Adolescent valuation of CARIES-QC-U: a child-centred preference-based measure of dental caries.

OBJECTIVES: This study develops an adolescent value set for a child-centred dental caries-specific measure of oral health-related quality of life (OHRQoL) based upon CARIES-QC (Caries Impacts and Experiences Questionnaire for Children). This study develops a new approach to valuing child health by eliciting adolescent preferences and anchoring these onto the 1-0 full health-dead QALY (quality adjusted life year) scale using ordinal adult preferences. METHODS: Two online surveys were created to elicit preferences for the CARIES-QC classification system. The first comprised best-worst scaling (BWS) tasks for completion by adolescents aged 11-16 years. The second comprised discrete choice experiment tasks with a duration attribute (DCETTO) for completion by adults aged over 18 years. Preferences were modelled using the conditional logit model. Mapping regressions anchored the adolescent BWS data onto the QALY scale using adult DCETTO values, since the BWS survey data alone cannot generate anchored values. RESULTS: 723 adolescents completed the BWS survey and 626 adults completed the DCETTO survey. The samples were representative of UK adolescent and adult populations. Fully consistent and robust models were produced for both BWS and DCETTO data. BWS preferences were mapped onto DCETTO values, resulting utility estimates for each health state defined by the classification system. CONCLUSION: This is the first measure with predetermined scoring based on preferences to be developed specifically for use in child oral health research, and uses a novel technique to generate a value set using adolescent preferences. The estimates can be used to generate QALYs in economic evaluations of interventions to improve children's oral health.

Everyone else is using it, so why isn't the UK? Silver diamine fluoride for children and young people.

INTRODUCTION: Silver diamine fluoride (SDF) is used to prevent and arrest caries across the globe, particularly in the developing world. Whilst its use in the Western World is increasing, it is not yet routinely used in the United Kingdom, nor is it advocated by our national guidelines. OBJECTIVES: To explore the literature surrounding the use of SDF, and consider the reasons why SDF has not yet been widely adopted in the United Kingdom (UK). DISCUSSION: There is a growing evidence base for the use of SDF for the arrest and prevention of dental caries in the primary and permanent dentition. Potential side effects include staining of carious tooth structure, but in some cases this is acceptable to parents. There is no evidence for the cost effectiveness of SDF, although it may be a reasonably cost-effective option. CONCLUSION: SDF is perhaps not yet widely adopted in the UK due to a perceived parental concern about its staining effect. With a growing evidence base and reportedly higher efficacy than fluoride varnish for caries prevention and arrest, SDF has the potential to play an important role in managing dental disease in children and young people in both primary and secondary care.

Effect of 6% hydroxyethyl starch 130/0.4 on kidney and haemostatic function in cardiac surgical patients: a randomised controlled trial.

Whether third-generation hydroxyethyl starch solutions provoke kidney injury or haemostatic abnormalities in patients having cardiac surgery remains unclear. We tested the hypotheses that intra-operative administration of a third-generation starch does not worsen postoperative kidney function or haemostasis in cardiac surgical patients compared with human albumin 5%. This triple-blind, non-inferiority, clinical trial randomly allocated patients aged 40-85 who underwent elective aortic valve replacement, with or without coronary artery bypass grafting, to plasma volume replacement with 6% starch 130/0.4 vs. 5% human albumin. Our primary outcome was postoperative urinary neutrophil gelatinase-associated lipocalin concentrations, a sensitive and early marker of postoperative kidney injury. Secondarily, we evaluated urinary interleukin-18; acute kidney injury using creatinine RIFLE criteria, coagulation measures, platelet count and function. Non-inferiority (delta 15%) was assessed with correction for multiple comparisons. We enrolled 141 patients (69 starch, 72 albumin) as planned. Results of the primary analysis demonstrated that postoperative urine neutrophil gelatinase-associated lipocalin (median (IQR [range])) was slightly lower with hydroxyethyl starch (5 (1-68 [0-996]) ng.ml-1 ) vs. albumin (5 (2-74 [0-1604]) ng.ml-1 ), although not non-inferior [ratio of geometric means (95%CI) 0.91 (0.57, 1.44); p = 0.15] due to higher than expected variability. Urine interleukin-18 concentrations were reduced, but interleukin-18 and kidney injury were again not non-inferior. Of 11 individual coagulation measures, platelet count and function, nine were non-inferior to albumin. Two remaining measures, thromboelastographic R value and arachidonic acid-induced platelet aggregation, were clinically similar but with wide confidence intervals. Starch administration during cardiac surgery produced similar observed effects on postoperative kidney function, coagulation, platelet count and platelet function compared with albumin, though greater than expected variability and wide confidence intervals precluded the conclusion of non-inferiority. Long-term mortality and kidney function appeared similar between starch and albumin.

