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.

Improved accuracy of co-morbidity coding over time after the introduction of ICD-10 administrative data.

BACKGROUND: Co-morbidity information derived from administrative data needs to be validated to allow its regular use. We assessed evolution in the accuracy of coding for Charlson and Elixhauser co-morbidities at three time points over a 5-year period, following the introduction of the International Classification of Diseases, 10th Revision (ICD-10), coding of hospital discharges.METHODS: Cross-sectional time trend evaluation study of coding accuracy using hospital chart data of 3'499 randomly selected patients who were discharged in 1999, 2001 and 2003, from two teaching and one non-teaching hospital in Switzerland. We measured sensitivity, positive predictive and Kappa values for agreement between administrative data coded with ICD-10 and chart data as the 'reference standard' for recording 36 co-morbidities.RESULTS: For the 17 the Charlson co-morbidities, the sensitivity - median (min-max) - was 36.5% (17.4-64.1) in 1999, 42.5% (22.2-64.6) in 2001 and 42.8% (8.4-75.6) in 2003. For the 29 Elixhauser co-morbidities, the sensitivity was 34.2% (1.9-64.1) in 1999, 38.6% (10.5-66.5) in 2001 and 41.6% (5.1-76.5) in 2003. Between 1999 and 2003, sensitivity estimates increased for 30 co-morbidities and decreased for 6 co-morbidities. The increase in sensitivities was statistically significant for six conditions and the decrease significant for one. Kappa values were increased for 29 co-morbidities and decreased for seven.CONCLUSIONS: Accuracy of administrative data in recording clinical conditions improved slightly between 1999 and 2003. These findings are of relevance to all jurisdictions introducing new coding systems, because they demonstrate a phenomenon of improved administrative data accuracy that may relate to a coding 'learning curve' with the new coding system.

Genome-wide association study of co-occurring anxiety in major depression.

OBJECTIVES: Co-morbidity between depression and anxiety disorders is common. In this study we define a quantitative measure of anxiety by summating four anxiety items from the SCAN interview in a large collection of major depression (MDD) cases to identify genes contributing to this complex phenotype. METHODS: A total of 1522 MDD cases dichotomised according to those with at least one anxiety item scored (n = 1080) and those without anxiety (n = 442) were analysed, and also compared to 1588 healthy controls at a genome-wide level, to identify genes that may contribute to anxiety in MDD. RESULTS: For the quantitative trait, suggestive evidence of association was detected for two SNPs, and for the dichotomous anxiety present/absent ratings for three SNPs at genome-wide level. In the genome-wide analysis of MDD cases with co-morbid anxiety and healthy controls, two SNPs attained P values of < 5 × 10⁻⁶. Analysing candidate genes, P values ≤ 0.0005 were found with three SNPs for the quantitative trait and three SNPs for the dichotomous trait. CONCLUSIONS: This study provides an initial genome-wide assessment of possible genetic contribution to anxiety in MDD. Although suggestive evidence of association was found for several SNPs, our findings suggest that there are no common variants strongly associated with anxious depression.

Co-trimoxazole prophylaxis is associated with reduced risk of incident tuberculosis in participants in the Swiss HIV Cohort Study.

Co-trimoxazole reduces mortality in HIV-infected adults with tuberculosis (TB), and in vitro data suggest potential antimycobacterial activity of co-trimoxazole. We aimed to evaluate whether prophylaxis with co-trimoxazole is associated with a decreased risk of incident TB in Swiss HIV Cohort Study (SHCS) participants. We determined the incidence of TB per 1,000 person-years from January 1992 to December 2012. Rates were analyzed separately in participants with current or no previous antiretroviral treatment (ART) using Poisson regression adjusted for CD4 cell count, sex, region of origin, injection drug use, and age. A total of 13,431 cohort participants contributed 107,549 person-years of follow-up: 182 patients had incident TB-132 (73%) before and 50 (27%) after ART initiation. The multivariable incidence rate ratios for cumulative co-trimoxazole exposure per year for persons with no previous ART and current ART were 0.70 (95% confidence interval [CI], 0.55 to 0.89) and 0.87 (95% CI, 0.74 to 1.0), respectively. Co-trimoxazole may prevent the development of TB among HIV-positive persons, especially among those with no previous ART.

