High-throughput gender identification of Accipitridae eagles with real-time PCR using TaqMan probes.

The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.

A facile method to functionalize gold nano-tripods with high suspension stability in an aqueous environment.

Here we aim to develop a facile emulsion-based method to prepare tripod gold nanoparticles (AuNPs) with high suspension stability in an aqueous environment. A gyroid-structured polymer template formed by the hydrolysis of a degradable block copolymer, polystyrene (PS)-b-poly(l-lactide), is used for the fabrication of AuNPs. Also, a successful emulsification of dichloromethane (DCM) in the aqueous phase is developed by using thiolated polyethylene glycol (PEG-SH) as the stabilizer. Subsequently, the nanohybrids of PS/Au can be fabricated by templated electroless plating, and then selectively dissolving in the DCM dispersive phase. Most interestingly, a dedicated process for the simultaneous release of the tripod AuNPs from the dissolution of PS associated with PEG-SH at the interface of the emulsion is achieved, giving PEG-SH-functionalized tripod AuNPs dispersed in the aqueous phase, which significantly improves the suspension stabilization of tripod AuNPs. The in situ temperature-programmed electrospray-differential mobility analysis provides a quantitative, statistical analysis of mobility diameter, dynamic shape factor, polydispersity, and colloidal stability.

Validation of Spilornis cheela hoya TaqMan probes for potential gender identification of many Accipitridae species.

The objective of this study was to test the hypothesis that genders of Accipitridae species, with the same or similar sequences to our previously proposed Spilornis cheela hoya (S. c. hoya) chromo-helicase-DNA binding protein (CHD)-W-specific and CHD-ZW-common TaqMan probes, can be successfully determined. Eight species of Accipitridae with known genders were collected. After PCR, TA cloning, sequencing, and alignment analyses, sequence length differences of Griffiths P2/P8 PCR amplicons between CHD-Z and CHD-W genes ranged from 2 to 19 bp for these Accipitridae species, and they were unsolved in 3% agarose gel. Using our previous proposed S. c. hoya TaqMan probes, the genders of Circaetus gallicus, completely homologous to the sequences for these CHD probes, were successfully identified. With one nucleotide difference to S. c. hoya CHD-W-specific probe, gender identification of Accipiter gularis, Accipiter soloensis, Accipiter trivirgatus, Accipiter virgatus, and Butastur indicus were validated. With two nucleotide differences in the CHD-W-specific probe and one nucleotide difference in the CHD-ZW-common probe, Pernis ptilorhyncus also performed well for gender identification. In conclusion, the S. c. hoyaCHD probes, coupled with the Griffiths P2/P8 primers, were validated to provide accurate and high-throughput gender identification for many Accipitridae species.

Once-daily vs. twice-daily intrapleural urokinase treatment of complicated parapneumonic effusion in paediatric patients: A randomised, prospective study.

To evaluate the effective dose frequency (once daily vs. twice daily) of intrapleural urokinase treatment in children who required tube thoracostomy for drainage of a complicated parapneumonic effusion, we designed a randomised prospective study in a tertiary medical centre in Taiwan. From June 2002 to January 2005, 30 paediatric patients with complicated parapneumonic effusion who had received chest tube drainage were randomised 1 : 1 to the once-daily (urokinase 5000-6000 IU/kg/dose) or twice-daily (urokinase 2500-3000 IU/kg/dose) treatment. We compared clinical manifestations and outcomes in both groups. There were no differences in pleural effusion characteristics between the groups. Six patients had Streptococcus pneumoniae, one had Staphylococcus aureus, one had Group A Streptococcus, and 22 had unknown pathogens. There were no significant differences between the once- vs. twice-daily group in the amount of drained pleural fluid (564.9 +/- 422.1 ml vs. 560.5 +/- 198.6 ml, respectively), fever duration after chest tube insertion (4.3 +/- 3.2 days vs. 5.3 +/- 2.7 days), or total admission days (14.3 +/- 3.9 days vs. 14.6 +/- 3.0 days) (p > 0.05 for all). Only two patients (one in each group) required the surgery. Thus, we found that both once- and twice-daily administration of urokinase were similarly efficacious, and resulted in good clinical outcomes. Both obviated the need for surgery in most (93%) cases of pneumonia with complicated parapneumonic effusion in this series. A larger, multicentre study is necessary to verify our findings.

Human hepatic alcohol and aldehyde dehydrogenases: genetic polymorphism and activities.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the major enzymes responsible for the metabolism of ethanol in the body. Both exhibit genetic polymorphism in racial populations. To determine hepatic ethanol metabolizing activities in relation to genetic polymorphism, a total of 23 surgical specimens were investigated. The expression patterns of ADH and ALDH isoenzymes were identified by means of agarose isoelectric focusing, and the activities were assayed spectrophotometrically. At 33 mM ethanol, pH 7.5, the activities in the liver with the homozygous phenotype ADH2 1-1 and ADH2 2-2 and the heterozygous phenotype ADH2 1-2 were determined to be 2.9 +/- 0.7, 16.0 +/- 2.5, and 13.6 +/- 1.0 U/g tissue, respectively. The activities of the ALDH2-active and ALDH2-inactive phenotypes at 200 microM acetaldehyde were determined to be 1.06 +/- 0.13 and 0.71 +/- 0.07 U/g tissue, respectively. These findings indicate that human hepatic ethanol-metabolizing activities differ significantly with respect to polymorphism at both the ADH2 and ALDH2 loci. The results suggest that this genetically determined differential hepatic activity may influence drinking behavior and the development of alcoholism among Orientals.