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The discovery of small RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi-associated RNAs (piRNAs) has led to new challenges in the selective detection of RNAs. Many noncoding RNAs act as post-translational regulators of gene expression and are involved in the regulation of cell proliferation or apoptosis, but are difficult to amplify, label, and detect. Standard microarray detection procedures involve pre-hybridization labeling or enzymatic 3'-labeling by polymerase-catalyzed extension. Dual labeling would improve the fidelity of detection, but no polymerases for 5'-extension are known. Here we report a novel labeling method for RNAs bearing natural 5'-phosphate groups, such as miRNAs, based on enzyme-free ligation of a biotin- or fluorophore-labeled oligonucleotide to the 5' termini. The method uses in situ activation of the natural 5'-phosphate groups in these RNAs and was optimized to give near-quantitative conversion in solution. With use of biotin- or fluorophore-bearing labeling strands, different miRNA sequences were detected on microarrays with little background fluorescence. In combination with an established method of enzymatic on-chip labeling at the 3' termini, highly selective detection of related miRNAs was achieved by dual recognition at both termini, even in the case of miRNAs differing in only one nucleotide.

In climate and weather models, the quantitative description of aerosol and cloud processes relies on simplified assumptions. This contributes major uncertainties to the prediction of global and regional climate change. Therefore, models need good parameterizations for heterogeneous ice nucleation by atmospheric aerosols. Here the authors present a new parameterization of immersion freezing on desert dust particles derived from a large number of experiments carried out at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) cloud chamber facility. The parameterization is valid in the temperature range between −12° and −36°C at or above water saturation and can be used in atmospheric models that include information about the dust surface area. The new parameterization was applied to calculate distribution maps of ice nuclei during a Saharan dust event based on model results from the regional-scale model Consortium for Small-Scale Modelling–Aerosols and Reactive Trace Gases (COSMO-ART). The results were then compared to measurements at the Taunus Observatory on Mount Kleiner Feldberg, Germany, and to three other parameterizations applied to the dust outbreak. The aerosol number concentration and surface area from the COSMO-ART model simulation were taken as input to different parameterizations. Although the surface area from the model agreed well with aerosol measurements during the dust event at Kleiner Feldberg, the ice nuclei (IN) number concentration calculated from the new surface-area-based parameterization was about a factor of 13 less than IN measurements during the same event. Systematic differences of more than a factor of 10 in the IN number concentration were also found among the different parameterizations. Uncertainties in the modeled and measured parameters probably both contribute to this discrepancy and should be addressed in future studies.

Denileukin Diftitox (ONTAK(®), DAB(389) IL-2) is a recombinant DNA-derived fusion protein depleting cells that express high-affinity IL-2 receptor. Important cell targets are CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(reg)). Elimination of immunosuppressive T(reg) by Denileukin Diftitox may provide a way to modulate immune tolerance following stem cell transplantation. Here, we combined T(reg) depletion with a vaccination approach to induce donor-specific immune reactions. To investigate this approach we chose the mixed chimerism canine stem cell transplantation model which represents a high state of tolerance between two hematopoietic systems. The aim was therefore to induce a graft versus hematopoiesis effect thereby converting mixed to full donor chimerism. Dog leukocyte antigen identical siblings that had developed a stable mixed chimerism after non-myeloablative stem cell transplantation received a single dose of Denileukin Diftitox (18μg/kg, i.v.) followed by several cell-lysate vaccinations. Host peripheral blood mononuclear cell lysates combined with CpG-ODN, and Montanide(®) ISA 51 were locally applied. In vitro studies demonstrated that canine T(reg) are a target of Denileukin Diftitox. The suppression of T-cell proliferation by T(reg) was abolished by addition of Denileukin Diftitox (10nM). An increase of proliferation of median 300% (range: 200%-425%) was observed. No change in donor chimerism was observed after administration of Denileukin Diftitox and vaccination. This study highlights that application of Denileukin Diftitox resulted in a depletion of T(reg) followed by an increase of immune response in vitro. This effect could not be confirmed in vivo even if the immune system was stimulated by vaccinations.

We explored the impact of exposure to an obesogenic diet (High Fat-High Sucrose; HFS) during the post-weaning period on sweet preference and behaviors linked to reward and anxiety. All rats were fed chow. In addition a HFS-transient group had access to this diet for 10days from post-natal (PN) day 22 and a HFS-continuous group continued access until adult. Behavioral tests were conducted immediately after PN 32 (adolescence) or after PN 60 (adult) and included: the condition place preference (CPP) test for chocolate, sugar and saccharin preference (anhedonia), the elevated plus maze (anxiety-like behavior) and the locomotor response to quinpirole in the open field. Behavior was unaltered in adult rats in the HFS-transient group, suggesting that a short exposure to this obesogenic food does not induce long-term effects in food preferences, reward perception and value of palatable food, anxiety or locomotor activity. Nevertheless, rats that continued to have access to HFS ate less chocolate during CPP training and consumed less saccharin and sucrose when tested in adolescence, effects that were attenuated when these rats became adult. Moreover, behavioral effects linked to transient HFS exposure in adolescence were not sustained if the rats did not remain on that diet until adult. Collectively our data demonstrate that exposure to fat and sucrose in adolescence can induce immediate reward hypofunction after only 10days on the diet. Moreover, this effect is attenuated when the diet is extended until the adult period, and completely reversed when the HFS diet is removed.