The bZIP transcription factor Rca1p is a central regulator of a novel CO₂ sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

A New Approach for the Carbon Monoxide (CO) Exposure Diagnosis: Measurement of Total CO in Human Blood Versus Carboxyhemoglobin (HbCO).

The aim of the study is to present the application of a headspace-gas chromatography-mass spectrometry (HS-GC-MS) method for the determination of the carbon monoxide (CO) blood concentration and to compare it with carboxyhemoglobin (HbCO) saturation. In postmortem cases, the HbCO measured by spectrophotometry frequently leads to inaccurate results due to inadequate samples or analyses. The true role of CO intoxication in the death of a person could be misclassified. The estimation of HbCO from HS-GC-MS CO measurements provides helpful information by determining the total CO levels (CO linked to hemoglobin (HbCO) and CO dissociated from hemoglobin). The CO concentrations were converted in HbCO saturation levels to define cutoff blood CO values. CO limits were defined as less than 1 μmol/mL for living persons, less than 1.5 μmol/mL for dead persons without CO exposure, and greater than 3 μmol/mL for dead persons with clear CO poisoning.

Distribution and risk factors for Plasmodium and helminth co-infections: a cross-sectional survey among children in Bagamoyo district, coastal region of Tanzania.

BACKGROUND: Plasmodium and soil transmitted helminth infections (STH) are a major public health problem, particularly among children. There are conflicting findings on potential association between these two parasites. This study investigated the Plasmodium and helminth co-infections among children aged 2 months to 9 years living in Bagamoyo district, coastal region of Tanzania. METHODS: A community-based cross-sectional survey was conducted among 1033 children. Stool, urine and blood samples were examined using a broad set of quality controlled diagnostic methods for common STH (Ascaris lumbricoides, hookworm, Strongyloides stercoralis, Enterobius vermicularis, Trichuris trichura), schistosoma species and Wuchereria bancrofti. Blood slides and malaria rapid diagnostic tests (mRDTs) were utilized for Plasmodium diagnosis. RESULTS: Out of 992 children analyzed, the prevalence of Plasmodium infection was 13% (130/992), helminth 28.5% (283/992); 5% (50/992) had co-infection with Plasmodium and helminth. The prevalence rate of Plasmodium, specific STH and co-infections increased significantly with age (p < 0.001), with older children mostly affected except for S. stercoralis monoinfection and co-infections. Spatial variations of co-infection prevalence were observed between and within villages. There was a trend for STH infections to be associated with Plasmodium infection [OR adjusted for age group 1.4, 95% CI (1.0-2.1)], which was more marked for S. stercoralis (OR = 2.2, 95% CI (1.1-4.3). Age and not schooling were risk factors for Plasmodium and STH co-infection. CONCLUSION: The findings suggest that STH and Plasmodium infections tend to occur in the same children, with increasing prevalence of co-infection with age. This calls for an integrated approach such as using mass chemotherapy with dual effect (e.g., ivermectin) coupled with improved housing, sanitation and hygiene for the control of both parasitic infections.

Simulated Patient as Co-facilitators: Benefits and Challenges of the Interprofessional Team OSCE (Submission #9350).

