High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS Treatment: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS: TREATMENT: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION: First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

Perturbation expansions of multilocus fixation probabilities for frequency-dependent selection with applications to the Hill-Robertson effect and to the joint evolution of helping and punishment.

Natural populations are of finite size and organisms carry multilocus genotypes. There are, nevertheless, few results on multilocus models when both random genetic drift and natural selection affect the evolutionary dynamics. In this paper we describe a formalism to calculate systematic perturbation expansions of moments of allelic states around neutrality in populations of constant size. This allows us to evaluate multilocus fixation probabilities (long-term limits of the moments) under arbitrary strength of selection and gene action. We show that such fixation probabilities can be expressed in terms of selection coefficients weighted by mean first passages times of ancestral gene lineages within a single ancestor. These passage times extend the coalescence times that weight selection coefficients in one-locus perturbation formulas for fixation probabilities. We then apply these results to investigate the Hill-Robertson effect and the coevolution of helping and punishment. Finally, we discuss limitations and strengths of the perturbation approach. In particular, it provides accurate approximations for fixation probabilities for weak selection regimes only (Ns < or = 1), but it provides generally good prediction for the direction of selection under frequency-dependent selection.

Design and evaluation of a panel os single-nucleotide polymorphisms in microRNA genomic regions for association studies in human disease

MicroRNAs (miRNA) are recognized posttranscriptional gene repressors involved in the control of almost every biological process. Allelic variants in these regions may be an important source of phenotypic diversity and contribute to disease susceptibility. We analyzed the genomic organization of 325 human miRNAs (release 7.1, miRBase) to construct a panel of 768 single-nucleotide polymorphisms (SNPs) covering approximately 1 Mb of genomic DNA, including 131 isolated miRNAs (40%) and 194 miRNAs arranged in 48 miRNA clusters, as well as their 5-kb flanking regions. Of these miRNAs, 37% were inside known protein-coding genes, which were significantly associated with biological functions regarding neurological, psychological or nutritional disorders. SNP coverage analysis revealed a lower SNP density in miRNAs compared with the average of the genome, with only 24 SNPs located in the 325 miRNAs studied. Further genotyping of 340 unrelated Spanish individuals showed that more than half of the SNPs in miRNAs were either rare or monomorphic, in agreement with the reported selective constraint on human miRNAs. A comparison of the minor allele frequencies between Spanish and HapMap population samples confirmed the applicability of this SNP panel to the study of complex disorders among the Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 in the CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showing geographical allelic frequency variation among the four HapMap populations, probably because of differences in natural selection. The designed miRNA SNP panel could help to identify still hidden links between miRNAs and human disease.

Meta-analysis of gene-level associations for rare variants based on single-variant statistics.

Meta-analysis of genome-wide association studies (GWASs) has led to the discoveries of many common variants associated with complex human diseases. There is a growing recognition that identifying "causal" rare variants also requires large-scale meta-analysis. The fact that association tests with rare variants are performed at the gene level rather than at the variant level poses unprecedented challenges in the meta-analysis. First, different studies may adopt different gene-level tests, so the results are not compatible. Second, gene-level tests require multivariate statistics (i.e., components of the test statistic and their covariance matrix), which are difficult to obtain. To overcome these challenges, we propose to perform gene-level tests for rare variants by combining the results of single-variant analysis (i.e., p values of association tests and effect estimates) from participating studies. This simple strategy is possible because of an insight that multivariate statistics can be recovered from single-variant statistics, together with the correlation matrix of the single-variant test statistics, which can be estimated from one of the participating studies or from a publicly available database. We show both theoretically and numerically that the proposed meta-analysis approach provides accurate control of the type I error and is as powerful as joint analysis of individual participant data. This approach accommodates any disease phenotype and any study design and produces all commonly used gene-level tests. An application to the GWAS summary results of the Genetic Investigation of ANthropometric Traits (GIANT) consortium reveals rare and low-frequency variants associated with human height. The relevant software is freely available.

Development and Validation of Decision Rules to Guide Frequency of Monitoring CD4 Cell Count in HIV-1 Infection before Starting Antiretroviral Therapy.

