What are the components that contribute to variability in echocardiographic measurements in aortic stenosis?

INTRODUCTION Echocardiography is the standard clinical approach for quantification of the severity of aortic stenosis (AS). A comprehensive examination of its overall reproducibility and the simultaneous estimation of its variance components by multiple operators, readers, probe applications, and beats have not been undertaken. METHOD AND RESULTS Twenty-seven subjects with AS were scanned over 7 months in the echo-department by a median of 3 different operators. From each patient and each operator multiple runs of beats from multiple probe positions were stored for later analysis by multiple readers. The coefficient of variation was 13.3%, 15.9%, 17.6%, and 20.2% for the aortic peak velocity (Vmax), and velocity time integral (VTI), and left ventricular outflow tract (LVOT) Vmax and VTI respectively. The largest individual contributors to the overall variability were the beat-to-beat variability (9.0%, 9.3%, 9.5%, 9.4% respectively) and that of inability of an individual operator to precisely apply the probe to the same position twice (8.3%, 9.4%, 12.9%, 10.7% respectively). The tracing (inter-reader) and reader (inter-reader), and operator (inter-operator) contribution were less important. CONCLUSIONS Reproducibility of measurements in AS is poorer than often reported in the literature. The source of this variability does not appear, as traditionally believed, to result from a lack of training or operator and reader specific factors. Rather the unavoidable beat-to-beat biological variability, and the inherent impossibility of applying the ultrasound probe in exactly the same position each time are the largest contributors. Consequently, guidelines suggesting greater standardisation of procedures and further training for sonographers are unlikely to result in an improvement in precision. Clinicians themselves should be wary of relying on even three-beat averages as their expected coefficient of variance is 10.3% for the peak velocity at the aortic valve.

Molecular variability of Meningococcal antigens in carriage and disease strains and in other Neisseria species

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

Intra- and interspecific competition in western barbastelle bats (Barbastella barbastellus, SCHREBER 1774)

In the present thesis I examined individual and sex-specific habitat use and site fidelity in the western barbastelle bat, Barbastella barbastellus, using data from a four-year monitoring in a Special Area of Conservation in Rhineland-Palatinate, Germany. The western barbastelle occurs in central and southern Europe from Portugal to the Caucasus, but is considered to be rare in large parts of its range. Up to now, long-term field studies to assess interannual site fidelity and the possible effects of intra- and interspecific competition have not been studied in this species. Nevertheless, such data provide important details to estimate the specific spatial requirements of its populations, which in turn can be incorporated in extended conservation actions. I used radio-telemetry, home range analyses und automated ultrasound detection to assess the relation between landscape elements and western barbastelle bats and their roosts. In addition, I estimated the degree of interspecific niche overlap with two selected forest-dwelling bat species, Bechstein's bat (Myotis bechsteinii) and the brown long-eared bat (Plecotus auritus). Intra- and interannual home range overlap analyses of female B. barbastellus revealed that fidelity to individual foraging grounds, i.e. a traditional use of particular sites, seems to effect the spatial distribution of home ranges more than intraspecific competition among communally roosting females. The results of a joint analysis of annual maternity roost selection and flight activities along commuting corridors highlight the necessity to protect roost complexes in conjunction with commuting corridors. Using radio-tracking data and an Euclidean distance approach I quantified the sex-specific and individual habitat use by female and male western barbastelle bats within their home ranges. My data indicated a partial sexual segregation in summer habitats. Females were found in deciduous forest patches and preferably foraged along linear elements within the forest. Males foraged closer to forest edges and in open habitats. Finally, I examined the resource partitioning between the western barbastelle bat and two syntopic bat species with a potential for interspecific competition due to similarities in foraging strategies, prey selection and roost preferences. Simultaneous radio-tracking of mixed-species pairs revealed a partial spatial separation of the three syntopic bat species along a gradient from the forest to edge habitats and open landscape. Long-eared bats were found close to open habitats which were avoided by the other two species. B. barbastellus preferred linear landscape elements (edge habitats) and forests, M. bechsteinii also preferred forest habitats. Only little overlap in terms of roost structure and tree species selection was found.

Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study

(1)H HR-MAS NMR spectroscopy was applied to apple tissue samples deriving from 3 different cultivars. The NMR data were statistically evaluated by analysis of variance (ANOVA), principal component analysis (PCA), and partial least-squares-discriminant analysis (PLS-DA). The intra-apple variability of the compounds was found to be significantly lower than the inter-apple variability within one cultivar. A clear separation of the three different apple cultivars could be obtained by multivariate analysis. Direct comparison of the NMR spectra obtained from apple tissue (with HR-MAS) and juice (with liquid-state HR NMR) showed distinct differences in some metabolites, which are probably due to changes induced by juice preparation. This preliminary study demonstrates the feasibility of (1)H HR-MAS NMR in combination with multivariate analysis as a tool for future chemometric studies applied to intact fruit tissues, e.g. for investigating compositional changes due to physiological disorders, specific growth or storage conditions.

