Codon volatility of hemagglutinin genes of H5N1 avian influenza viruses from different clades

Codon volatility is a method recently developed to estimate selective pressures on proteins on the basis of their synonymous codon usage. Volatility of a codon was defined as the fraction of single nucleotide substitutions that would be nonsynonymous. Higher volatility may indicate that the gene has been under more positive selection in the recent past. We analyzed volatility of hemagglutinin genes of H5N1 viruses in the recent outbreaks and observed differences in the volatility among viruses of different clades. The codon volatility of subclade 2.1 viruses from Indonesia was the lowest among all H5N1 clades and subclades. Time series analyses since the beginning of the epidemic in 2004 showed that codon volatility of subclade 2.1 has gradually decreased, while those of other major clades have been increasing. This may reflect differences in the recent evolution of these viruses.     

Association of Hb Q-Thailand with homozygous Hb E and heterozygous Hb Constant Spring in pregnancy

Hemoglobin (Hb) Q-Thailand [alpha74(EF3): Asp-->His] is an abnormal Hb found mainly in China and South-east Asian countries. Association of the alpha(Q-Thailand) allele with alpha-thalassemia has important implications in diagnosis. We report the hitherto undescribed conditions of this variant in two unrelated pregnant Thai women. Routine Hb analyses using high-performance liquid chromatography identified abnormal Hb migrating after Hb A(2) in addition to a homozygous Hb E in the proband 1 and to a heterozygous Hb Constant Spring (Hb CS) in the proband 2. Further alpha-globin gene analysis identified that the variant was caused by the GAC to CAC mutation at codon 74 of the alpha1-globin gene corresponding to the Hb Q-Thailand, detected in cis to the 4.2 kb deletional alpha-thalassemia 2 in both cases. Interaction of the alpha(Q-Thailand) with the beta(E) globin chains in the proband 1 leads to a Hb variant, namely the Hb QE. Family study of the proband 1 showed that her non-pregnant sister had the same genotype but her father was a double heterozygote for Hb E and Hb Q-Thailand in whom both Hb Q-Thailand and Hb QE were detected. Genotype-phenotype relationships observed in these families with complex hemoglobinopathies are presented and compared with those of simple homozygote for Hb E, heterozygote for Hb CS and heterozygote for Hb Q-Thailand found in other unrelated subjects. A simple DNA assay based on allele-specific polymerase chain reaction for simultaneous detection of the Hb Q-Thailand mutation and the 4.2 kb deletional alpha-thalassemia 2 determinant was developed and validated.     

Diagnosing pneumonia in rural Thailand: Digital cameras versus film digitizers for chest radiograph teleradiology.

BACKGROUND: Accurate surveillance for pneumonia requires standardized classification of chest radiographs. Digital imaging permits rapid electronic transfer of data to radiologists, and recent improvements in digital camera technology present high quality, yet cheaper, options. METHODS: We evaluated the comparative utility of digital camera versus film digitizer in capturing chest radiographs in a pneumonia surveillance system in rural Thailand using a panel of radiologists; the gold standard was the hard-copy radiograph. We calculated sensitivity and specificity and conducted a receiver operator characteristics (ROC) analysis. RESULTS: Of the 192 radiographs from patients with clinical pneumonia, 166 (86%) were classified as pneumonia on the hard copies. Sensitivity and specificity for identifying pneumonia were 89% and 73% for the camera and 90% and 65% for the digitizer. In the ROC analysis, there was no statistically significant difference in the area under the curve (camera, 0.86; film digitizer, 0.91, p = 0.29). The digital camera set cost 965 dollars compared to 3000 dollars for the film digitizer. CONCLUSION: Detection of pneumonia was not measurably compromised by using digital cameras compared with film digitizers. The 3-fold lower cost of the digital camera makes this technology an affordable and widely accessible alternative for surveillance systems, vaccine trials, and perhaps clinical use.     

