reaction (PCR) is not typically used for rapid testing because it is time consuming. We evaluated the utility of saliva as diagnostic materials for influenza virus infection by PCR-based assays. METHODS: Nasopharyngeal swabs and saliva were simultaneously collected from 144 patients and investigated by reverse transcription-quantitative PCR (RT-qPCR) and droplet-RT-PCR. RESULTS: Overall concordance of results from nasopharyngeal swabs and saliva were 95.8%. Influenza gene was detectable in less than 12min in saliva by the droplet-RT-PCR. Saliva as well as nasopharyngeal swabs contained more than 1×10(2) copies/µl of the influenza gene. About half of the patients provided positive results in nasopharyngeal swabs and saliva within 24h from the onset of the symptoms. CONCLUSION: The study demonstrates that saliva can be used as an alternative specimen source to nasopharyngeal swabs. When rapid PCR assay including RNA extraction to be full-automation in a miniaturized machine, point-of-care test based on PCR may be realized using saliva without restriction of materials.

Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instant-quality fluorescence in situ hybridization.

BACKGROUND: Acute promyelocytic leukemia (APL) with the PML-RARA fusion gene can be effectively cured using molecular-targeted therapies, which require both detection and quantification of the PML-RARA fusion gene. Here, we developed a rapid assay for identifying and measuring the PML-RARA fusion gene in patients with APL using droplet-reverse transcription-polymerase chain reaction (droplet-RT-PCR) and instant quality-fluorescence in situ hybridization (IQ-FISH). METHODS: RNA for droplet-RT-PCR and fixed-cell suspensions for IQ-FISH were prepared from five patients with APL and three controls. We evaluated the amplification efficiency and reaction time with droplet-RT-PCR and signal clarity and hybridization time with IQ-FISH. RESULTS: The reaction using droplet-RT-PCR was completed in 26min. The PML-RARA fusion gene was detected in all samples from the five patients. IQ-FISH yielded clear signals after 1h of hybridization. There were no significant differences in signal clarity or positive signal ratios between IQ-FISH and conventional FISH. CONCLUSIONS: Simultaneous droplet-RT-PCR and IQ-FISH, in addition to morphological examination of blood smears, can be used to diagnose patients as having APL within 4h based on molecular/cytogenetic results. Rapid diagnosis can allow effective therapies to be started promptly.

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

BACKGROUND: Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. METHODS: The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. RESULTS: One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. CONCLUSIONS: One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

BACKGROUND: Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. METHODS: SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). RESULTS: Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. CONCLUSION: The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

A new high-speed droplet-real-time polymerase chain reaction method can detect bovine respiratory syncytial virus in less than 10 min.

The polymerase chain reaction (PCR) has been widely used for diagnosis of infectious diseases of domestic animals. Rapid detection of respiratory pathogens of cattle is useful for making therapeutic decisions. Therefore, we developed a new genetic-based method called droplet-real-time PCR, which can detect bovine respiratory syncytial virus (BRSV) within 10 min. Our droplet-real-time PCR markedly reduced the reaction time of reverse transcription-PCR while maintaining the same sensitivity as conventional real-time PCR, and it can be used as a rapid assay for detection of BRSV. Furthermore, our method is potentially applicable for rapid diagnosis of almost all infectious diseases, including highly pathogenic avian influenza virus.

DNA microarray analysis of transcriptional responses of rats housed on solid and grid cage flooring.

We performed DNA microarray analysis on the white blood cells (WBCs) of rats housed on solid and grid cage flooring. The expression levels of 50 genes were found to increase more than 2-fold in the WBCs on grid cage flooring, including many genes encoding proteins involved in inflammatory or immune responses. It is therefore suggested that the health and welfare of laboratory rats is likely to be improved by housing rats on solid floors.

Removal of maternal retinoic acid by embryonic CYP26 is required for correct Nodal expression during early embryonic patterning.

The abundance of retinoic acid (RA) is determined by the balance between its synthesis by retinaldehyde dehydrogenase (RALDH) and its degradation by CYP26. In particular, the dynamic expression of three CYP26 genes controls the regional level of RA within the body. Pregastrulation mouse embryos express CYP26 but not RALDH. We now show that mice lacking all three CYP26 genes manifest duplication of the body axis as a result of expansion of the Nodal expression domain throughout the epiblast. Mouse Nodal was found to contain an RA-responsive element in intron 1 that is highly conserved among mammals. In the absence of CYP26, maternally derived RA activates Nodal expression in the entire epiblast of pregastrulation embryos via this element. These observations suggest that maternal RA must be removed by embryonic CYP26 for correct Nodal expression during embryonic patterning.

CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse.

The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1-/-Cyp26c1-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.

Slab optical waveguide high-acidity sensor based on an absorbance change of protoporphyrin IX.

A sensitive and fast-responsive evanescent wave absorption sensor has been constructed for pH measurements in highly acidic ranges. This sensor is based on a pH-dependent color change of protoporphyrin IX (PPIX). For the sensitive detection, a visible attenuated total reflection spectrometer with a slab optical waveguide (SOWG) was laboratory-made, and the guiding layer surface was modified with a PPIX-immobilized acrylamide-based thin membrane. The sensing membrane with a thickness of approximately 1 mum was directly fabricated on the SOWG glass surface by copolymerization of acrylamide, N,N'-methylene bisacrylamide, and PPIX in the narrow space confined by a cover plate. PPIX possesses two double bonds in its structure, and so it can be covalently incorporated into the membrane. The response characteristics of the PPIX-immobilized optode membrane were explored using aqueous solutions with different concentrations of HNO(3) or HCl. The optode membrane provided characteristic Soret band absorption spectra depending on the hydrogen ion concentration; the absorbance at 410 nm increased with increasing the concentrations in the range of 0.15-2 M, corresponding to the range of pH -0.3 to 0.8. The absorption signal reached 90% of its final value within 10 s, while the absorption signal was quite readily returned to background level simply by passing 2 mL of distilled water through a flow cell with a volume of 16.5 muL placed on the SOWG. Due to the rapid response and reversibility, this sensor could be operated in a flow-through mode as well as in a conventional static mode, where deionized water was conveniently used as a carrier and conditioning solution. In terms of the stability and precision, this sensor showed no significant change in response even after 100 assays and after being stored in a dry condition for over 6 months. Relative standard deviations for 10 replicate measurements were less than 1.8% in the linear range, and the detection limit calculated from 3 times of the standard deviation was 0.02 pH unit.

The prepattern transcription factor Irx2, a target of the FGF8/MAP kinase cascade, is involved in cerebellum formation.

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Here we report characterization of Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes, that is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. Our data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates.

Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb.

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.