Phenotypic homogeneity and genotypic variability in a large series of congenital isolated ACTH-deficiency patients with TPIT gene mutations

Congenital isolated ACTH deficiency (IAD) is a rare disease characterized by low plasma ACTH and cortisol levels and preservation of all other pituitary hormones. This condition was poorly defined before we identified TPIT, a T-box transcription factor with a specific role in differentiation of the corticotroph lineage in mice and humans, as its principal molecular cause.

Cathepsin D primes caspase-8 activation by multiple intra-chain proteolysis

During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.

REMOVING TECHNICAL VARIABILITY IN RNA-SEQ DATA USING CONDITIONAL QUANTILE NORMALIZATION

The ability to measure gene expression on a genome-wide scale is one of the most promising accomplishments in molecular biology. Microarrays, the technology that first permitted this, were riddled with problems due to unwanted sources of variability. Many of these problems are now mitigated, after a decade’s worth of statistical methodology development. The recently developed RNA sequencing (RNA-seq) technology has generated much excitement in part due to claims of reduced variability in comparison to microarrays. However, we show RNA-seq data demonstrates unwanted and obscuring variability similar to what was first observed in microarrays. In particular, we find GC-content has a strong sample specific effect on gene expression measurements that, if left uncorrected, leads to false positives in downstream results. We also report on commonly observed data distortions that demonstrate the need for data normalization. Here we describe statistical methodology that improves precision by 42% without loss of accuracy. Our resulting conditional quantile normalization (CQN) algorithm combines robust generalized regression to remove systematic bias introduced by deterministic features such as GC-content, and quantile normalization to correct for global distortions.

Distribution and genetic variability among Campylobacter spp. isolates from different animal species and humans in Switzerland

In Switzerland, a national database with 1028 Campylobacter isolates from poultry, pigs, cats, dogs, cattle, humans, zoo animals and water has been created. The database contains the genetic fingerprint and background information of each Campylobacter isolate. Dominant species could be identified in the different sources with a majority of Campylobacter jejuni in poultry (73%), humans (79%), cattle (95%), zoo animals (40%) and water (100%), of Campylobacter coli in pigs (72%), and of Campylobacter upsaliensis/helveticus in cats and dogs (55%). The comparison of three genotyping methods, amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis and restriction fragment length polymorphism, revealed that AFLP allows discrimination between the different Campylobacter species and is the most appropriate method to distinguish specific strains within the same species. Genotyping analysis demonstrated that the Campylobacter population is heterogeneous among the different sources and that no dominant clone is spread in the country. Genotyping and the resulting database are useful tools to trace back future Campylobacter infections.

Intra-annual radial growth and water relations of trees - implications towards a growth mechanism

There is a missing link between tree physiological and wood-anatomical knowledge which makes it impossible mechanistically to explain and predict the radial growth of individual trees from climate data. Empirical data of microclimatic factors, intra-annual growth rates, and tree-specific ratios between actual and potential transpiration (T PET−1) of trees of three species (Quercus pubescens, Pinus sylvestris, and Picea abies) at two dry sites in the central Wallis, Switzerland, were recorded from 2002 to 2004 at a 10 min resolution. This included the exceptionally hot and dry summer of 2003. These data were analysed in terms of direct (current conditions) and indirect impacts (predispositions of the past year) on growth. Rain was found to be the only factor which, to a large extent, consistently explained the radial increment for all three tree species at both sites and in the short term as well. Other factors had some explanatory power on the seasonal time-scale only. Quercus pubescens built up much of its tree ring before bud break. Pinus sylvestris and Picea abies started radial growth 1–2 weeks after Quercus pubescens and this was despite the fact that they had a high T PET−1 before budburst and radial growth started. A high T PET−1 was assumed to be related to open stomata, a very high net CO2 assimilation rate, and thus a potential carbon (C)-income for the tree. The main period of radial growth covered about 30–70% of the productive days of a year. In terms of C-allocation, these results mean that Quercus pubescens depended entirely on internal C-stores in the early phase of radial growth and that for all three species there was a long time period of C-assimilation which was not used for radial growth in above-ground wood. The results further suggest a strong dependence of radial growth on the current tree water relations and only secondarily on the C-balance. A concept is discussed which links radial growth over a feedback loop to actual tree water-relations and long-term affected C-storage to microclimate.