A systematic review of the quality and scope of economic evaluations in child oral health research.

BACKGROUND: Economic evaluations provide policy makers with information to facilitate efficient resource allocation. To date, the quality and scope of economic evaluations in the field of child oral health has not been evaluated. Furthermore, whilst the involvement of children in research has been actively encouraged in recent years, the success of this movement in dental health economics has not yet been explored. This review aimed to determine the quality and scope of published economic evaluations applied to children's oral health and to consider the extent of children's involvement. METHODS: The following databases were searched: CINAHL, Cochrane Library, Econlit, EThOS, MEDLINE, NHS EED, OpenGrey, Scopus, Web of Science. Full economic evaluations, relating to any aspect of child oral health, published after 1997 were included and appraised against the Drummond checklist and the Consolidated Health Economic Evaluation Reporting Standards by a team of four calibrated reviewers. Data were also extracted regarding children's involvement and the outcome measures used. RESULTS: Two thousand seven hundred fifteen studies were identified, of which 46 met the inclusion criteria. The majority (n = 38, 82%) were cost-effectiveness studies, with most focusing on the prevention or management of dental caries (n = 42, 91%). One study quantified outcomes in Quality Adjusted Life Years (QALYs), and one study utilised a child-reported outcome measure. The mean percentage of applicable Drummond checklist criteria met by the studies in this review was 48% (median = 50%, range = 0-100%) with key methodological weaknesses noted in relation to discounting of costs and outcomes. The mean percentage of applicable CHEERS criteria met by each study was 77% (median = 83%, range = 33-100%), with limited reporting of conflicts of interest. Children's engagement was largely overlooked. CONCLUSIONS: There is a paucity of high-quality economic evaluations in the field of child oral health. This deficiency could be addressed through the endorsement of standardised economic evaluation guidelines by dental journals. The development of a child-centred utility measure for use in paediatric oral health would enable researchers to quantify outcomes in terms of quality adjusted life years (QALYs) whilst promoting child-centred research.

Caries prevalence, clinical consequences and self-reported pain experienced by children living in the West Bank.

AIM: To investigate both caries prevalence and clinical consequences experienced by deprived children in the West Bank, using a child-centred approach. MATERIALS AND METHODS: Children were invited by their social workers to attend free dental screening sessions held across clinics in the north of the West Bank. Data were collected using the dmft/DMFT and pufa/PUFA indices. Dental pain was reported by children using the Wong-Baker FACES® pain scale. Data were analysed using SPSS Version 22.0. RESULTS: Data were collected for 177 children aged 4 to 18 years. Caries prevalence was 95.5% with only eight children presenting clinically caries-free. The sample had a dmft of 3.88, and DMFT of 3.44. The Care Index was calculated at 0.1 (mft/dmft). Clinical consequences of caries were identified in 64% of the sample, with a mean pufa score of 2.12, and a PUFA score of 0.55. Dental pain was experienced by 45% of children. CONCLUSION: Deprived children living in the West Bank experience high levels of untreated dental caries, with significant clinical consequences and self-reported pain.

Developing and Applying the Propensity Score to Make Causal Inferences: Variable Selection and Stratification.

This Monte Carlo simulation examined the effects of variable selection (combinations of confounders with four patterns of relationships to outcome and assignment to treatment) and number of strata (5, 10, or 20) in propensity score analyses. The focus was on how the variations affected the average effect size compared to quasi-assignment without adjustment for bias. Results indicate that if a propensity score model does not include variables strongly related to both outcome and assignment, not only will bias not decrease, but it may possibly increase. Furthermore, models that include a variable highly related to assignment to treatment but do not also include a variable highly related to the outcome could increase bias. In regards to the number of strata, results varied depending on the propensity score model and sample size. In 75% of the models that resulted in a significant reduction in bias, quintiles outperformed the other stratification schemes. In fact, the richer that the propensity score model was (i.e., including multiple covariates of varying relationships to the outcome and to assignment to treatment), the more likely that the model required fewer strata to balance the covariates. In models without that same richness, additional strata were necessary. Finally, the study suggests that when developing a rich propensity score model with stratification, it is crucial to examine the strata for overlap.

