Twenty-four chromosome FISH in human IVF embryos reveals patterns of post-zygotic chromosome segregation and nuclear organisation.

Fluorescence in situ hybridisation (FISH) was first applied on in vitro fertilisation (IVF) embryos for the preimplantation genetic diagnosis of sex, then chromosome translocations and later for chromosome copy number (PGS). Because of the controversy surrounding PGS diagnostically, it has been replaced by array-based approaches; however, FISH remains a powerful tool for investigating mechanisms of both post-zygotic segregation error and nuclear organisation, especially if most or all of the chromosomes in the karyotype can be analysed. The purpose of this study was to develop and apply a 24 chromosome FISH assay to investigate chromosome-specific rates of gain and loss, nuclear organisation patterns and the veracity of the original PGS result in days 5-6 human embryos. Analysis of 17 embryos by this newly developed approach gave strong signals for all chromosomes; it revealed chromosome copy number for each human chromosome per cell for each embryo and the nuclear address of the (mostly centromeric) loci probed. As all embryos were surplus to IVF requirements for both transfer and freezing (and many had an abnormal PGS indication) expected high levels of chromosome abnormalities were seen and no single nucleus displayed a normal complement; all were mosaic. Certain patterns emerged, however, namely that chromosome loss was more common than gain and apparent mitotic non-disjunction. Moreover, the centromeric probes tended preferentially to occupy the nuclear centre. Where we had a prior day 3 biopsy PGS result, it was confirmed, in part, by 24 colour FISH in most but not all cases.

Is there a paternal age effect for aneuploidy?

Finding a positive association between paternal age and the incidence of aneuploidy is not difficult. A cursory analysis however reveals that any association is indirect, brought about by a close correlation between paternal age and maternal age. Approaches for dissecting out the confounding age effects of the mother has led to a lively exchange among epidemiologists, with perhaps a consensus for the absence of a paternal age effect, at least for trisomy 21. Molecular studies revealed the relatively minor contribution of paternal errors to trisomy, but even research on the paternally derived trisomies alone has been inconclusive; thus studies focussed directly on the sperm heads. Human-hamster fusion assays were superseded by FISH for establishing any possible link between age and the proportion of disomic sperm in an ejaculate. Despite innumerable microscope hours however, although convincing studies suggesting an age effect for disomies 1, 9, 18 and 21 and the sex chromosomes are in the literature, others failed to notice any association for these or other chromosomes. It is biologically plausible that chromosomal non-disjunction errors should increase with age. Male reproductive hormone production, testicular morphology and semen parameters all decline slowly with age and paternal age is implicated in congenital birth defects, such as achondroplasia and Apert syndromes and also linked to compromised DNA repair mechanisms. Despite several decades of epidemiological and molecular cytogenetic studies, however, we are still not close to a definitive answer of whether or not there is a paternal age effect for aneuploidy. In this review we conclude by questioning the efficacy of FISH because of difficulties in detecting nullisomy and because of evidence that the centromeres (from which most sperm-FISH probes are derived) cluster at the nuclear centre. Array-based approaches may well supersede FISH in addressing the question of a paternal age effect; for now, however, the jury is still out.

Nuclear organization in human sperm: preliminary evidence for altered sex chromosome centromere position in infertile males.

BACKGROUND: Many genetic defects with a chromosomal basis affect male reproduction via a range of different mechanisms. Chromosome position is a well-known marker of nuclear organization, and alterations in standard patterns can lead to disease phenotypes such as cancer, laminopathies and epilepsy. It has been demonstrated that normal mammalian sperm adopt a pattern with the centromeres aligning towards the nuclear centre. The purpose of this study was to test the hypothesis that altered chromosome position in the sperm head is associated with male infertility. METHODS: The average nuclear positions of fluorescence in-situ hybridization signals for three centromeric probes (for chromosomes X, Y and 18) were compared in normoozoospermic men and in men with compromised semen parameters. RESULTS: In controls, the centromeres of chromosomes X, Y and 18 all occupied a central nuclear location. In infertile men the sex chromosomes appeared more likely to be distributed in a pattern not distinguishable from a random model. CONCLUSIONS: Our findings cast doubt on the reliability of centromeric probes for aneuploidy screening. The analysis of chromosome position in sperm heads should be further investigated for the screening of infertile men.

Antibiotics for bronchiolitis in children.