Black carbon (BC) and mineral dust are among the most abundant insoluble aerosol components in the atmosphere. When released, most BC and dust particles are externally mixed with other aerosol species. Through coagulation with particles containing soluble material and condensation of gases, the externally mixed particles may obtain a liquid coating and be transferred into an internal mixture. The mixing state of BC and dust aerosol particles influences their radiative and hygroscopic properties, as well as their ability of forming ice crystals. We introduce the new aerosol microphysics submodel MADE-in, implemented within the ECHAM/MESSy Atmospheric Chemistry global model (EMAC). MADE-in is able to track mass and number concentrations of BC and dust particles in their different mixing states, as well as particles free of BC and dust. MADE-in describes these three classes of particles through a superposition of seven log-normally distributed modes, and predicts the evolution of their size distribution and chemical composition. Six out of the seven modes are mutually interacting, allowing for the transfer of mass and number among them. Separate modes for the different mixing states of BC and dust particles in EMAC/MADE-in allow for explicit simulations of the relevant aging processes, i.e. condensation, coagulation and cloud processing. EMAC/MADE-in has been evaluated with surface and airborne measurements and mostly performs well both in the planetary boundary layer and in the upper troposphere and lowermost stratosphere.

Background: Obesity and diabetes in mice can be modified by dietary variables. Here we systematically analysed the effect of the sucrose and fat content and of the fat quality in New Zealand Obese mice, a mouse model of the metabolic syndrome. Results: Male NZO mice fed a semi-purified diet with sucrose exhibited an identical weight gain and diabetes incidence as controls without sucrose. In contrast, mice on a chow diet gained weight more slowly and developed diabetes approximately 10 weeks later than those on the semi-purified diet (energy density 3.05 vs. 3.85 kcal/g; fibre content 12.9 vs. 4.7 %). In a second experimental series, neither the fat content (10 vs. 40% of the total energy) nor the quality of the fat (lard, safflower oil, or fish oil) of semi-purified diets modified weight gain. However, diabetes started approximately 2 weeks earlier and appeared more severe (blood glucose 30 vs. 20 mmol/l at week 13) in the high-fat diet group (energy density 4.58 kcal/g; fibre content 5.7 %). Conclusions: Obesity in NZO mice develops independent of the dietary sucrose or fat content, and of the fat quality. However, the dietary fat content accelerates the onset of diabetes without enhancing adiposity. In contrast, chow diet exerts an anti-adipogenic/anti-diabetogenic effect that appears to be due to its lower caloric density and/or its higher fibre content.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

Hadi Al-Hasani and colleagues report that a frameshift deletion in Tbc1d1 in lean SJL mice underlies a quantitative trait locus conferring protection against high-fat diet-induced obesity. TBC1D1 seems to influence metabolism by regulating fatty acid uptake and oxidation in skeletal muscle cells. We previously identified Nob1 as a quantitative trait locus for high-fat diet–induced obesity and diabetes in genome-wide scans of outcross populations of obese and lean mouse strains. Additional crossbreeding experiments indicated that Nob1 represents an obesity suppressor from the lean Swiss Jim Lambert (SJL) strain. Here we identify a SJL-specific mutation in the Tbc1d1 gene that results in a truncated protein lacking the TBC Rab–GTPase-activating protein domain. TBC1D1, which has been recently linked to human obesity, is related to the insulin signaling protein AS160 and is predominantly expressed in skeletal muscle. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, whereas overexpression of TBC1D1 had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation and reduced glucose uptake in isolated skeletal muscle. Our data strongly suggest that mutation of Tbc1d1 suppresses high-fat diet–induced obesity by increasing lipid use in skeletal muscle.

We included a parameterisation of the emissions of pollen grains into the comprehensive model system COSMO-ART. In addition, a detailed density distribution of birch trees within Switzerland was derived. Based on these new developments, we carried out numerical simulations of the dispersion of pollen grains for an episode that occurred in April 2006 over Switzerland and the adjacent regions. Since COSMO-ART is based on the operational forecast model of the German Weather Service, we are presenting a feasibility study of daily pollen forecast based on methods which have been developed during the last two decades for the treatment of anthropogenic aerosol. A comparison of the model results and very detailed pollen counts documents the current possibilities and the shortcomings of the method and gives hints for necessary improvements.