Clinicopathologic and molecular characterization of myeloid neoplasms with isolated t(6;9)(p23;q34).

INTRODUCTION: The t(6;9)(p23;q34);DEK-NUP214 [t(6;9)] abnormality is found in 0.7-1.8% of patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). FLT3-ITD mutations are detected in t(6;9) patients. The t(6;9) abnormality is associated with poor outcomes. We studied the clinicopathologic and molecular profiles of patients with AML/MDS carrying t(6;9). METHODS: We collected clinical data of nine patients with AML/MDS with isolated t(6;9) (median age = 41 years; male/female = 4/5) and genotyped DNAs using whole exome, Sanger, and targeted sequencing. RESULTS: Our cohort was characterized by frequent multilineage dysplasia (56%), absence of phospho-STAT3/STAT5 expression, presence of myeloid markers (CD13, CD33, CD34, CD117, HLA-DR) with an aberrant expression of CD7, and poor outcome (median survival of 20 months). Although basophilia has been described in association with t(6;9), we observed lack of marrow basophilia in our cohort. Molecularly, 83% (5/6) of patients with AML/MDS with t(6;9) were characterized by at least one somatic mutation. Among them, four patients showed multiple mutations. FLT3-ITD mutations were detected in 33% of patients (2/6); 80% (4/5) of mutant patients died even after hematopoietic stem cell transplantation. CONCLUSION: Our data demonstrated that AML/MDS patients with t(6;9) have diverse molecular mutations regardless of the presence of FLT3 mutations, which may contribute to their poor survival outcomes.

Validation of a panel of ADAMTS13 assays for diagnosis of thrombotic thrombocytopenic purpura: activity, functional inhibitor, and autoantibody test.

INTRODUCTION: Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. METHODS: Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone). RESULTS: A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%. CONCLUSION: ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study.

Unusual extrinsic staining following microabrasion in a girl with amelogenesis imperfecta.

BACKGROUND: Developmental defects of enamel (DDE), such as amelogenesis imperfecta (AI), may present with tooth discolouration that is of aesthetic concern to the affected individual. Children and young people with DDE may therefore seek dental interventions to improve their dental appearance. The most commonly employed approaches include microabrasion, bleaching and/or placement of composite resin veneers. CASE REPORT: A 13-year-old girl with hypomature AI requested treatment for the 'marks' on her teeth which were having a negative impact on her social interactions. Clinical examination revealed generalised dense white opacities, and a microabrasion approach was performed on 11, 12 and 13 using a commercial preparation of 6.6 % hydrochloric acid. Concerningly, the girl's father phoned the next day reporting that his daughter's teeth had turned 'orange'. An urgent review revealed that the treated teeth had indeed become an orange colour. Further enquiry found that the patient had eaten a tomato pizza immediately after her dental treatment and this was believed to have caused the severe extrinsic staining. The patient was provided with a 16 % carbamide peroxide preparation for night-time use in a laboratory-made tray. A 2-week review revealed complete resolution of the staining. FOLLOW-UP: Direct composite resin restorations were subsequently provided for the girl's maxillary anterior teeth to achieve an optimal cosmetic result and she has remained pleased with her dental appearance. CONCLUSION: Clinicians should be aware of the potential for extrinsic staining following microabrasion or tooth bleaching. Patients should be advised against consuming coloured food and drink for at least 48 h after their treatment.

Parents' Experiences of Toothbrushing with Children: A Qualitative Study.