BACKGROUND: Bronchiolitis is a serious, potentially life-threatening respiratory illness commonly affecting young babies. It is most often caused by Respiratory Syncytial Virus (RSV). The diagnosis is usually made on clinical grounds (especially tachypnoea and wheezing in a child less than two years of age). Antibiotics are not recommended for bronchiolitis unless there is concern about complications such as secondary bacterial pneumonia. Despite this, they are used at rates of 34 to 99% in uncomplicated cases. OBJECTIVES: To evaluate the use of antibiotics for bronchiolitis. SEARCH STRATEGY: We searched the following electronic databases: the Cochrane Central Register of Controlled Trials (CENTRAL) which includes the Acute Respiratory Infection Groups' specialised register, the Database of Abstracts of Reviews of Effects (DARE) (The Cochrane Library Issue 3, 2006); MEDLINE (January 1966 to August Week 2, 2006); EMBASE (1990 to March 2006); and Current Contents (2001 to September 2006). SELECTION CRITERIA: Types of studies: single or double blind randomised controlled trials comparing antibiotics to placebo in the treatment of bronchiolitis. TYPES OF PARTICIPANTS: children under the age of two years diagnosed with bronchiolitis using clinical criteria (including respiratory distress preceded by coryzal symptoms with or without fever). Types of interventions: oral, intravenous, intramuscular or inhaled antibiotics versus placebo. Types of outcome measures: primary clinical outcomes: time for the resolution of symptoms/signs (pulmonary markers: respiratory distress; wheeze; crepitations; oxygen saturation; and fever). SECONDARY OUTCOMES: hospital admissions; time to discharge from hospital; re-admissions; complications/adverse events developed; and radiological findings. DATA COLLECTION AND ANALYSIS: All data were analysed using Review Manager software, version 4.2.7. MAIN RESULTS: One study met our inclusion criteria. It randomised children presenting clinically with bronchiolitis to either ampicillin or placebo. The main outcome measure was duration of illness and death. There was no significant difference between the two groups for length of illness and there were no deaths in either group. AUTHORS' CONCLUSIONS: This review found no evidence to support the use of antibiotics for bronchiolitis. This results needs to be treated with caution given only one RCT justified inclusion. It is unlikely that simple RCTs of antibiotics against placebo for bronchiolitis will be undertaken in future. Research to identify a possible small subgroup of patients presenting with bronchiolitis-like symptoms who may benefit from antibiotics may be justified. Otherwise, research may be better focussed on determining the reasons for clinicians to use antibiotics so readily for bronchiolitis, and ways of reducing their anxiety, and therefore their use of antibiotics for bronchiolitis.

Malaria vectors in a traditional dry zone village in Sri Lanka.

Malaria transmission by anopheline mosquitoes was studied in a traditional tank-irrigation-based rice-producing village in the malaria-endemic low country dry zone of northcentral Sri Lanka during the period August 1994-February 1997. Adult mosquitoes were collected from human and bovid bait catches, bovid-baited trap huts, indoor catches, and pit traps. Mosquito head-thoraces were tested for the presence of Plasmodium falciparum and P. vivax, and blood-engorged abdomens for the presence of human blood by ELISAs. House surveys were done at two-day intervals to record cases of blood film-confirmed malaria among the villagers. A total of 7,823 female anophelines representing 14 species were collected. Trends in anopheline abundance were significantly correlated with rainfall of the preceding month in An. annularis, An. barbirostris, An. subpictus, An. vagus, and An. varuna, but were not significant in An. culicifacies and An. peditaeniatus. Malaria parasite infections were seen in seven mosquito species, with 75% of the positive mosquitoes containing P. falciparum and 25% P. vivax. Polymorph PV247 was recorded from a vector (i.e., An. varuna) for the first time in Sri Lanka. Computations of mean number of infective vector (MIV) rates using abundance, circumsporozoite (CS) protein rate, and human blood index (HBI) showed the highest rate in An. culicifacies. A malaria outbreak occurred from October 1994 to January 1995 in which 45.5% of village residents experienced at least a single disease episode. Thereafter, malaria incidence remained low. Anopheles culicifacies abundance lagged by one month correlated positively with monthly malaria incidence during the outbreak period, and although this species ranked fifth in terms of abundance, infection was associated with a high MIV rate due to a high CS protein rate and HBI. Abundance trends in other species did not correlate significantly with malaria. It was concluded that An. culicifacies was epidemiologically the most important vector in the study area.

Household responses to malaria and their costs: a study from rural Sri Lanka.

A study of the cost of malaria at the household level, community perceptions, preventive measures and illness behaviour linked to the disease was undertaken in 5 villages in the dry zone of Sri Lanka. The surveyed community had a high knowledge of malaria, although side effects of antimalarial drugs were often confused with symptoms of the disease. The community sought prompt diagnosis and treatment at 'western-type' facilities, with 84% making use of government facilities as their first choice and 16% preferring private facilities. The preventive measures used were burning coils (54% of families) and special leaves (69% of families), and 93% of the families had their houses sprayed with insecticides. Average direct expenditure on a single malaria episode was $3 US, with some families spending more than 10% of the annual household net income per episode. The highest expenditure was on special diets for the sick person, to neutralize the perceived heating effect of the disease and its treatment.