Early autonomic dysfunction in patients with diabetes mellitus assessed by spectral analysis of heart rate and blood pressure variability

Patients with diabetes mellitus (DM) often have alterations of the autonomic nervous system (ANS), even early in their disease course. Previous research has not evaluated whether these changes may have consequences on adaptation mechanisms in DM, e.g. to mental stress. We therefore evaluated whether patients with DM who already had early alterations of the ANS reacted with an abnormal regulatory pattern to mental stress. We used the spectral analysis technique, known to be valuable and reliable in the investigation of disturbances of the ANS. We investigated 34 patients with DM without clinical evidence of ANS dysfunction (e.g. orthostatic hypotension) and 44 normal control subjects (NC group). No patients on medication known to alter ANS responses were accepted. The investigation consisted of a resting state evaluation and a mental stress task (BonnDet). In basal values, only the 21 patients with type 2 DM were different in respect to body mass index and systolic blood pressure. In the study parameters we found significantly lower values in resting and mental stress spectral power of mid-frequency band (known to represent predominantly sympathetic influences) and of high-frequency and respiration bands (known to represent parasympathetic influences) in patients with DM (types 1 and 2) compared with NC group (5.3 +/- 1.2 ms2 vs. 6.1 +/- 1.3 ms2, and 5.5 +/- 1.6 ms2 vs. 6.2 +/- 1.5 ms2, and 4.6 +/- 1.7 ms2 vs. 6.2 +/- 1.5 ms2, for resting values respectively; 4.7 +/- 1.4 ms2 vs. 5.9 +/- 1.2 ms2, and 4.6 +/- 1.9 ms2 vs. 5.6 +/- 1.7 ms2, and 3.7 +/- 2.1 ms2 vs. 5.6 +/- 1.7 ms2, for stress values respectively; M/F ratio 6/26 vs. 30/14). These differences remained significant even when controlled for age, sex, and body weight. However, patients with DM type 2 (and significantly higher body weight) showed only significant values in mental stress modulus values. There were no specific group effects in the patients with DM in adaptation mechanisms to mental stress compared with the NC group. These findings demonstrate that power spectral examinations at rest are sufficiently reliable to diagnose early alterations in ANS in patients with DM. The spectral analysis technique is sensitive and reliable in investigation of ANS in patients with DM without clinically symptomatic autonomic dysfunction.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

Effects of verum acupuncture compared to placebo acupuncture on quantitative EEG and heart rate variability in healthy volunteers

OBJECTIVES: The aim of this single-blind randomized crossover study was to evaluate specific effects of manual acupuncture on central and vegetative nervous system activity measured by quantitative electroencephalography (qEEG) and heart rate variability (HRV). DESIGN: Twenty (20) healthy volunteers (mean: 25.2 +/- 3.6 years) were monitored simultaneously using a qEEG system and a 12-channel electrocardiogram recorder during verum acupuncture (VA) at acupuncture point Large Intestine 4 (Hegu) (LI4) or placebo acupuncture (PA) at a sham point. RESULTS: In the EEG conduction of the occipital area, needle stimulation in VA increased alpha1-frequency significantly, and the ratio alpha1/theta was shifted to the benefit of alpha1 over all electrodes. The HRV parameters showed a significant increase of the low frequency/high frequency (HF) ratio during the first minute of stimulation in VA, indicating an initial increase of sympathetic activation. However, an increase of HF power in the minute after stimulation followed by a decrease in heart rate suggests delayed vagal activation. De qi (a sensation that is typical of acupuncture needling) occurred in 16 subjects during VA and in 9 volunteers during PA (80% versus 45%). CONCLUSIONS: Manual stimulation on LI4 seems to lead to specific changes in alpha EEG-frequency and in HRV parameters. A linear relationship between the HRV parameters and the alpha EEG band might point to a specific modulation of cerebral function by vegetative effects during acupuncture.