Globally, dental caries is one of the most prevalent diseases and is more common in children living in deprived areas. Dental caries is preventable, and guidance in the United Kingdom recommends parental supervised brushing (PSB): a collection of behaviors-including twice-daily toothbrushing with fluoridated toothpaste-that should begin upon eruption of the first tooth (approximately 6 to 12 mo of age) and for which children need to be helped or supervised by an adult until at least 7 y of age. The aim of this study was to explore parents' experiences of toothbrushing with their young children and to establish barriers and facilitators to PSB at individual, interpersonal, and environmental levels according to the theoretical domains framework. Qualitative semistructured interviews guided by the framework were conducted with 27 parents of young children (<7 y) in 2 deprived areas of the United Kingdom. Framework analysis was used. Parents were not aware of national guidance concerning their active involvement in toothbrushing; however, they did have detailed knowledge of toothbrushing practices for children, and their intentions were to brush their children's teeth themselves twice every day as part of a family routine. Nonetheless, parents' difficulties experienced in managing their children's challenging behavior and the environmental context of their stressful lives meant that many parents adopted a role of simply reminding their children to brush or watching them brush. As such, the main barriers to PSB among parents living in deprived areas were skills in managing their children's behavior and environmental influences on family life. The results of our study have clear implications for the development of appropriate interventions to address the modifiable barriers to improve parental adoption of PSB. Knowledge Transfer Statement: The results of this study will be used to develop a behavior change intervention to encourage parental supervised brushing. The intervention-which is likely to be delivered through health practitioners rather than dental teams-will be developed to reduce dental caries among young children and will require evaluation in terms of its clinical and cost effectiveness.

Distinct iron architecture in SF3B1-mutant myelodysplastic syndrome patients is linked to an SLC25A37 splice variant with a retained intron.

Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/flow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe(2+) in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a specific isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe(2+) into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

Ruxolitinib leads to improvement of pulmonary hypertension in patients with myelofibrosis.

Pulmonary hypertension (PH) is a frequently under recognized complication of myelofibrosis (MF). The pathophysiology of PH in MF is unknown and no definitive therapies have been established. We studied 15 patients with MF-associated PH and compared their echocardiographic and PH relevant biomarkers (nitric oxide (NO), N-terminal pro-hormone of brain natriuretic peptide (NT-pro BNP), von Willebrand antigen (vWB), ristocetin-cofactor activity (RCA) and uric acid (UA)) pre- and post-ruxolitinib treatment. Ruxolitinib decreased the plasma levels of NT-pro BNP (73%; P=0.043), UA (60%), vWB (86%) and RCA (73%; P=0.036). Improvements in echocardiographic findings were also seen in 66% of patients (P=0.022). Furthermore, marked increase in NO compared with baseline (69.75 vs 40.1 picomolar (pM); P=0.001) was observed post-ruxolitinib therapy, whereas no changes were noted with conventional therapies. Treatment with ruxolitinib also resulted in the reduction of key cytokines (tumor necrosis factor alpha, interleukin-4 (IL-4), IL-6 and IL-8) and induction of interferon-gamma. Animal studies further supported the role of ruxolitinib in the induction of NO levels. In conclusion, aberrant Janus kinase (JAK)-signal transducer and activator of transcription signaling in MF may mediate PH through dysregulation of NO and cytokine levels, which can be restored by therapy with JAK inhibitors suggesting that inhibition of this pathway is a novel target for the management of patients with PH.

Cadmium stress alters gene expression of DNA mismatch repair related genes in Arabidopsis seedlings.

Cadmium (Cd) is a non essential element, and is a widespread environmental pollutant. Exposure to Cd can result in a variety of adverse health effects in plant and humans. In the current study, Arabidopsis seedlings were used as a bio-indicator of Cd pollution. Seedlings were grown on MS media containing 0-6.0 mg L(-1) Cd for 18 days, and the gene expression patterns were used to link increased Cd exposure with progressive biological effects. Reduction of total soluble protein content in shoots of the Arabidopsis seedlings occurred with increase in Cd concentrations. For the gene expression patterns, seven genes known to be involved in cell division and DNA mismatch repair (MMR) system were investigated by semi-quantitative RT-PCR, and normalized using 18S rRNA gene expression. Expression of the proliferating cell nuclear antigen 2 (atPCNA 2), MutS 3 homolog (atMSH 3) and MutL1 homolog (atMLH1) genes in shoots of Arabidopsis was strongly induced by exposure to 0.75 mg L(-1) Cd, but were repressed by other Cd concentrations whereas exposure to 0.75-6 mg L(-1) of Cd resulted in a decreased expression of atPCNA1, atMSH 2, 6 and 7 genes independently of any observable biological effects, including survival, fresh weight and chlorophyll level of shoots. This work demonstrated that specific gene expression changes could serve as useful molecular biomarkers indicative of Cd exposure and related biological effects.