PIP: Knowledge of community perceptions of malaria, treatment-seeking behaviors, and the household costs of illness is essential to the planning of sustainable malaria control interventions. These factors were investigated in household surveys conducted in 5 villages in Sri Lanka's dry zone in February 1995. 95% of the 216 families interviewed regarded malaria as their main health problem, followed by diarrhea and eye diseases. Malaria was ranked as the third most important community concern, following lack of water for cultivation and poverty. Overall knowledge of malaria's causes, symptoms, and treatment was high, although the side effects of antimalarial drugs were often confused with symptoms of the disease. Reported for the 3-month period preceding the survey were 178 episodes of malaria involving 160 families. Home treatment with paracetamol was the first step taken in 85% of cases; however, all families sought prompt treatment from Western-type facilities, especially government hospitals (48%) and mobile clinics (30%). No families consulted indigenous or ayurvedic physicians, despite their availability in the area. This utilization of prompt, effective treatment is presumed to reflect high knowledge about malaria, the perceived seriousness of the disease, and good transport facilities in the area. The total direct expenditure on a single episode of malaria averaged US $3; the median net annual income was $258. Special foods for malaria patients aimed at neutralizing the perceived heating effect of malaria and antimalarial drugs (e.g., sodas, oranges) were the most costly item. Adults lost an average of 7.8 days of work per episode and those accompanying them to health facilities lost 2.9 days. Preventive measures used by families included bed nets (23%), mosquito coils (54%), and insecticides (93%).

Anopheline (Diptera:Culicidae) breeding in a traditional tank-based village ecosystem in north central Sri Lanka.

A 13-mo survey of immature anopheline mosquitoes breeding in surface water habitats was done at Mahameegaswewa village within the Huruluwewa watershed in north central Sri Lanka as part of a multidisciplinary study on malaria epidemiology. The watershed is representative of the ancient small tank-based irrigation network that still forms an important component of the rice production system in the low elevation dry zone. In total, 3,818 immatures representing 12 species were obtained from 2,940 samples taken from 5 larval habitats within the village ecosystem. Anopheles varuna Iyengar and An. culicifacies Giles were the most abundant species collected. Peak abundance in both species occurred in August-October. Anopheles barbirostris Van der Wulp and An. peditaeniatus Leicester also were abundant, but neither these nor the other anophelines attained comparable abundance to An. varuna and An. culicifacies. A clear progression in breeding habitat use from stream bed to tank bed and drainage area pools was seen in An. culicifacies during the premonsoon period. Environmental management measures to reduce or modify these habitats could potentially decrease malaria. transmission.

Dihydroxylated metabolites of cannabinol formed by rat liver in vitro.

Cannabinol (CBN) was metabolized in vitro by a 10,000 g supernatant from rat liver. After removal of unchanged CBN and its monohydroxylated metabolites four dihydroxylated metabolites were isolated. By nuclear magnetic resonance and mass spectrometry the compounds were identified as 1'',7-dihydroxy-CBN. Side chain hydroxylation occurred predominantly at C-4'' anc C-3''.

Chromatographic separation of cannabinoids and their monooxygenated derivatives.

delta1-Tetrahydrocannabinol, delta6-tetrahydrocannabinol, cannabinol, cannabidiol and several of their monooxygenated derivatives have been separated from each other by a combination of liquid, thin-layer and gas chromatography. Retention volumes (on Sephadex LH-20), Rf values and retention times can be recorded, and may provide guidance in the separation and identification of these known cannabinoids.

Oxygen kinetics of frozen cytochrome oxidase. The capacity of the oxygen pocket.

Low-temperature kinetics of the reaction between O2 and cytochrome oxidase suggest the existence of an O2 pocket of limited capacity in membrane-bound cytochrome oxidase, and one of larger capacity in purified cytochrome oxidase. A model is proposed to explain the difference in capacity of the pockets.

Spectroscopic forms of carbonmonoxi-cytochrome oxidase.

A systematic study of the errors of low-temperature recording of kinetics of the cytochrome oxidase-CO reaction had identified the classic devitrification process of Keilin & Hartree [(1950) Nature (London)165, 504-505]. The methodology described here minimizes this effect, and the computation methods afford appropriate ways of detecting a residual effect. Thus it has been possible to identify that absorption difference spectra and kinetics of the reaction of fully reduced or half-reduced cytochrome oxidase with CO indicate only one spectroscopic form of the respective carbonmonoxi-cytochrome oxidase.

The effect of measuring light photolysis on the kinetics of reaction of reduced cytochrome a3 and carbon monoxide.

The kinetics of reaction of reduced cytochrome a3 and CO are re-investigated by non-linear optimization techniques. When photolysis by the monitoring light is taken into account, the experimental data are best fitted by a single-intermediate mechanism.