Hypothesis: The quality of care for chronic patients depends on the collaborative skills of the healthcare providers.1,2 The literature lacks reports of the use of simulation to teach collaborative skills in non-acute care settings. We posit that simulation offers benefits for supporting the development of collaborative practice in non-acute settings. We explored the benefits and challenges of using an Interprofessional Team - Objective Structured Clinical Examination (IT-OSCE) as a formative assessment tool. IT-OSCE is an intervention which involves an interprofessional team of trainees interacting with a simulated patient (SP) enabling them to practice collaborative skills in non-acute care settings.5 A simulated patient are people trained to portray patients in a simulated scenario for educational purposes.6,7 Since interprofessional education (IPE) ultimately aims to provide collaborative patient-centered care.8,9 We sought to promote patient-centeredness in the learning process. Methods: The IT-OSCE was conducted with four trios of students from different professions. The debriefing was co-facilitated by the SP with a faculty. The participants were final-year students in nursing, physiotherapy and medicine. Our research question focused on the introduction of co-facilitated (SP and faculty) debriefing after an IT-OSCE: 1) What are the benefits and challenges of involving the SP during the debriefing? and 2) To evaluate the IT-OSCE, an exploratory case study was used to provide fine grained data 10, 11. Three focus groups were conducted - two with students (n=6; n=5), one with SPs (n=3) and one with faculty (n=4). Audiotapes were transcribed for thematic analysis performed by three researchers, who found a consensus on the final set of themes. Results: The thematic analysis showed little differentiation between SPs, student and faculty perspectives. The analysis of transcripts revealed more particularly, that the SP's co-facilitation during the debriefing of an IT-OSCE proved to be feasible. It was appreciated by all the participants and appeared to value and to promote patient-centeredness in the learning process. The main challenge consisted in SPs feedback, more particularly in how they could report accurate observations to a students' group rather than individual students. Conclusion: In conclusion, SP methodology using an IT-OSCE seems to be a useful and promising way to train collaborative skills, aligning IPE, simulation-based team training in a non-acute care setting and patient-centeredness. We acknowledge the limitations of the study, especially the small sample and consider the exploration of SP-based IPE in non-acute care settings as strength. Future studies could consider the preparation of SPs and faculty as co-facilitators. References: 1. Borrill CS, Carletta J, Carter AJ, et al. The effectiveness of health care teams in the National Health Service. Aston centre for Health Service Organisational Research. 2001. 2. Reeves S, Lewin S, Espin S, Zwarenstein M. Interprofessional teamwork for health and social care. Oxford: Wiley-Blackwell; 2010. 3. Issenberg S, McGaghie WC, Petrusa ER, Gordon DL, Scalese RJ. Features and uses of high-fidelity medical simulations that lead to effective learning - a BEME systematic review. Medical Teacher. 2005;27(1):10-28. 4. McGaghie W, Petrusa ER, Gordon DL, Scalese RJ. A critical review of simulation-based medical education research: 2003-2009. Medical Education. 2010;44(1):50-63. 5. Simmons B, Egan-Lee E, Wagner SJ, Esdaile M, Baker L, Reeves S. Assessment of interprofessional learning: the design of an interprofessional objective structured clinical examination (iOSCE) approach. Journal of Interprofessional Care. 2011;25(1):73-74. 6. Nestel D, Layat Burn C, Pritchard SA, Glastonbury R, Tabak D. The use of simulated patients in medical education: Guide Supplement 42.1 - Viewpoint. Medical teacher. 2011;33(12):1027-1029. Disclosures: None (C) 2014 by Lippincott Williams & Wilkins, Inc.

Discrimination of cortical laminae using MEG.

Typically MEG source reconstruction is used to estimate the distribution of current flow on a single anatomically derived cortical surface model. In this study we use two such models representing superficial and deep cortical laminae. We establish how well we can discriminate between these two different cortical layer models based on the same MEG data in the presence of different levels of co-registration noise, Signal-to-Noise Ratio (SNR) and cortical patch size. We demonstrate that it is possible to make a distinction between superficial and deep cortical laminae for levels of co-registration noise of less than 2mm translation and 2° rotation at SNR>11dB. We also show that an incorrect estimate of cortical patch size will tend to bias layer estimates. We then use a 3D printed head-cast (Troebinger et al., 2014) to achieve comparable levels of co-registration noise, in an auditory evoked response paradigm, and show that it is possible to discriminate between these cortical layer models in real data.

Which subgroup of patients with rheumatoid arthritis benefits from switching to rituximab versus alternative anti-tumour necrosis factor (TNF) agents after previous failure of an anti-TNF agent?

