Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish beta A and beta E alleles. Competing 15mer primers were a fluorescein-labeled complement to beta A and a rhodamine-labeled complement to beta E, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine- labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha 3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.     

Association between developmental defects of enamel and dental caries in schoolchildren

Despite improvement, dental caries is still the main public oral health problem worldwide and the major cause of pain, tooth loss and chewing difficulties in children and adolescents; and it impacts negatively on oral health-related quality of life. A cross-sectional study of a multistage representative sample of 8–12-year-old Brazilian school children was carried out in order to investigate the association between enamel defects and dental caries. Children's mothers completed a questionnaire about socio-demographic and behavioural characteristics at home. Firth's bias reduced logistic regression models were undertaken to assess the association between the main exposure (enamel defects) and caries experience. The prevalence of any enamel defect was 64.0%; the prevalence of diffuse opacities, demarcated opacities and enamel hypoplasia was 35.0%, 29.5% and 3.7%, respectively. The prevalence of dental caries was 32.4%, with mean DMFT of 0.6 (SD, 1.2). Dental caries experience was more common among children who had enamel hypoplasia in their posterior teeth (OR = 2.79; 95% CI: 1.05, 6.51) than among those with none. In anterior teeth, there was no association. Enamel hypoplasia appears to be an important risk factor for dental caries.     

Differential cholinergic activation of G proteins in rat and mouse brainstem: relevance for sleep and nociception

Murine models are increasingly used for investigations of sleep, yet no previous studies have characterized cholinergic activation of guanine nucleotide binding proteins (G proteins) in mouse brainstem nuclei known to regulate sleep. This study used in vitro [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiography to test the hypothesis that muscarinic cholinergic receptors activate G proteins in C57BL/6J (B6) mouse brainstem. The nuclei studied are homologous to those known in rat and cat to modulate sleep and nociception. In B6 mouse, carbachol significantly increased specific binding of [(35)S]GTPgammaS in the pontine reticular nucleus, caudal part (79%); pontine reticular nucleus, oral part (131%); laterodorsal tegmental nucleus (56%); pedunculopontine tegmental nucleus (86%); dorsal raphe nucleus (53%); dorsal medial periaqueductal gray (54%); and ventrolateral periaqueductal gray (52%) when compared with basal binding. Carbachol-induced G protein activation was concentration-dependent and blocked by atropine, demonstrating mediation by muscarinic receptors. G protein activation by carbachol was heterogeneous across B6 mouse brainstem nuclei. Comparison of [(35)S]GTPgammaS binding between mouse and rat revealed different magnitudes of G protein activation in the pontine reticular formation. In the same pontine reticular formation area of B6 mouse where in vitro treatment with carbachol activates G proteins, in vivo microinjection of cholinomimetics causes a rapid eye movement sleep-like state. These data provide the first direct measurement of muscarinic receptor-activated G proteins in B6 mouse brainstem nuclei known in other species to regulate sleep.     

Correlation between right ventricular indices and clinical improvement in epoprostenol treated pulmonary hypertension patients

The aim of this study was to evaluate which parameter of right ventricular (RV) echocardiographic best mirrors the clinical status of patients with pulmonary arterial hypertension. Patients with pulmonary arterial hypertension on epoprostenol therapy were identified via hospital registry. Twenty patients, (16 females, 4 males) were included in the study, 9 with primary pulmonary hypertension and 11 with other diseases. Echocardiograms before therapy and at 22.7 (+/-9.3) months into therapy were compared. The right ventricular myocardial performance index (RVMPI) was measured as the sum of the isometric contraction time and the isometric relaxation time divided by right ventricular ejection time. Other measures included peak tricuspid regurgitation jet velocity (TRV), pulmonary artery systolic pressure (PASP), pulmonary valve velocity time integral (PVVTI), PASP/PVVTI (as an index of total pulmonary resistance) and symptoms by New York Heart Association (NYHA) functional class. Echo parameters of right ventricular function were analyzed in patients, before and during therapy. There was significant improvement of NYHA class in patients following epoprostenol therapy (P < 0.0001). Peak tricuspid regurgitant jet velocity (pre 4.2 +/- 0.6 m/sec, post 3.8 +/- 0.7 m/sec, P = 0.02) and PASP/PVVTI (pre 6.7 +/- 3.3 mmHg/m per second, post 4.8 +/- 2.2 mmHg/m per second, P < 0.0001) were significantly improved during treatment. RVMPI did not improve (pre 0.6 +/- 0.3, post 0.6 +/- 0.3, P = 0.54). Changes in NYHA class did not correlate with changes in RVMPI (P = 0.33) or changes in PASP/PVVTI (P = 0.58). Despite significant improvements in TRV, PASP/PVVTI, and NYHA class, there was no significant change in RVMPI on epoprostenol therapy. Changes in right ventricular indices were not correlated with changes in NYHA class.     

