Testing many treatments within a single protocol over 10 years at MRC Clinical Trials Unit at UCL: Multi-arm, multi-stage platform, umbrella and basket protocols

There is real need to change how we do some of our clinical trials, as currently the testing and development process is too slow, too costly and too failure-prone often we find that a new treatment is no better than the current standard. Much of the focus on the development and testing pathway has been in improving the design of phase I and II trials. In this article, we present examples of new methods for improving the design of phase III trials (and the necessary lead up to them) as they are the most time-consuming and expensive part of the pathway. Key to all these methods is the aim to test many treatments and/or pose many therapeutic questions within one protocol

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains

Tobacco smoking and somatic mutations in human bronchial epithelium

Tobacco smoking causes lung cancer, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA. The profound effects of tobacco on the genome of lung cancer cells are well-documented, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4–14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0–6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis

Transcriptome characterization and polymorphism detection between subspecies of big sagebrush (Artemisia tridentata)

<p>Abstract</p> <p>Background</p> <p>Big sagebrush (<it>Artemisia tridentata</it>) is one of the most widely distributed and ecologically important shrub species in western North America. This species serves as a critical habitat and food resource for many animals and invertebrates. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is a serious threat to big sagebrush ecosystem sustainability. Lack of genomic data has limited our understanding of the evolutionary history and ecological adaptation in this species. Here, we report on the sequencing of expressed sequence tags (ESTs) and detection of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in subspecies of big sagebrush.</p> <p>Results</p> <p>cDNA of <it>A. tridentata </it>sspp. <it>tridentata </it>and <it>vaseyana </it>were normalized and sequenced using the 454 GS FLX Titanium pyrosequencing technology. Assembly of the reads resulted in 20,357 contig consensus sequences in ssp. <it>tridentata </it>and 20,250 contigs in ssp. <it>vaseyana</it>. A BLASTx search against the non-redundant (NR) protein database using 29,541 consensus sequences obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e^-15). A total of 20,952 SNPs and 119 polymorphic SSRs were detected between the two subspecies. SNPs were validated through various methods including sequence capture. Validation of SNPs in different individuals uncovered a high level of nucleotide variation in EST sequences. EST sequences of a third, tetraploid subspecies (ssp. <it>wyomingensis</it>) obtained by Illumina sequencing were mapped to the consensus sequences of the combined 454 EST assembly. Approximately one-third of the SNPs between sspp. <it>tridentata </it>and <it>vaseyana </it>identified in the combined assembly were also polymorphic within the two geographically distant ssp. <it>wyomingensis </it>samples.</p> <p>Conclusion</p> <p>We have produced a large EST dataset for <it>Artemisia tridentata</it>, which contains a large sample of the big sagebrush leaf transcriptome. SNP mapping among the three subspecies suggest the origin of ssp. <it>wyomingensis </it>via mixed ancestry. A large number of SNP and SSR markers provide the foundation for future research to address questions in big sagebrush evolution, ecological genetics, and conservation using genomic approaches.</p

Single-nucleotide polymorphism discovery by high-throughput sequencing in sorghum

<p>Abstract</p> <p>Background</p> <p>Eight diverse sorghum (<it>Sorghum bicolor </it>L. Moench) accessions were subjected to short-read genome sequencing to characterize the distribution of single-nucleotide polymorphisms (SNPs). Two strategies were used for DNA library preparation. Missing SNP genotype data were imputed by local haplotype comparison. The effect of library type and genomic diversity on SNP discovery and imputation are evaluated.</p> <p>Results</p> <p>Alignment of eight genome equivalents (6 Gb) to the public reference genome revealed 283,000 SNPs at ≥82% confirmation probability. Sequencing from libraries constructed to limit sequencing to start at defined restriction sites led to genotyping 10-fold more SNPs in all 8 accessions, and correctly imputing 11% more missing data, than from semirandom libraries. The SNP yield advantage of the reduced-representation method was less than expected, since up to one fifth of reads started at noncanonical restriction sites and up to one third of restriction sites predicted <it>in silico </it>to yield unique alignments were not sampled at near-saturation. For imputation accuracy, the availability of a genomically similar accession in the germplasm panel was more important than panel size or sequencing coverage.</p> <p>Conclusions</p> <p>A sequence quantity of 3 million 50-base reads per accession using a <it>Bsr</it>FI library would conservatively provide satisfactory genotyping of 96,000 sorghum SNPs. For most reliable SNP-genotype imputation in shallowly sequenced genomes, germplasm panels should consist of pairs or groups of genomically similar entries. These results may help in designing strategies for economical genotyping-by-sequencing of large numbers of plant accessions.</p