BACKGROUND: Patients with rheumatoid arthritis (RA) with an inadequate response to TNF antagonists (aTNFs) may switch to an alternative aTNF or start treatment from a different class of drugs, such as rituximab (RTX). It remains unclear in which clinical settings these therapeutic strategies offer most benefit. OBJECTIVE: To analyse the effectiveness of RTX versus alternative aTNFs on RA disease activity in different subgroups of patients. METHODS: A prospective cohort study of patients with RA who discontinued at least one aTNF and subsequently received either RTX or an alternative aTNF, nested within the Swiss RA registry (SCQM-RA) was carried out. The primary outcome, longitudinal improvement in 28-joint count Disease Activity Score (DAS28), was analysed using multivariate regression models for longitudinal data and adjusted for potential confounders. RESULTS: Of the 318 patients with RA included; 155 received RTX and 163 received an alternative aTNF. The relative benefit of RTX varied with the type of prior aTNF failure: when the motive for switching was ineffectiveness to previous aTNFs, the longitudinal improvement in DAS28 was significantly better with RTX than with an alternative aTNF (p = 0.03; at 6 months, -1.34 (95% CI -1.54 to -1.15) vs -0.93 (95% CI -1.28 to -0.59), respectively). When the motive for switching was other causes, the longitudinal improvement in DAS28 was similar for RTX and alternative aTNFs (p = 0.40). These results were not significantly modified by the number of previous aTNF failures, the type of aTNF switches, or the presence of co-treatment with a disease-modifying antirheumatic drug. CONCLUSION: This observational study suggests that in patients with RA who have stopped a previous aTNF treatment because of ineffectiveness changing to RTX is more effective than switching to an alternative aTNF.

Protective effects of hypercapnic acidosis on ventilator-induced lung injury.

To investigate whether respiratory acidosis modulates ventilator-induced lung injury (VILI), we perfused (constant flow) 21 isolated sets of normal rabbit lungs, ventilated them for 20 min (pressure controlled ventilation [PCV] = 15 cm H(2)O) (Baseline) with an inspired CO(2) fraction adjusted for the partial pressure of CO(2) in the perfusate (PCO(2) approximately equal to 40 mm Hg), and then randomized them into three groups. Group A (control: n = 7) was ventilated with PCV = 15 cm H(2)O for three consecutive 20-min periods (T1, T2, T3). In Group B (high PCV/normocapnia; n = 7), PCV was given at 20 (T1), 25 (T2), and 30 (T3) cm H(2)O. The targeted PCO(2) was 40 mm Hg in Groups A and B. Group C (high PCV/hypercapnia; n = 7) was ventilated in the same way as Group B, but the targeted PCO(2) was approximately equal to 70 to 100 mm Hg. The changes (from Baseline to T3) in weight gain (Delta WG: g) and in the ultrafiltration coefficient (Delta K(f) = gr/min/ cm H(2)O/100g) and the protein and hemoglobin concentrations in bronchoalveolar lavage fluid (BALF) were used to assess injury. Group B experienced a significantly greater Delta WG (14.85 +/- 5.49 [mean +/- SEM] g) and Delta K(f) (1.40 +/- 0.49 g/min/cm H(2)O/100 g) than did either Group A (Delta WG = 0.70 +/- 0.43; Delta K(f) = 0.01 +/- 0.03) or Group C (Delta WG = 5.27 +/- 2.03 g; Delta K(f) = 0.25 +/- 0.12 g/min/cm H(2)O/ 100 g). BALF protein and hemoglobin concentrations (g/L) were higher in Group B (11.98 +/- 3.78 g/L and 1.82 +/- 0.40 g/L, respectively) than in Group A (2.92 +/- 0.75 g/L and 0.38 +/- 0.15 g/L) or Group C (5.71 +/- 1.88 g/L and 1.19 +/- 0.32 g/L). We conclude that respiratory acidosis decreases the severity of VILI in this model.

Factors determining the spontaneous activation of splenic dendritic cells in culture.

Dendritic cells (DCs) serve as a link between the innate and adaptive immune systems. The activation state of DCs is crucial in this role. However, when DCs are isolated from lymphoid tissues, purified and placed in culture they undergo 'spontaneous' activation. The basis of this was explored, using up-regulation of DC surface MHC II, CD40, CD80 and CD86 as indicators of DC activation. No evidence was found for DC damage during isolation or for microbial products causing the activation. The culture activation of spleen DCs differed from that of Langerhans cells when released from E-cadherin-mediated adhesions, since E-cadherin was not detected and activation still occurred with β-catenin null DCs. Much of the activation could be attributed to DC-DC interactions. Although increases in surface MHC II levels occurred under all culture conditions tested, the increase in expression of CD40, CD80 and CD86 was much less under culture conditions where such interactions were minimised. DC-to-DC contact under the artificial conditions of high DC concentration in culture induced the production of soluble factors and these, in turn, induced the up-regulation of co-stimulatory molecules on the DC surface.

