Dose-related Mutagenic and Clastogenic Effects of Benzo[b]fluoranthene in Mouse Somatic Tissues Detected by Duplex Sequencing and the Micronucleus Assay.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that originate from the incomplete combustion of organic materials. We investigated the clastogenicity and mutagenicity of benzo[ b ]fluoranthene (BbF), one of 16 priority PAHs, in MutaMouse males after a 28-day oral exposure. BbF causes robust dose-dependent increases in micronucleus frequency in peripheral blood, indicative of chromosome damage. Duplex Sequencing (DS), an error-corrected sequencing technology, reveals that BbF induces dose-dependent increases in mutation frequencies in bone marrow (BM) and liver. Mutagenicity is increased in intergenic relative to genic regions, suggesting a role for transcription-coupled repair of BbF-induced DNA damage. At higher doses, the maximum mutagenic response to BbF is higher in liver, which has a lower mitotic index but higher metabolic capacity than BM; however, mutagenic potency is comparable between the two tissues. BbF induces primarily C:G>A:T mutations, followed by C:G>T:A and C:G>G:C, indicating that BbF metabolites mainly target guanines and cytosines. The mutation spectrum of BbF correlates with cancer mutational signatures associated with tobacco exposure, supporting its contribution to the carcinogenicity of combustion-derived PAHs in humans. Overall, BbF's mutagenic effects are similar to benzo[ a ]pyrene, a well-studied mutagenic PAH. Our work showcases the utility of DS for effective mutagenicity assessment of environmental pollutants. Synopsis: We used Duplex Sequencing to study the mutagenicity of benzo[ b ]fluoranthene across the mouse genome. Dose-dependent changes in mutation frequency and spectrum quantify its role in PAH-induced carcinogenicity.

Spatiotemporal resonances in a microfluidic system.

We report on the experimental observation of a spatiotemporal resonance phenomenon, in which a temporal excitation locks with a spatial pattern in an open flow system. The observation is made in a microfluidic system. We obtain the expected regimes--mixing and resonant patterns--in qualitative agreement with the theory. As an application, we realized a dual system particle extraction-micromixer.

Improving agglutination tests by working in microfluidic channels.

Latex agglutination tests are used for the diagnosis of diseases in man and animals. They are generally simple, cheap, and do not require sophisticated equipment, nor highly specialized skills. In this Technical Note, we put latex agglutination tests in a microfluidic format. The experiment is performed in PDMS (polydimethylsiloxane) microchannels, using streptavidin-coated superparamagnetic beads and a magnetic field. The target molecule is biotinylated protein A. By taking full advantage of the microfluidic conditions (scaling down of the detection volume and controlled action of the shear flow), we achieved an analytical sensitivity of 10 fmol l(-1)(several hundreds of fg ml(-1)) and a fast response (a few minutes) ; the test is also quantitative. Performances of agglutination tests can thus be improved by orders of magnitude by adapting them to a microfluidic format; this comes in addition to the usual advantages offered by this technology (integration, high throughput etc.).

Chaotic mixing in cross-channel micromixers.

In this article we concentrate on a particular micromixer that exploits chaotic trajectories to achieve mixing. The micromixer we consider here is a cross-channel intersection, in which a main stream is perturbed by an oscillatory flow, driven by an external source. Depending on the amplitude and frequency of the oscillatory flow, one obtains wavy and chaotic regimes, reminiscent of a tendril-whorl mapping. The chaotic states, in which material lines are stretched and folded, favour mixing. A spatiotemporal resonance phenomenon, in which the material-line deformation is transient, is shown. An experiment using soft lithography and integrated valves, in which the resonant states are revealed, is described. From a practical viewpoint, the cross-channel micromixer offers a variety of regimes, which can be exploited to mix fluids or separate particles of different sizes. In the context of microsystems, it can be viewed as a 'smart' elementary system.

Electrokinetically driven microfluidic chips with surface-modified chambers for heterogeneous immunoassays.