Evaluation of flood risk in response to climate variability

Standard procedures for forecasting flood risk (Bulletin 17B) assume annual maximum flood (AMF) series are stationary, meaning the distribution of flood flows is not significantly affected by climatic trends/cycles, or anthropogenic activities within the watershed. Historical flood events are therefore considered representative of future flood occurrences, and the risk associated with a given flood magnitude is modeled as constant over time. However, in light of increasing evidence to the contrary, this assumption should be reconsidered, especially as the existence of nonstationarity in AMF series can have significant impacts on planning and management of water resources and relevant infrastructure. Research presented in this thesis quantifies the degree of nonstationarity evident in AMF series for unimpaired watersheds throughout the contiguous U.S., identifies meteorological, climatic, and anthropogenic causes of this nonstationarity, and proposes an extension of the Bulletin 17B methodology which yields forecasts of flood risk that reflect climatic influences on flood magnitude. To appropriately forecast flood risk, it is necessary to consider the driving causes of nonstationarity in AMF series. Herein, large-scale climate patterns—including El Niño-Southern Oscillation (ENSO), Pacific Decadal Oscillation (PDO), North Atlantic Oscillation (NAO), and Atlantic Multidecadal Oscillation (AMO)—are identified as influencing factors on flood magnitude at numerous stations across the U.S. Strong relationships between flood magnitude and associated precipitation series were also observed for the majority of sites analyzed in the Upper Midwest and Northeastern regions of the U.S. Although relationships between flood magnitude and associated temperature series are not apparent, results do indicate that temperature is highly correlated with the timing of flood peaks. Despite consideration of watersheds classified as unimpaired, analyses also suggest that identified change-points in AMF series are due to dam construction, and other types of regulation and diversion. Although not explored herein, trends in AMF series are also likely to be partially explained by changes in land use and land cover over time. Results obtained herein suggest that improved forecasts of flood risk may be obtained using a simple modification of the Bulletin 17B framework, wherein the mean and standard deviation of the log-transformed flows are modeled as functions of climate indices associated with oceanic-atmospheric patterns (e.g. AMO, ENSO, NAO, and PDO) with lead times between 3 and 9 months. Herein, one-year ahead forecasts of the mean and standard deviation, and subsequently flood risk, are obtained by applying site specific multivariate regression models, which reflect the phase and intensity of a given climate pattern, as well as possible impacts of coupling of the climate cycles. These forecasts of flood risk are compared with forecasts derived using the existing Bulletin 17B model; large differences in the one-year ahead forecasts are observed in some locations. The increased knowledge of the inherent structure of AMF series and an improved understanding of physical and/or climatic causes of nonstationarity gained from this research should serve as insight for the formulation of a physical-casual based statistical model, incorporating both climatic variations and human impacts, for flood risk over longer planning horizons (e.g., 10-, 50, 100-years) necessary for water resources design, planning, and management.

Drug-specific in vitro release of IL-2, IL-5, IL-13 and IFN-gamma in patients with delayed-type drug hypersensitivity

BACKGROUND: The most prevalent drug hypersensitivity reactions are T-cell mediated. The only established in vitro test for detecting T-cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug-sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well-documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T-cell sensitization to drugs. METHODS: Peripheral blood mononuclear cell of 10 patients, five allergic to beta-lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17-plex bead-based immunoassay kits. RESULTS: Among the 17 cytokines/chemokines analysed, interleukin-2 (IL-2), IL-5, IL-13 and interferon-gamma (IFN-gamma) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide- and beta-lactam-reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients. CONCLUSION: The measurement of IL-2, IL-5, IL-13 or IFN-gamma or a combination thereof might be a useful in vitro tool for detection of T-cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.

Intra- and inter-individual variation of BIS-index and Entropy during controlled sedation with midazolam/remifentanil and dexmedetomidine/remifentanil in healthy volunteers: an interventional study

INTRODUCTION: We studied intra-individual and inter-individual variability of two online sedation monitors, BIS and Entropy, in volunteers under sedation. METHODS: Ten healthy volunteers were sedated in a stepwise manner with doses of either midazolam and remifentanil or dexmedetomidine and remifentanil. One week later the procedure was repeated with the remaining drug combination. The doses were adjusted to achieve three different sedation levels (Ramsay Scores 2, 3 and 4) and controlled by a computer-driven drug-delivery system to maintain stable plasma concentrations of the drugs. At each level of sedation, BIS and Entropy (response entropy and state entropy) values were recorded for 20 minutes. Baseline recordings were obtained before the sedative medications were administered. RESULTS: Both inter-individual and intra-individual variability increased as the sedation level deepened. Entropy values showed greater variability than BIS(R) values, and the variability was greater during dexmedetomidine/remifentanil sedation than during midazolam/remifentanil sedation. CONCLUSIONS: The large intra-individual and inter-individual variability of BIS and Entropy values in sedated volunteers makes the determination of sedation levels by processed electroencephalogram (EEG) variables impossible. Reports in the literature which draw conclusions based on processed EEG variables obtained from sedated intensive care unit (ICU) patients may be inaccurate due to this variability. TRIAL REGISTRATION: clinicaltrials.gov Nr. NCT00641563.