Multiplex allele-specific PCR assay for differential diagnosis of Hb S, Hb D-Punjab and Hb Tak

BACKGROUND: Apart from hemoglobin (Hb) E, Hb D-Punjab [beta121(GH4)Glu-Gln] and Hb Tak [beta147Term-Thr] are the two most common beta-chain variants among the Asian population. These two Hb variants have similar alkaline electrophoretic mobilities and HPLC profiles as those of the Hb S [beta6(A3)Glu-Val]. Differential diagnosis of these clinically relevant hemoglobinopathies is therefore problematic. Direct detection of the beta-globin gene mutations would be another diagnostic alternative. METHODS: A simultaneous DNA diagnosis of the three Hb variants was developed based on the multiplex allele-specific polymerase chain reaction (PCR) approach. The method was validated on 10 carriers of Hb D-Punjab, 5 carriers of Hb Tak, 2 carriers of Hb S and 50 normal individuals of Thai origin. RESULTS: The three abnormal Hbs could be correctly diagnosed with the simultaneous PCR approach, and a complete concordance with results using other established methods was obtained. CONCLUSIONS: The multiplex allele-specific PCR approach developed should prove useful in complementing routine Hb analysis for differential diagnosis of these three common Hb variants and should facilitate a program of hemoglobinopathy screening in the region.     

In vitro evaluation of aerosol bronchodilator delivery during noninvasive positive pressure ventilation: effect of ventilator settings and nebulizer position

OBJECTIVE: Respiratory failure due to exacerbation of obstructive lung disease has been successfully treated with noninvasive positive pressure ventilation (NPPV). However, there have been no reports of factors affecting aerosol delivery during NPPV. Our objective was to determine the effect of ventilator settings and nebulizer position on albuterol delivery during NPPV. DESIGN: Bench model study. SETTING: University laboratory. SUBJECTS: None. INTERVENTIONS: A Respironics BiPAP S/T-D30 with a standard circuit was attached to a lung model simulating spontaneous breathing. Inspiratory/expiratory pressures of 10/5, 15/5, 20/5, 15/10, 20/10, and 25/10 cm H2O were tested at respiratory rates of 10 and 20/min. A nebulizer was filled with 5 mg of albuterol in 4 mL of solution, driven with 8 L/min oxygen, and placed at either a proximal (ventilator outlet) or distal (between leak port and lung model connection) position. Albuterol delivery was estimated by measuring the amount of the albuterol collected on a filter placed at the inlet of the lung model. MEASUREMENT AND MAIN RESULTS: Albuterol delivery varied from 5.2 +/- 0.4% to 24.5 +/- 1.3% of the nominal dose and was significantly affected by the position of the nebulizer, respiratory rate, and BiPAP settings (p <.001 in each case). The greatest albuterol delivery was observed with the nebulizer operating at the distal position and a respiratory rate of 20/min. At this respiratory rate and nebulizer placement, albuterol delivery increased with increasing inspiratory pressure levels and decreased as expiratory pressure levels were increased. Nebulizer flow did not affect function of the ventilator. CONCLUSIONS: At optimum nebulizer position (between the leak port and patient connection) and ventilator settings (high inspiratory pressure and low expiratory pressure), as much as 25% of the nominal albuterol dose may be delivered during NPPV.     

Heliox delivery with noninvasive positive pressure ventilation: a laboratory study