This article presents the first example of a microfluidic chip for heterogeneous bioassays using a locally immobilized biospecific layer and operated electrokinetically. The reaction chamber has picoliter dimensions and is integrated into a network of microchannels etched in glass. The high affinity of protein A (PA) for rabbit immunoglobulin G (rIgG) was exploited for chip testing, with PA being immobilized on microchannel walls and fluorescently labeled (Cy5) rIgG serving as sample. It was possible to operate the chip in an immunoaffinity chromatographic manner, using electrokinetically pumped solutions. Concentration of antibody from dilute solution onto the solid phase was demonstrated, with signal gains of approximately 30 possible. A dose-response curve for Cy5-rIgG was obtained for concentrations down to 50 nM, for an incubation time of 200 s. The flexibility of chip layout was demonstrated for competitive immunoassay of rIgG, using both a combined sample/tracer incubation and sequential addition of these solutions. With assay times generally below 5 min for this unoptimized device, the microfluidic approach described shows great potential for many high-throughput screening applications.

Pharmacokinetics of isoniazid under fasting conditions, with food, and with antacids.

STUDY OBJECTIVES: To determine the intra- and intersubject variability in and the effects of food or antacids on the pharmacokinetics of isoniazid (INH). DESIGN: Randomized, four-period cross-over Phase I study in 14 healthy male and female volunteers. Subjects ingested single doses of INH 300 mg under fasting conditions twice, with a high-fat meal, and with aluminum-magnesium antacid. They also received standard doses of rifampin, pyrazinamide, and ethambutol. RESULTS: Serum was collected for 48 hours, and assayed by high performance liquid chromatography (HPLC). Data were analyzed using noncompartmental methods and a compartmental analysis using nonparametric expectation maximization. Both fasting conditions produced similar results: a mean INH Cmax of 5.53 +/- 2.92 microg/ml, Tmax of 1.02 +/- 1.10 hours, and AUC0-infinity of 20.16 +/- 12.45 microg x hr/ml. These findings are similar to those reported previously. Antacids did not alter these parameters significantly (Cmax of 5.62 +/- 2.53 microg/ml, Tmax of 0.71 +/- 0.56 hours, and AUC0-infinity of 20.27 +/- 11.39 microg x hr/ml). In contrast, the high-fat meal recommended by the Food and Drug Administration reduced INH Cmax by 51% (2.73 +/- 1.70 microg/ml), nearly doubled Tmax (1.93 +/- 1.61 hours), and reduced AUC0-infinity by 12% (17.72 +/- 10.32 microg x hr/ml). CONCLUSIONS: These changes in Cmax, Tmax, and AUC0-infinity can be avoided by giving INH on an empty stomach whenever possible.

Pharmacodynamics of recombinant human DNase I in serum.

Recombinant human deoxyribonuclease I (rhDNase) may be an effective therapeutic for the treatment of systemic lupus erythematosus (SLE). The pharmacodynamics of rhDNase in serum was investigated using two activity assays: one based on hydrolysis of a radiolabelled phage DNA and the other based on hydrolysis of human chromatin. The concentration of endogenous immunoreactive DNase in sera from 16 normal subjects was 3.2 +/- 1.4 ng/ml (mean +/- s.d.); however, low levels or no nuclease activity were detected in the same sera, suggesting the presence of DNase inhibitors. We assessed the ability of rhDNase to degrade DNA in undiluted serum, since the observed inhibition of endogenous DNase was reversed upon dilution. Addition of rhDNase to undiluted serum at a concentration of 50-100 ng/ml was necessary for degradation of radiolabelled phage DNA. The activity of rhDNase added to serum from normal subjects and SLE patients was similar. rhDNase degraded human chromatin and chromatin/anti-DNA immune complexes in serum with similar potency (EC50 approximately 100-200 ng/ml). A 500-fold variation in the chromatin/anti-DNA stoichiometry did not significantly affect the digestion of these immune complexes by rhDNase in buffer. These results indicate that a minimum rhDNase concentration of 50-100 ng/ml in serum was required to achieve detectable catalytic activity and that the presence of antibodies to DNA did not inhibit the degradation of DNA/anti-DNA immune complexes.

Problem-based learning in a health sciences librarianship course.