Expression of fission yeast cdc25 driven by the wheat ADP-glucose pyrophosphorylase large subunit promoter reduces pollen viability and prevents transmission of the transgene in wheat.

Cell number was to be measured in wheat (Triticum aestivum) endosperm expressing Spcdc25 (a fission yeast cell-cycle regulator) controlled by a supposedly endosperm-specific promoter, AGP2 (from the large subunit of ADP glucose pyrophosphorylase). Wheat was transformed by biolistics either with AGP2::GUS or AGP2::Spcdc25. PCR and RT-PCR checked integration and expression of the transgene, respectively. In cv. Chinese Spring, AGP2::GUS was unexpectedly expressed in carpels and pollen, as well as endosperm. In cv. Cadenza, three AGP2::Spcdc25 plants, AGP2::Spcdc25.1, .2 and .3, were generated. Spcdc25 expression was detected in mature leaves of AGP2::Spcdc25.1/.3 which exhibited abnormal spikes, 50% pollen viability and low seed set per plant; both were small compared with the nonexpressing and normal AGP2::Spcdc25.2. Spcdc25 was not transmitted to the T(1) in AGP2::Spcdc25.1 or .3, which developed normally. Spcdc25 was PCR-positive in AGP2::Spcdc25.2, using primers for a central portion, but not with primers for the 5' end, of the ORF, indicating a rearrangement; Spcdc25 was not expressed in either T(0) or T(1). The AGP2 promoter is not tissue-specific and Spcdc25 expression disrupted reproduction.

The Arabidopsis CDC25 induces a short cell length when overexpressed in fission yeast: evidence for cell cycle function.

The putative mitotic inducer gene, Arath;CDC25 cloned in Arabidopsis thaliana, was screened for cell cycle function by overexpressing it in Schizosaccharomyces pombe (fission yeast). The expression pattern of Arath;CDC25 was also examined in different tissues of A. thaliana. Fission yeast was transformed with plasmids pREP1 and pREP81 with the Arath;CDC25 gene under the control of the thiamine-repressible nmt promoter. Using reverse transcription-polymerase chain reaction (RT-PCR), the expression of Arath;CDC25 was examined in seedlings, flower buds, mature leaves and stems of A. thaliana; actin (ACT2) was used as a control. In three independent transformants of fission yeast, cultured in the absence of thiamine (T), pREP1::Arath;CDC25 induced a highly significant reduction in mitotic cell length compared with wild type, pREP::Arath;CDC25 +T, and empty vector (pREP1 +/- T). The extent of cell shortening was greater using the stronger pREP1 compared with the weaker pREP81. However, Arath;CDC25 was expressed at low levels in all tissues examined. The data indicate that Arath;CDC25 can function as a mitotic accelerator in fission yeast. However, unlike other plant cell cycle genes, expression of Arath;CDC25 was not enhanced in rapidly dividing compared with non-proliferative Arabidopsis tissues.

Differential expression of putative floral genes in Pharbitis nil shoot apices cultured on glucose compared with sucrose.

If, following an inductive treatment of 2 d of continuous darkness, shoot apices of Pharbitis nil are cultured 1 d later on White's medium supplemented with 2% sucrose, they cannot form carpels, but they can if they are cultured on 2% glucose. It was hypothesized that the differential effect of these sugars was because of differential expression of carpel-specific genes. Partial cDNA homologues to the Arabidopsis genes, LEAFY (PnLFY), AGAMOUS (PnAG1/2), and CRABS CLAWS (PnCRC1/2) were cloned. PnLFY was expressed in the shoot apex 1 d following the start of induction and remained higher than in non-induced apices for a further 6 d before exhibiting a major peak of expression on day 7. Peaks of expression of PnAG1 and PnAG2 spanned days 7-11, coinciding with the appearance of stamens and then carpels. The Pharbitis 'PnCRC2' showed greatest homology to Arabidopsis YABBY2 (PnYABBY). Its expression peaked on day 8 when the carpels first appeared. 'PnCRC1' showed greatest homology to Arabidopsis FILAMENTOUS (PnFIL). Its expression was approximately the same in inductive and non-inductive treatments. Apart from PnFIL these partial cDNAs could be used as markers to test the hypothesis concerning differential effects of sucrose and glucose. Cultured shoot apices from induced plants were sampled at weekly intervals. All four genes were expressed more strongly in the glucose compared with the sucrose treatment, most notably at day 17. A more intensive sampling (days 15-19) indicated that PnLFY and PnYABBY exhibited much higher expression on glucose compared with sucrose, most notably on days 15-16 and days 18-19.