Carbachol in the pontine reticular formation of C57BL/6J mouse decreases acetylcholine release in prefrontal cortex

The prefrontal cortex and brainstem modulate autonomic and arousal state control but the neurotransmitter mechanisms underlying communication between prefrontal cortex and brainstem remain poorly understood. This study examined the hypothesis that microdialysis delivery of carbachol to the pontine reticular formation (PRF) of anesthetized C57BL/6J (B6) mouse modulates acetylcholine (ACh) release in the frontal association cortex. Microdialysis delivery of carbachol (8.8 mM) to the PRF caused a significant (P<0.01) decrease (-28%) in ACh release in the frontal association cortex, a significant (P<0.01) decrease (-23%) in respiratory rate, and a significant (P<0.01) increase (223%) in time to righting after anesthesia. Additional in vitro studies used the [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) assay to test the hypothesis that muscarinic cholinergic receptors activate guanine nucleotide binding proteins (G proteins) in the frontal association cortex and basal forebrain. In vitro treatment with carbachol (1 mM) caused a significant (P<0.01) increase in [(35)S]GTPgammaS binding in the frontal association cortex (62%) and basal forebrain nuclei including medial septum (227%), vertical (210%) and horizontal (165%) limbs of the diagonal band of Broca, and substantia innominata (127%). G protein activation by carbachol was concentration-dependent and blocked by atropine, indicating that the carbachol-stimulated [(35)S]GTPgammaS binding was mediated by muscarinic cholinergic receptors. Together, the in vitro and in vivo data show for the first time in B6 mouse that cholinergic neurotransmission in the PRF can significantly alter ACh release in frontal association cortex, arousal from anesthesia, and respiratory rate.     

Invasive fungal rhinosinusitis in immunocompromised patients

INTRODUCTION: Invasive fungal rhinosinusitis (IFR) is one of the most important causes of morbidity and mortality in immunocompromised patients, principally those with cellular immunodeficiency, with mortality ranging from 50 to 80%. Prophylaxis and early diagnosis increase the chances of successful treatment. STUDY DESIGN: Clinical prospective randomized study. AIM: To present cases of IFR and to compare them with data reported in the literature. MATERIAL AND METHODS: Analysis of eleven cases of IFR confirmed by pathologist examination. RESULTS: Aspergillus was found to be the most prevalent pathogen. Symptoms ranged from high fever in most cases to nasal discharge, ulceration of the nasal mucosa, headache and periorbital edema. CONCLUSION: The combination of amphotericin B and endoscopic surgery, associated or not with Caldwell-Luc surgery, showed good results. The use of liposomal amphotericin B also presented a satisfactory outcome.     

Virginia Cleland: an oral history by her colleagues and mentorees

PURPOSE: The purpose of this study, an oral history, was to create a documented account of how Virginia Cleland was experienced by her colleagues and those she mentored. DESIGN: Data were collected for 8 months by snowball sampling of colleagues and mentorees. Nineteen individuals from across the United States were contacted and interviewed via telephone. METHODS: An oral history method was used to allow selected colleagues and mentorees of Cleland to talk about their experiences with her as a nurse scholar, academic, advocate for women, and visionary. The following 5 predominant themes were identified: (1) keeping boundaries clear; (2) "outspoken" leadership; (3) intense commitment to potential growth of others; (4) pragmatic, creative ideas, and action; and (5) equal rights for women. CONCLUSIONS: The oral history method is an effective way to demonstrate the characteristics of nurse leaders who contribute to successes in their careers. Nurses are given the opportunity to evaluate the qualities of leadership, especially in turbulent and challenging times.