Problem-based learning (PBL) has been adopted by many medical schools in North America. Because problem solving, information seeking, and lifelong learning skills are central to the PBL curriculum, health sciences librarians have been actively involved in the PBL process at these medical schools. The introduction of PBL in a library and information science curriculum may be appropriate to consider at this time. PBL techniques have been incorporated into a health sciences librarianship course at the School of Library and Information Science (LIS) at the University of Wisconsin-Milwaukee to explore the use of this method in an advanced Library and Information Science course. After completion of the course, the use of PBL has been evaluated by the students and the instructor. The modified PBL course design is presented and the perceptions of the students and the instructor are discussed.

How to do it: aortic and mitral valve replacement through aortotomy.

Combined replacement of the aortic and mitral values is a relatively common operation. Occasionally it is feasible to replace the mitral value through the aortic root. This remarkable approach eliminates the need for a second atrial incision, and in reoperation it avoids extensive dissection and release of adhesions. Injury to the heart is minimized and the incidence of perioperative bleeding is reduced. The exposure is excellent and the operation is performed safely and expeditiously.

Direct repair of right atrial rupture after blunt chest trauma without cardiopulmonary bypass.

Tamponade from free wall rupture of the cardiac chambers following blunt thoracic trauma is relatively frequent. Diagnosis requires a high index of suspicion and is rapidly confirmed by echocardiography. Emergent surgery is always mandatory despite apparent stable vital signs. We report a successful repair of a lacerated right atrium without cardiopulmonary bypass (CPB), saved in extremis after undue in-hospital delay.

Review of microscopic studies on the fetal and neonatal kidney.

The developing mammalian kidney has been studied by light microscopic, electron microscopic, immunohistochemical, and autoradiographic techniques. The microscopic studies have been conducted on in vivo samples and in vitro samples. The cellular biology and molecular biology of the developmental steps have been clarified, but more investigations are needed. Information has also been collected concerning the influence of the environment on the microscopic development of the kidney.

Homozygotes and heterozygotes for ciliary neurotrophic factor null alleles do not show earlier onset of Huntington's disease.

The CAG repeat number on Huntington's disease (HD) chromosomes accounts for about 60% of the variance in the age at onset of HD. However, distinct familial factors may also influence the age at onset. HD is associated with loss of medium-sized GABA-ergic striatal output neurons. Intracerebral administration of human ciliary neurotrophic factor (CNTF) protects striatal output neurons in excitotoxic rodent and primate models of HD induced by intrastriatal quinolinic acid injection. We have examined the effect of a common null mutation in the human CNTF gene on the age of onset of HD using a multiple regression approach that takes into account the CAG repeat number on HD chromosomes. We failed to detect an earlier onset of HD in nine homozygotes and 71 heterozygotes with this CNTF mutation compared with 203 homozygotes with wild-type alleles.

Renin and angiotensin II receptor expression in the brains of DES-treated Syrian hamsters.

BACKGROUND: The renin angiotensin system (RAS) promotes vasoconstriction. Expression of RAS is induced by different factors. METHODS: In this study, forebrain sections of hamster brains were studied by immunohistochemical methods to determine the location of renin-positive and angiotensin II receptor-positive cells. The brain sections were obtained from diethylstilbesterol- (DES-) treated hamsters, adult non-DES-treated hamsters, elderly non-DES-treated hamsters, neonatal hamsters, and 15-day fetal hamsters. Circulating renin activity was determined for all but the neonatal and 15-day fetal hamsters. RESULTS: Renin-positive and angiotensin II receptor-positive vascular smooth muscle cells were observed in DES-treated hamsters. No positive cells were observed in neonatal, 15-day fetal, and adult non-DES-treated hamsters. Some expression was observed in elderly hamsters. CONCLUSIONS: Therefore, focal expression of the renin angiotensin system in brain vasculature was induced by the synthetic estrogen DES.

Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease.

