Differential gene expression of two extreme honey bee (Apis mellifera) colonies showing varroa tolerance and susceptibility.

Varroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is the most serious pest threatening the apiculture industry. In our honey bee breeding programme, two honey bee colonies showing extreme phenotypes for varroa tolerance/resistance (S88) and susceptibility (G4) were identified by natural selection from a large gene pool over a 6-year period. To investigate potential defence mechanisms for honey bee tolerance to varroa infestation, we employed DNA microarray and real time quantitative (PCR) analyses to identify differentially expressed genes in the tolerant and susceptible colonies at pupa and adult stages. Our results showed that more differentially expressed genes were identified in the tolerant bees than in bees from the susceptible colony, indicating that the tolerant colony showed an increased genetic capacity to respond to varroa mite infestation. In both colonies, there were more differentially expressed genes identified at the pupa stage than at the adult stage, indicating that pupa bees are more responsive to varroa infestation than adult bees. Genes showing differential expression in the colony phenotypes were categorized into several groups based on their molecular functions, such as olfactory signalling, detoxification processes, exoskeleton formation, protein degradation and long-chain fatty acid metabolism, suggesting that these biological processes play roles in conferring varroa tolerance to naturally selected colonies. Identification of differentially expressed genes between the two colony phenotypes provides potential molecular markers for selecting and breeding varroa-tolerant honey bees.

Developmental dysplasia of the hip: open reduction as a risk factor for substantial osteonecrosis.

BACKGROUND: Kalamchi and MacEwen (K&M) described a four-group scheme for classifying osteonecrosis (ON) following treatment for developmental dysplasia of the hip (DDH). However, the four groups can overlap in radiographic appearance, making assessment difficult. QUESTIONS/PURPOSES: We (1) describe a simplified K&M classification; (2) determined whether the simplified classification was reliable; and (3) assessed whether differences in the type of reduction or age at reduction resulted in different degrees of ON. PATIENTS AND METHODS: We retrospectively reviewed 300 patients with DDH treated with either open or closed reduction. We included 101 of these patients (133 involved hips). Intraobserver and interobserver reliability testing of the original and our simplified classification was performed. ON occurred in 64 hips (48%). Of these, 22 had original K&M Group I disease (classified as simplified Group A), and 42 had original K&M Groups II, III, or IV disease (classified as simplified Group B). The mean age of the patients at final followup was 12.4 years (range, 6-26.3 years). RESULTS: The interobserver reliability of the simplified classification was greater than that of the K&M classification (0.51 vs 0.33, respectively). Closed reduction after skin traction resulted in a lower incidence of Group B ON than open reduction, regardless of age at reduction. CONCLUSIONS: We propose a simplified and more reliable classification of ON after DDH. With the new classification we found type of reduction (closed with traction versus open without femoral shortening) but not age influenced the risk of ON. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.

The NHS breast screening programme (pathology) EQA: experience in recent years relating to issues involved in individual performance appraisal.

BACKGROUND: The original role of the National Health Service breast screening programme (pathology) external quality assessment (EQA) scheme was educational; it aimed to raise standards, reinforce use of common terminology, and assess the consistency of pathology reporting of breast disease in the UK. AIMS/METHODS: To examine the performance (scores) of pathologists participating in the scheme in recent years. The scheme has evolved to help identify poor performers, reliant upon setting an acceptable cutpoint. Therefore, the effects of different cutpoint strategies were evaluated and implications discussed. RESULTS/CONCLUSIONS: Pathologists who joined the scheme improved over time, particularly those who did less well initially. There was no obvious association between performance and the number of breast cancer cases reported each year. This is not unexpected because the EQA does not measure expertise, but was established to demonstrate a common level of performance (conformity to consensus) for routine cases, rather than the ability to diagnose unusual/difficult cases. A new method of establishing cutpoints using interquartile ranges is proposed. The findings also suggest that EQA can alter a pathologist's practice: those who leave the scheme (for whatever reason) have, on average, marginally lower scores. Consequently, with the cutpoint methodology currently used (which is common to several EQA schemes) there is the potential for the cutpoint to drift upwards. In future, individuals previously deemed competent could subsequently be erroneously labelled as poor performers. Due consideration should be given to this issue with future development of schemes.

Impact of a national external quality assessment scheme for breast pathology in the UK.

BACKGROUND: This article presents the results and observed effects of the UK National Health Service Breast Screening Programme (NHSBSP) external quality assurance scheme in breast histopathology. AIMS/METHODS: The major objectives were to monitor and improve the consistency of diagnoses made by pathologists and the quality of prognostic information in pathology reports. The scheme is based on a twice yearly circulation of 12 cases to over 600 registered participants. The level of agreement was generally measured using kappa statistics. RESULTS: Four main situations were encountered with respect to diagnostic consistency, namely: (1) where consistency is naturally very high-this included diagnosing in situ and invasive carcinomas (and certain distinctive subtypes) and uncomplicated benign lesions; (2) where the level of consistency was low but could be improved by making guidelines more detailed and explicit-this included histological grading; (3) where consistency could be improved but only by changing the system of classification-this included classification of ductal carcinoma in situ; and (4) where no improvement in consistency could be achieved-this included diagnosing atypical hyperplasia and reporting vascular invasion. Size measurements were more consistent for invasive than in situ carcinomas. Even in cases where there is a high level of agreement on tumour size, a few widely outlying measurements were encountered, for which no explanation is readily forthcoming. CONCLUSIONS: These results broadly confirm the robustness of the systems of breast disease diagnosis and classification adopted by the NHSBSP, and also identify areas where improvement or new approaches are required.