The policy-making structure of European regulatory networks and the domestic adoption of standards

European regulatory networks (ERNs) constitute the main governance instrument for the informal co-ordination of public regulation at the European Union (EU) level. They are in charge of co-ordinating national regulators and ensuring the implementation of harmonized regulatory policies across the EU, while also offering sector-specific expertise to the Commission. To this aim, ERNs develop 'best practices' and benchmarking procedures in the form of standards, norms and guidelines to be adopted in member states. In this paper, we focus on the Committee of European Securities Regulators and examine the consequences of the policy-making structure of ERNs on the domestic adoption of standards. We find that the regulators of countries with larger financial industries tend to occupy more central positions in the network, especially among newer member states. In turn, network centrality is associated with a more prompt domestic adoption of standards.

Proposal of a new classification for optimising outcome assessment following partial cricotracheal resections in severe pediatric subglottic stenosis.

OBJECTIVE: Creation of a patent subglottic airway after partial cricotracheal resection (PCTR) may not always result in successful decannulation due to associated parameters such as co-morbidity and/or glottic involvement. We classified patients after incorporating these additional parameters into the original Myer-Cotton classification to assess whether this could better predict the outcome measures after PCTR. METHODS: One hundred children with Myer-Cotton grade III or IV subglottic stenosis who underwent PCTR between 1978 and 2008 were identified from a prospectively collected database. The patients were classified into four groups based on the association of co-morbidity and/or glottic involvement. Delay in decannulation, revision open surgery and rates of decannulation were the outcome measures compared between the groups. RESULTS: There were 68 children with Myer-Cotton grade III and 32 children with grade IV stenosis. Based on the new classification, there were 36 children with isolated SGS, 31 with associated co-morbidity, 19 with associated glottic involvement and 14 children with both co-morbidity and glottic involvement. A trend towards less optimal results was noticed with the association of co-morbidity and/or glottic involvement. Statistical significance was reached for maximum decannulation failure in the group with both co-morbidity and glottic involvement. Delayed decannulation significantly correlated in the group with associated glottic involvement. CONCLUSION: This new classification is relatively simple and aimed at providing more accurate and uniform prognostic information to both patients and surgeons when dealing with the whole spectrum of severe SGS.

Characterization of tumor reactive CD8 T cells induced by peptide vaccination in melanoma patients

ABSTRACT¦Naturally acquired tumor-specific T-cells can be detected in most advanced cancer patients.¦Yet, they often fail to control or eliminate the disease, in contrast to many virus-specific CD8¦T lymphocytes. Therapeutic vaccines aim at inducing and boosting specific T-cells mediated¦immunity to reduce tumor burden. The properties of CD8 T-cells required for protection from¦infectious disease and cancer are only partially characterized.¦The objectives of this study were to assess effector functions, stage of differentiation and¦clonotype selection of tumor-reactive T lymphocytes following peptide vaccination in¦melanoma patients over time. Results were compared to protective viral-specific T-cell¦responses found in healthy individuals. We also characterized dominant versus low/non¦dominant T-cell clonotypes with the aim to further understand the in vivo function of each set¦of frequency-based specific T-cells.¦Here we developed and applied a novel approach for molecular and functional analysis of¦single T lymphocytes ex vivo. T-cell receptor (TCR) clonotype mapping revealed rapid¦selection and expansion of co-dominant T-cell clonotypes, which made up the majority of the¦highly differentiated "effector" T-cells, but only 25% of the less differentiated "effectormemory"¦cells, mostly composed of non-dominant clonotypes. Moreover, we show that¦advanced effector cell differentiation was indeed clonotype-dependent. Surprisingly, however,¦the acquisition of effector functions (cytokine production, killing) was clonotype-independent.¦Vaccination of melanoma patients with native peptide induced competent effector function in¦both dominant and non-dominant clonotypes, suggesting that most if not all clonotypes¦participating in a T-cell response have the potential to develop equal functional competence.¦In contrast, many T-cells remained poorly functional after vaccination with analog peptide,¦despite similar clonotype-dependent differentiation. Our findings show that the type of¦peptide vaccine has a critical influence on the selection and functional activation of the¦clonotypic T-cell repertoire. They also show that systematic assessment of individual T-cells¦identifies the cellular basis of immune responses, contributing to the rational development of¦vaccines.