Huntington disease (HD) is associated with abnormal expansions of a CAG repeat close to the 5' end of the IT15 gene. We have assembled a set of 293 HD subjects whose ages of onset were known and sized their HD CAG repeats. These repeats accounted for 69% of the variance of age of onset when we used the most parsimonious model, which relates the logarithm of age of onset to a function of CAG repeat number. Since other familial factors have been proposed to influence the age of onset of HD, we have examined a number of candidate loci. The CAG repeat number on normal chromosomes, the delta2642 polymorphism in the HD gene, and apolipoprotein E genotypes did not affect the age of onset of HD. Although mitochondrial energy production defects in HD have led to suggestions that variants in the mitochondrial genome may be associated with clinical variability in HD, this suggestion was not supported by our preliminary experiments that examined the DdeI mitochondrial restriction fragment length polymorphism at position 10,394. Excitotoxicity has been a favored mechanism to explain the cell death in HD, particularly since intrastriatal injection of excitatory amino acids in animals creates HD-like pathology. Accordingly, we investigated the GluR6 kainate receptor. Of the variance in the age of onset of HD that was not accounted for by the CAG repeats, 13% could be attributed to GluR6 genotype variation. These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD and highlight the potential importance of this process in other polyglutamine repeat expansion diseases.

Clinical presentation and patterns of regional cerebral atrophy related to the length of trinucleotide repeat expansion in patients with adult onset Huntington's disease.

We correlated trinucleotide CAG repeat numbers in the huntingtin gene with the regional brain atrophy and clinical phenotype in 23 adult autopsy cases of Huntington's disease (HD). CAG repeat number (39-56, mean 45.4 +/- 4.6) correlated inversely (P < 0.0001) with age at onset and death, but not with disease duration or initial symptoms. Cross-sectional areas of the striatum, pallidum, thalamus, amygdala, hippocampus, and the cortical grey and white matter within the frontal, temporal and parietal lobes at four levels (genu of the corpus callosum, amygdala, accumbens, hippocampus) were measured morphometrically from the coronal brain slices using image analysis. None of these morphometric variables correlated with number of CAG repeats. Thus, tissue atrophy in advanced HD is unrelated to the underlying genetic defect.

Analysis of spinocerebellar ataxia types 1, 2, 3, and 6, dentatorubral-pallidoluysian atrophy, and Friedreich's ataxia genes in spinocerebellar ataxia patients in the UK.

Accurate clinical diagnosis of the spinocerebellar ataxias (SCAs) can be difficult because of overlap in phenotype with other disorders and variation in clinical manifestations. Six SCA loci have been mapped and four disease causing genes identified, in addition to the causative gene for Friedreich's ataxia (FA). All of the identified mutations are expansions of trinucleotide repeat tracts. The SCA2 and SCA6 genes were published recently. The extent of the normal CAG size ranges at these loci and the relative frequencies of the known causes of SCA in the UK are not known. This study first investigated the normal size ranges of the SCA2 and SCA6 loci by genotyping control populations of West African and South African subjects, since African populations generally show the greatest allelic diversity. We found one allele larger than the previously determined normal range for SCA2, and our results at the SCA6 locus agreed with the previously reported normal range. The second component of the study assessed the relative frequencies of the SCA1, 2, 3, and 6, DRPLA, and FA trinucleotide repeat mutations in 146 patients presenting with SCA-like symptoms referred to genetic diagnostic laboratories in the UK. We detected mutations in 14% of patients referred with a diagnosis of autosomal dominant SCA, and in 15% of patients referred with spinocerebellar ataxia where we did not have sufficient family history data available to allow categorisation as familial or sporadic cases. Friedreich's ataxia accounted for 3% of the latter category of cases in our sample, but the most common causes of SCA were SCA2 and SCA6.

Engineering actin-resistant human DNase I for treatment of cystic fibrosis.

Human deoxyribonuclease I (DNase I), an enzyme recently approved for treatment of cystic fibrosis (CF), has been engineered to create two classes of mutants: actin-resistant variants, which still catalyze DNA hydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but still bind G-actin. Actin-resistant variants with the least affinity for actin, as measured by an actin binding ELISA and actin inhibition of [33P] DNA hydrolysis, resulted from the introduction of charged, aliphatic, or aromatic residues at Ala-114 or charged residues on the central hydrophobic actin binding interface at Tyr-65 or Val-67. In CF sputum, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelasticity as determined in sputum compaction assays. The reduced viscoelasticity correlated with reduced DNA length as measured by pulsed-field gel electrophoresis. In contrast, the active site variants H252A or H134A had no effect on altering either viscoelasticity or DNA length in CF sputum. The data from both the active site and actin-resistant variants demonstrate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin). The increased potency of the actin-resistant variants indicates that G-actin is a significant inhibitor of DNase I in CF sputum. These results further suggest that actin-resistant DNase I variants may have improved efficacy in CF patients.