Cross-Sectional Study into the Costs and Impact on Family Functioning of 4-Year-Old Children with Aggressive Behavior

Early-onset aggressive behavior is known for its negative developmental consequences, and the associated high costs for families, the health care system and wider society. Although the origins of aggressive behavior are to be found in early childhood, the costs incurred by aggressive behavior of young children have not been studied extensively. The present study aimed to investigate whether preschool children with a high level of aggressive behavior already differ in the generated amount of costs and impact on family functioning from children with lower levels of aggressive behavior. A population-based sample of 317 preschool children was divided into four groups with different levels of aggression (moderate, borderline, clinical). Parents filled out questionnaires to assess service use (lifetime and past 3 months) and impact on family functioning. Over the past 3 months as well as over the first 4 years of life, children with a clinical level of aggression were more costly than children with a low level of aggression (mean total costs over the past 3 months: low = €167,05 versus clinical € = 1034,83 and mean lifetime costs: low € = 817,37 versus clinical € = 1433,04), due to higher costs of services used by the child. In addition, families of children with a borderline or clinical level of aggressive behavior reported more impairment in their daily functioning than families of children with lower levels of aggression. The findings demonstrate that a high level of aggressive behavior results in high costs and impaired family functioning in the preschool years already

Effectiveness of preoperative staging in rectal cancer: digital rectal examination, endoluminal ultrasound or magnetic resonance imaging?

In rectal cancer, preoperative staging should identify early tumours suitable for treatment by surgery alone and locally advanced tumours that require therapy to induce tumour regression from the potential resection margin. Currently, local staging can be performed by digital rectal examination (DRE), endoluminal ultrasound (EUS) or magnetic resonance imaging (MRI). Each staging method was compared for clinical benefit and cost-effectiveness. The accuracy of high-resolution MRI, DRE and EUS in identifying favourable, unfavourable and locally advanced rectal carcinomas in 98 patients undergoing total mesorectal excision was compared prospectively against the resection specimen pathological as the gold standard. Agreement between each staging modality with pathology assessment of tumour favourability was calculated with the chance-corrected agreement given as the kappa statistic, based on marginal homogenised data. Differences in effectiveness of the staging modalities were compared with differences in costs of the staging modalities to generate cost effectiveness ratios. Agreement between staging and histologic assessment of tumour favourability was 94% for MRI (kappa=0.81, s.e.=0.05; kappa(W)=0.83), compared with very poor agreements of 65% for DRE (kappa=0.08, s.e.=0.068, kappa(W)=0.16) and 69% for EUS (kappa=0.17, s.e.=0.065, kappa(W)=0.17). The resource benefits resulting from the use of MRI rather than DRE was 67164 UK pounds and 92244 UK pounds when MRI was used rather than EUS. Magnetic resonance imaging dominated both DRE and EUS on cost and clinical effectiveness by selecting appropriate patients for neoadjuvant therapy and justifies its use for local staging of rectal cancer patients

Patient characteristics, comorbidities, and medication use for children with ADHD with and without a co-occurring reading disorder: A retrospective cohort study

<p>Abstract</p> <p>Background</p> <p>Children and adolescents with attention-deficit/hyperactivity disorder (ADHD) often have a co-occurring reading disorder (RD). The purpose of this research was to assess differences between children with ADHD without RD (ADHD-only) and those with ADHD and co-occurring RD (ADHD+RD).</p> <p>Methods</p> <p>Using data from the U.S. Thomson Reuter Marketscan^®Databases for the years 2005 through 2007, this analysis compared the medical records--including patient demographics, comorbidities, and medication use--of children (age < 18) with ADHD-only to those with ADHD+RD.</p> <p>Results</p> <p>Patients with ADHD+RD were significantly younger, more likely to have received a procedure code associated with formal psychological or non-psychological testing, and more likely to have been diagnosed with comorbid bipolar disorder, conduct disorder, or depression. They were no more likely to have received an antidepressant, anti-manic (bipolar), or antipsychotic, and were significantly less likely to have received a prescription for a stimulant medication.</p> <p>Conclusions</p> <p>Relying on a claims database, there appear to be differences in the patient characteristics, comorbidities, and medication use when comparing children with ADHD-only to those with ADHD+RD.</p