BACKGROUND: There is clinical interest in the use of heliox (helium-oxygen mixture) during noninvasive positive pressure ventilation (NPPV), but delivery of heliox with ventilators designed for NPPV has not been reported. We studied helium concentration ([He]) when an 80%:20% helium:oxygen mixture (heliox) was used with 5 NPPV ventilators (Knightstar, Quantum, BiPAP S/T-D30, Sullivan, and BiPAP Vision). METHODS: A simulated spontaneous breathing lung model was connected to the ventilators with a circuit incorporating a standard leak. Heliox flows of 0, 5, 10, and 18 L/min and oxygen flows of 0 and 10 L/min were titrated into the system at either a proximal position near the lung model or a distal position near the ventilator (titration method). Because the BiPAP Vision has an oxygen delivery module, it was also studied using heliox connected to the air inlet of an oxygen blender, with the blender outlet connected to the oxygen module of the ventilator (blender method). All ventilators were evaluated in spontaneous/timed mode at inspiratory/expiratory pressures of 10/5, 15/5, and 20/5 cm H(2)O. After 5 minutes, [He], oxygen concentration, and pressure in the lung model were recorded. RESULTS: Heliox flow, NPPV settings, site of heliox infusion, and type of ventilator significantly (p < 0.05) affected [He]. [He] was > 60% when heliox flow was 18 L/min in some combinations of settings. The BiPAP S/T-D30 and Quantum occasionally functioned erratically. The BiPAP Vision (blender method) ventilator performed erratically with heliox unless the exhalation port test was bypassed on startup. The addition of heliox flow had no important effect on inspiratory or expiratory positive airway pressure on those breaths during which the ventilators functioned correctly. CONCLUSION: Heliox flow was the most important determinant of [He] when using heliox with NPPV. With heliox there was a potential for ventilator malfunction in some conditions. The clinical implications of these findings remain to be determined.     

Effective screening for double heterozygosity of Hb E/α0-thalassemia

One hundred and forty-one blood samples of hemoglobin E (Hb E) carriers were collected to define suitable cutoff values of Hb E level, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) as screening indicators for detecting individuals with double heterozygosity for Hb E/α^o-thalassemia. Based on the values that gave 100% sensitivity, Hb E < 26%, MCV < 74 fl, and MCH < 24 pg were selected. Further validation of these selected values in additional 152 heterozygous Hb E pregnant women revealed 100% sensitivity, 86.2% specificity, and a 25.9% positive predictive value (PPV) for using Hb E cutoff point only, meanwhile, 100% sensitivity, 83.4% specificity, and 22.6% PPV were achieved for the MCV cutoff point. In addition, 100% sensitivity, 86.3% specificity, and 25.9% PPV were gained for the MCH cutoff point. Combining Hb E level with either MCV or MCH cutoff points, the specificity and PPV were increased to 95.1% and 50.0%, respectively. It is concluded that Hb E level < 26%, MCV < 74 fl, and MCH < 24 pg could be used for screening α^o-thalassemia in heterozygous Hb E. However, to improve specificity and PPV of the tests, a combination of Hb E level < 26% with either MCV < 74 fl or MCH < 24 pg is recommended.     

Expression and localization of receptor protein tyrosine phosphatase β and its ligand pleiotrophin in the submandibular gland of mice

ObjectivesThe family of receptor protein tyrosine phosphatase β (RPTPβ) is composed of 4 splice variants and thought to play roles in the neural migration and outgrowth. Several ligands including the growth factor pleiotrophin (PTN) bind to RPTPβ and inhibit its phosphatase activity, thereby activating cellular signalling pathways. We examined the expression and localization of RPTPβ and its ligands in the submandibular gland (SMG) of mice, which is known for a prominent sexual dimorphism in the duct system.DesignThe homogenates and tissue sections of male and female mouse SMG were analysed with RT-PCR, Western blotting, and immunohistochemistry.ResultsThe short receptor type of RPTPβ (RPTPβ-S) was dominantly expressed in the SMG, and the male gland had significantly higher levels of RPTPβ-S expression than the female gland. In the male, RPTPβ-S was localized predominantly in intercalated duct (ID) cells, but was not found in granular convoluted tubule (GCT) cells or acinar cells. In the female, weaker reactivity was demonstrated in both ID and striated duct (SD) cells. Of the known ligands for RPTPβ, PTN was expressed in the SMG, without sexual difference in levels. In the male, PTN was localized in ID cells as well as in cells located in the distal ends of GCT that are in close vicinity to the ID, whereas in the female PTN was colocalized with RPTPβ-S throughout ID and SD cells.ConclusionsThese results indicated that the distribution of RPTPβ-S and its ligand PTN has a close relation to the sexual dimorphism in the duct system of mouse SMG.