An evaluation of 18S rDNA approaches for the study of fungal diversity in grassland soils.

Fungal community structure and diversity in two types of agricultural grassland soil were investigated by amplified 18S ribosomal DNA restriction analysis (ARDRA) and 18S ribosomal DNA sequence analysis. These two grassland sites represent a species-rich old hay meadow and an agriculturally improved site with low floristic diversity. Two primer sets were used in combination to amplify approximately 550 bp of rDNA from three major fungal groups, the zygomycetes, basidiomycetes, and ascomycetes, and clone libraries were created for each site. 18S ARDRA was used to analyze 170 rDNA clones, and three diversity indices were calculated. A small-scale culturing analysis was also carried out and the most common isolates analyzed using ARDRA and sequence analysis. The soil fungal community revealed by the rDNA approaches was significantly different from that produced by this limited culture-based analysis. Twenty-eight soil-derived clones were sequenced, and many represented fungal taxa rarely reported in culture-based studies. The PCR-based techniques detected differences in diversity between the two fungal communities and changes in patterns of dominance that paralleled higher plant diversity. The results suggest that 18S rDNA-based approaches are a useful tool for initial screening of fungal communities, and that they represent a more comprehensive picture of the community than plate culturing.

Diversity, genetic structure and evidence of outcrossing in British populations of the rock fern Adiantum capillus-veneris using microsatellites.

Microsatellites were isolated and a marker system was developed in the fern Adiantum capillus-veneris. Polymorphic markers were then used to study the genetic diversity and structure of populations within the UK and Ireland where this species grows at the northern edge of its range, requiring a specific rock habitat and limited to a few scattered populations. Three dinucleotide loci detected a high level of diversity (23 alleles and 28 multilocus genotypes) across the UK and Ireland, with nearly all variation partitioned among rather than within populations. Of 17 populations represented by multiple samples, all except four were monomorphic. Heterozygosity was detected in three populations, all within Glamorgan, Wales (UK), showing evidence of outcrossing. We make inferences on the factors determining the observed levels and patterns of genetic variation and the possible evolutionary history of the populations.

Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line.

It was examined whether ethylene induces programmed cell death in a cell cycle-specific manner. Following synchronization of the tobacco TBY-2 cell line with aphidicolin and its subsequent removal, ethylene was injected into the head space of 300 cm(3) culture flasks at 0 h or 3.5 h later and cells were sampled for 26 h. There were significant increases in cell mortality at G(2)/M in both the 0 h and 3.5 h ethylene treatments, and for the latter treatment, another peak in S-phase. The effect at G(2)/M was greater in the 3.5 h treatment, but was ameliorated by the simultaneous addition of silver nitrate (1.2 microM). In addition, the 3.5 h ethylene treatment resulted in a 1 h delay in the characteristic rise in the mitotic index following aphidicolin-induced synchrony. The addition of silver nitrate alone (1.2 microM), also delayed the entry of cells into mitosis but had no effect on cell cycle length compared with the controls (14 h throughout all treatments) but it induced a peak of mortality 2.5 h after its addition. Nuclear shrinkage was also a characteristic feature of dying cells at G(2)/M. Using Apoptag, an in situ apoptosis detection kit, nuclear DNA fragmentation was observed in the TBY-2 cells which were often isolated on the end of a filament of normal cells. In the 3.5 h ethylene treatment, a marked increase was noted in the percentage of such cells at the G(2)/M transition compared with the controls. Hence, the data show cell death occurring at a major phase transition of the cell cycle and the observations of nuclear shrinkage, isolation of dying cells and nuclear DNA fragmentation suggest a programmed mechanism of cell death exacerbated by ethylene treatment.