Modification and inheritance of pleiotropic cross resistance and collateral sensitivity in Saccharomyces cerevisiae.

A meiotic segregant (oliPR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oliPR1 has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethlphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oliPR1 phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested. Resistant diploids that produced four resistant ascospores were the result of mitotic recombination prior to meiosis. A partial revertant phenotype (sensitive to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was shown to be due to a single nuclear gene causing partial suppression of oliPR1. Anaerobic pretreatment, 37degrees and 0.5 MKC1 were observed to reduce the growth of oliPR1 when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,-chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho--] state. A revertant of oliPR1 (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin and neomycin. Continuous monitoring of oxygen uptake by the revertant afteranaerobic pretreatment revealed that anaerbiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oliPR1 is a mutation resulting in the alteration of plasma membrane permeability to many diverse inhibitors.

Effects of Abscisic Acid Metabolites and Analogs on Freezing Tolerance and Gene Expression in Bromegrass (Bromus inermis Leyss) Cell Cultures.

Optical isomers and racemic mixtures of abscisic acid (ABA) and the ABA metabolites abscisyl alcohol (ABA alc), abscisyl aldehyde (ABA ald), phaseic acid (PA), and 7[prime]hydroxyABA (7[prime]OHABA) were studied to determine their effects on freezing tolerance and gene expression in bromegrass (Bromus inermis Leyss) cell-suspension cultures. A dihydroABA analog (DHABA) series that cannot be converted to PA was also investigated. Racemic ABA, (+)-ABA, ([plus or minus])-DHABA, and (+)-DHABA were the most active in inducing freezing tolerance, (-)-ABA, ([plus or minus])-7[prime]OHBA, (-)-DHABA, ([plus or minus])-ABA ald, and ([plus or minus])-ABA alc had a moderate effect, and PA was inactive. If the relative cellular water content decreased below 82%, dehydrin gene expression increased. Except for (-)-ABA, increased expression of dehydrin genes and increased accumulation of responsive to ABA (RAB) proteins were linked to increased levels of frost tolerance. PA had no effect on the induction of RAB proteins; however, ([plus or minus])- and (+)-DHABA were both active, which suggests that PA is not involved in freezing tolerance. Both (+)-ABA and (-)-ABA induced dehydrin genes and the accumulation of RAB proteins to similar levels, but (-)-ABA was less effective than (+)-ABA at increasing freezing tolerance. The (-)-DHABA analog was inactive, implying that the ring double bond is necessary in the (-) isomers for activating an ABA response.

Comparison of Dehydrin Gene Expression and Freezing Tolerance in Bromus inermis and Secale cereale Grown in Controlled Environments, Hydroponics, and the Field.

There have been very few reports on the expression of stress-responsive genes in field-grown material. A barley dehydrin cDNA was used to investigate the expression of dehydrin-like transcripts after low-temperature and abscisic acid-induced acclimation of bromegrass (Bromus inermis Leyss) suspension cells and of bromegrass and rye (Secale cereale) plants grown in the field and under controlled environmental conditions. Field-acclimated plants accumulated high levels of dehydrin transcripts and were very freezing tolerant. Plants grown in pots and hydroponics under controlled environments also accumulated dehydrin transcripts and showed increased freezing tolerance. Simulation of a combined drought and freezing stress in pots resulted in expression of dehydrin-like transcripts comparable to those observed in field-acclimated material.

A new method of testing for mitogen-induced lymphocyte stimulation: measurement of the percentage of growing cells and of some aspects of their cell kinetics with an electronic particle counter.

Human lymphocytes were cultured with or without added PHA for periods not exceeding one day so that none of the cells had entered the first mitotic division. The size distribution of the cells was measured with an electronic particle counter and the results were collected in a multichannel analyser. An iterative stochastic model was developed to estimate the proportion of responding cells and their growth characteristics. This mathematical model was based on a few simple assumptions about the pattern of cell growth and was considered to fulfill basic requirements of plausibility and parsimony. The technique described in this paper makes measurement of the absolute percentage of responding cells and their average growth rate in mitogen-induced lymphocyte stimulation tests possible in routine diagnostic laboratories for the first time.

A simple method for determining the extent of cellular contamination in peripheral blood lymphocyte preparations.

A method is described for calculating, from the size distribution of the cells, the extent of cellular contamination of lymphocyte concentrates prepared from venous blood. The precision of this method was checked by direct comparison with differential leucocyte counts obtained by direct microscopy on 29 samples chosen because they showed a wide range of intensity of contamination. By virtue of its technical simplicity, the method has proved useful in a diagnostic service laboratory for checking the purity of lymphocyte preparations either before performing lymphocyte function tests or after a period of 24 h in tissue culture.