Phenotypic characterization of individuals with 30-40 CAG repeats in the Huntington disease (HD) gene reveals HD cases with 36 repeats and apparently normal elderly individuals with 36-39 repeats.

Abnormal CAG expansions in the IT-15 gene are associated with Huntington disease (HD). In the diagnostic setting it is necessary to define the limits of the CAG size ranges on normal and HD-associated chromosomes. Most large analyses that defined the limits of the normal and pathological size ranges employed PCR assays, which included the CAG repeats and a CCG repeat tract that was thought to be invariant. Many of these experiments found an overlap between the normal and disease size ranges. Subsequent findings that the CCG repeats vary by 8 trinucleotide lengths suggested that the limits of the normal and disease size ranges should be reevaluated with assays that exclude the CCG polymorphism. Since patients with between 30 and 40 repeats are rare, a consortium was assembled to collect such individuals. All 178 samples were reanalyzed in Cambridge by using assays specific for the CAG repeats. We have optimized methods for reliable sizing of CAG repeats and show cases that demonstrate the dangers of using PCR assays that include both the CAG and CCG polymorphisms. Seven HD patients had 36 repeats, which confirms that this allele is associated with disease. Individuals without apparent symptoms or signs of HD were found at 36 repeats (aged 74, 78, 79, and 87 years), 37 repeats (aged 69 years), 38 repeats (aged 69 and 90 years), and 39 repeats (aged 67, 90, and 95 years). The detailed case histories of an exceptional case from this series will be presented: a 95-year-old man with 39 repeats who did not have classical features of HD. The apparently healthy survival into old age of some individuals with 36-39 repeats suggests that the HD mutation may not always be fully penetrant.

Surgery in native valve endocarditis: indications, results and risk factors.

Seventy-nine patients (mean age 49 years) underwent valve replacement or repair for active (58.2%) or healed (41.8%) native valve endocarditis between 1976 and 1992. The most common indication for surgery was congestive heart failure (73.4%), followed by multiple systemic emboli (21.5%). Emergency operation was necessary in 27.8% of the cases. Operative mortality was 3.8% (3 patients) and late mortality 15.1% (12 patients). Streptococci were the most common infecting agents (41.8%), followed by Staphylococcus aureus (11.4%). No organisms were isolated in 27 cases (34.2%). Follow-up spanned 379.8 patient-years with a maximum of 15.8 years. Fifteen late valve-related events (periprosthetic leak, recurrent endocarditis, thrombo-embolic events and hemolysis) and 20 other late complications (anticoagulant-related hemorrhage, arrhythmias or congestive heart failure) occurred in 22 patients. The linearized rate for all late complications is 5.8% per patient-year. The influence of eight preoperative variables on overall mortality and late valve-related complications was assessed: age, valve(s) affected, active or healed infection, bacteriology, annular abscess, emergency or elective surgery, preoperative renal function and NYHA class. Only Staphylococcus aureus (P = 0.0012) was a significant predictor of late valve-related complications. Furthermore, no difference in survival or in valve-related complications was found between the active and healed infections.

Mutation size and age at onset in Huntington's disease.

The mutation responsible for Huntington's disease is a polymorphic (CAG)n repeat sequence which is expanded on affected chromosomes. The number of repeat units observed on 229 affected chromosomes varied from 27 to 102, while the control chromosomes showed a range of 7 to 34 repeats. There was a highly significant relationship between the size of the expanded region and age at onset, larger mutations being associated with earlier onset. This association was strongest in those with onset before 25 years of age but less clear cut with later onset, and is therefore unlikely to be useful for predicting age at onset in the context of presymptomatic testing.