Background: Although CD4 cell count monitoring is used to decide when to start antiretroviral therapy in patients with HIV-1 infection, there are no evidence-based recommendations regarding its optimal frequency. It is common practice to monitor every 3 to 6 months, often coupled with viral load monitoring. We developed rules to guide frequency of CD4 cell count monitoring in HIV infection before starting antiretroviral therapy, which we validated retrospectively in patients from the Swiss HIV Cohort Study.Methodology/Principal Findings: We built up two prediction rules ("Snap-shot rule" for a single sample and "Track-shot rule" for multiple determinations) based on a systematic review of published longitudinal analyses of CD4 cell count trajectories. We applied the rules in 2608 untreated patients to classify their 18 061 CD4 counts as either justifiable or superfluous, according to their prior >= 5% or < 5% chance of meeting predetermined thresholds for starting treatment. The percentage of measurements that both rules falsely deemed superfluous never exceeded 5%. Superfluous CD4 determinations represented 4%, 11%, and 39% of all actual determinations for treatment thresholds of 500, 350, and 200x10(6)/L, respectively. The Track-shot rule was only marginally superior to the Snap-shot rule. Both rules lose usefulness for CD4 counts coming near to treatment threshold.Conclusions/Significance: Frequent CD4 count monitoring of patients with CD4 counts well above the threshold for initiating therapy is unlikely to identify patients who require therapy. It appears sufficient to measure CD4 cell count 1 year after a count > 650 for a threshold of 200, > 900 for 350, or > 1150 for 500x10(6)/L, respectively. When CD4 counts fall below these limits, increased monitoring frequency becomes advisable. These rules offer guidance for efficient CD4 monitoring, particularly in resource-limited settings.

Intraspecific competition reveals conditional fitness effects of single gene polymorphism at the Arabidopsis root growth regulator BRX.

Intraspecific genetic variation for morphological traits is observed in many organisms. In Arabidopsis thaliana, alleles responsible for intraspecific morphological variation are increasingly being identified. However, the fitness consequences remain unclear in most cases. Here, the fitness effects of alleles of the BRX gene are investigated. A brx loss-of-function allele, which was found in a natural accession, results in a highly branched but poorly elongated root system. Comparison between the control accession Sav-0 and an introgression of brx into this background (brxS) indicated that, surprisingly, brx loss of function did not negatively affect fitness in pure stands. However, in mixed, well-watered stands brxS performance and reproductive output decreased significantly, as the proportion of Sav-0 neighbors increased. Additional comparisons between brxS and a brxS line that was complemented by a BRX transgene confirmed a direct effect of the loss-of-function allele on plant performance, as indicated by restored competitive ability of the transgenic genotype. Further, because plant height was very similar across genotypes and because the experimental setup largely excluded shading effects, the impaired competitiveness of the brx loss-of-function genotype likely reflects below-ground competition. In summary, these data reveal conditional fitness effects of a single gene polymorphism in response to intraspecific competition in Arabidopsis.

Frequency and Type of Side Effects of Immunomodulating Medication in the Swiss Inflammatory Bowel Disease Cohort

Background: Medical treatment of inflammatory bowel disease (IBD) is becoming more and more complex, as several classes of immuno-modulating drugs (IMD) are often used simultaneously. Thus, the probability of adverse effects is greatly increased. Most studies reporting on adverse effects focus on single therapy, and studies providing a global survey of side effects for multiple treatments are lacking. Aim: To assess the type and frequency of adverse events in IBD patients treated with single and multiple IMD therapy. Methods: Analysis of data from the Swiss IBD Cohort Study (SIBDCS) that collects data on a large sample of IBD patients from hospitals and private practices across Switzerland. The following IMD categories were analyzed: 5-ASA, azathioprine (Aza), 6-mercaptopurine (6-MP), methotrexate (MTX), anti-TNF (infliximab, adalimumab, certolizumab-pegol), cyclosporine, tacrolimus, and steroids. The following side effects were assessed: hepatitis, pancreatitis, leucopenia, thrombopenia, nephritis, allergic reaction, pneumonitis, infections (including tuberculosis), osteoporosis, abdominal pain/diarrhea (unrelated to IBD activity), cataract, diabetes, exanthema, hirsutism, lupus-like syndrome, myalgias, depression/psychosis, tumor development. Results: A total of 1,961 patients were analyzed (977 [50%] female, mean age 42.1 ± 14.4 years): 1,119 with Crohn's disease (CD), 800 with ulcerative colitis (UC), and 42 with indeterminate colitis (IC). Three-hundred eighteen (16.2%) patients were not treated with any of the above-mentioned medications, while 650 (33.2%), 569 (29%) and 424 (21.6%) patients had one-, two-, and three- or more- IMD therapy, respectively. Of the 1,643 patients treated with IMD, 535 (32.6%) patients reported at least one side effect. We found a significant correlation between the number of drugs used by a patient and the frequency of side effects (17.4% side effects for one drug, 29% for 2 drugs, and 60.6% for three or more drugs, p < 0.001). The frequency of side effects for the different IMD classes were as follows: 5-ASA (n = 980 treated patients) 10.8%, Aza/6-MP (n = 636) 51.9% (pancreatitis in 57 = 9%, hepatitis in 17 = 2.7% of treated patients), MTX (n = 146) 42.5% (hepatitis in 4 = 2.7% of treated patients), anti-TNF (n = 255) 23.1%, cyclosporine (n = 49) 10.2%, tacrolimus (n = 5) 20%, steroids (systemic or topical, n = 1,150) 9.6%. Conclusion: IBD treatment is associated with a significant number of side effects. A direct correlation between the number of IMD used simultaneously and the frequency of side effects was observed. The results of this study indicate that treating physicians should be vigilant for the occurrence of side effects in IBD patients under single and/or multiple drug therapy.

Development and Validation of Single Nucleotide Polymorphisms (SNPs) Markers from Two Transcriptome 454-Runs of Turbot (Scophthalmus maximus) Using High-Throughput Genotyping

The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genesrelated to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while Ш were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot

Positive effects of an angiotensin II type 1 receptor antagonist in Camurati-Engelmann disease: a single case observation.

Camurati-Engelmann disease is characterized by hyperostosis of the long bones and the skull, muscle atrophy, severe limb pain, and progressive joint contractures in some patients. It is caused by heterozygous mutations in the transforming growth factor β1 (TGFβ1) believed to result in improper folding of the latency-associated peptide domain of TGFβ1 and thus in increased or deregulated bioactivity. Losartan, an angiotensin II type 1 receptor antagonist, has been found to downregulate the expression of TGFβ type 1 and 2 receptors. Clinical trials with losartan have shown a benefit in Marfan syndrome, while trials are underway for Duchenne muscular dystrophy and other myopathies associated with TGFβ1 signaling. We hypothesized that due to its anti-TGFβ1 activity, losartan might be beneficial in Camurati-Engelmann disease. This report concerns a boy who presented at age 13 years with severe limb pain and difficulty in walking. Clinical and radiographic evaluation results were compatible with Camurati-Engelmann disease and the diagnosis was confirmed by mutation analysis (c.652C > T [p.Arg218Cys]). The boy underwent an experimental treatment with losartan at a dosage of 50 mg/day, orally. During the treatment period of 18 months, the intensity and frequency of limb pain decreased significantly (as shown by a pain diary), and muscle strength improved, allowing the boy to resume walking and climbing stairs. No obvious side effects were observed. We cautiously conclude that TGFβ1 inhibition with losartan deserves further evaluation in the clinical management of Camurati-Engelmann disease.

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

A quasi-randomized group trial of a brief alcohol intervention on risky single occasion drinking among secondary school students.

OBJECTIVES: To show the effectiveness of a brief group alcohol intervention. Aims of the intervention were to reduce the frequency of heavy drinking occasions, maximum number of drinks on an occasion and overall weekly consumption. METHODS: A cluster quasi-randomized control trial (intervention n = 338; control n = 330) among 16- to 18-year-old secondary school students in the Swiss Canton of Zürich. Groups homogeneous for heavy drinking occasions (5+/4+ drinks for men/women) consisted of those having medium risk (3-4) or high risk (5+) occasions in the past 30 days. Groups of 8-10 individuals received two 45-min sessions based on motivational interviewing techniques. RESULTS: Borderline significant beneficial effects (p < 0.10) on heavy drinking occasions and alcohol volume were found 6 months later for the medium-risk group only, but not for the high-risk group. None of the effects remained significant after Bonferroni corrections. CONCLUSIONS: Group intervention was ineffective for all at-risk users. The heaviest drinkers may need more intensive treatment. Alternative explanations were iatrogenic effects among the heaviest drinkers, assessment reactivity, or reduction of social desirability bias at follow-up through peer feedback.

Collective and single-particle excitations of a trapped Bose gas

The density of states of a Bose-condensed gas confined in a harmonic trap is investigated. The predictions of Bogoliubov theory are compared with those of Hartree-Fock theory and of the hydrodynamic model. We show that the Hartree-Fock scheme provides an excellent description of the excitation spectrum in a wide range of energy, revealing a major role played by single-particle excitations in these confined systems. The crossover from the hydrodynamic regime, holding at low energies, to the independent-particle regime is explicitly explored by studying the frequency of the surface mode as a function of their angular momentum. The applicability of the semiclassical approximation for the excited states is also discussed. We show that the semiclassical approach provides simple and accurate formulas for the density of states and the quantum depletion of the condensate.

Malaria haplotype frequency estimation.

We present a Bayesian approach for estimating the relative frequencies of multi-single nucleotide polymorphism (SNP) haplotypes in populations of the malaria parasite Plasmodium falciparum by using microarray SNP data from human blood samples. Each sample comes from a malaria patient and contains one or several parasite clones that may genetically differ. Samples containing multiple parasite clones with different genetic markers pose a special challenge. The situation is comparable with a polyploid organism. The data from each blood sample indicates whether the parasites in the blood carry a mutant or a wildtype allele at various selected genomic positions. If both mutant and wildtype alleles are detected at a given position in a multiply infected sample, the data indicates the presence of both alleles, but the ratio is unknown. Thus, the data only partially reveals which specific combinations of genetic markers (i.e. haplotypes across the examined SNPs) occur in distinct parasite clones. In addition, SNP data may contain errors at non-negligible rates. We use a multinomial mixture model with partially missing observations to represent this data and a Markov chain Monte Carlo method to estimate the haplotype frequencies in a population. Our approach addresses both challenges, multiple infections and data errors.