OBJECTIVE: We have previously reported that a high-fat, carbohydrate-free diet prevents diabetes and β-cell destruction in the New Zealand Obese (NZO) mouse strain. Here we investigated the effect of diets with and without carbohydrates on obesity and development of β-cell failure in a second mouse model of type 2 diabetes, the db/db mouse. RESULTS: When kept on a carbohydratecontaining standard (SD; with (w/w) 5.1, 58.3, and 17.6% fat, carbohydrates and protein, respectively) or high-fat diet (HFD; 14.6, 46.7 and 17.1%), db/db mice developed severe diabetes (blood glucose >20 mmol/l, weight loss, polydipsia and polyurea) associated with a selective loss of pancreatic β-cells, reduced GLUT2 expression in the remaining β-cells, and reduced plasma insulin levels. In contrast, db/db mice kept on a high-fat, carbohydrate-free diet (CFD; with 30.2 and 26.4% (w/w) fat or protein) did not develop diabetes and exhibited near-normal, hyperplastic islets in spite of a morbid obesity (fat content >60%) associated with hyperinsulinaemia. CONCLUSION: These data indicate that in genetically different mouse models of obesity-associated diabetes, obesity and dietary fat are not sufficient, and dietary carbohydrates are required, for β-cell destruction.

Background DNA methylation is an essential nuclear process associated with genomic functions such as transcription factor binding and the regulation of gene expression. DNA methylation patterns can also serve as potential biomarkers for disease progression and response to therapy. However, the full dynamics of DNA methylation across daily physiologic events have not been fully elucidated. Objective We sought to study how ingesting a standardized meal acutely affects peripheral blood DNA methylation. Design We performed an observational study in healthy men (n = 26) on DNA methylation and gene expression in whole blood before and 160 min after the ingestion of a standardized meal. Cytosine-phosphate-guanine (CpG) methylation was assayed on the HumanMethylation450k microarray, and gene expression was measured with the Human Gene 2.1 ST Array. Results Differential methylation after food intake was detected in 13% of the analyzed probes (63,207 CpG probes) at a 5% false discovery rate (FDR). This effect was driven by changes in leukocyte fractions as estimated from comparisons against methylation datasets generated from sorted leukocytes. When methylation values were adjusted for estimated leukocyte fractions, 541 probes were observed to be altered in the postprandial state (5% FDR). Conclusions Apparent alterations in DNA methylation 160 min after meal ingestion mainly reflect changes in the estimated leukocyte population in whole blood. These results have major methodologic implications for genome-wide methylation studies because they highlight the strong underlying effects of changes in leukocyte fractions on CpG methylation patterns as well as the potential importance of meal-standardized sampling procedures for future investigations when alterations in white blood cell fractions are unavailable. This trial was registered at clinicaltrials.gov as LSF008786.

Context: Shift workers are at increased risk of metabolic morbidities. Clock genes are known to regulate metabolic processes in peripheral tissues, eg, glucose oxidation. Objective: This study aimed to investigate how clock genes are affected at the epigenetic and transcriptional level in peripheral human tissues following acute total sleep deprivation (TSD), mimicking shift work with extended wakefulness. Intervention: In a randomized, two-period, two-condition, crossover clinical study, 15 healthy men underwent two experimental sessions: x sleep (2230–0700 h) and overnight wakefulness. On the subsequent morning, serum cortisol was measured, followed by skeletal muscle and subcutaneous adipose tissue biopsies for DNA methylation and gene expression analyses of core clock genes (BMAL1, CLOCK, CRY1, PER1). Finally, baseline and 2-h post-oral glucose load plasma glucose concentrations were determined. Main Outcome Measures: In adipose tissue, acute sleep deprivation vs sleep increased methylation in the promoter of CRY1 (+4%; P = .026) and in two promoter-interacting enhancer regions of PER1 (+15%; P = .036; +9%; P = .026). In skeletal muscle, TSD vs sleep decreased gene expression of BMAL1 (−18%; P = .033) and CRY1 (−22%; P = .047). Concentrations of serum cortisol, which can reset peripheral tissue clocks, were decreased (2449 ± 932 vs 3178 ± 723 nmol/L; P = .039), whereas postprandial plasma glucose concentrations were elevated after TSD (7.77 ± 1.63 vs 6.59 ± 1.32 mmol/L; P = .011). Conclusions: Our findings demonstrate that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in key metabolic tissues. Tissue-specific clock alterations could explain why shift work may disrupt metabolic integrity as observed herein.

Birch trees produce large amounts of highly allergenic pollen grains that are distributed by wind and impact human health by causing seasonal hay fever, pollen-related asthma, and other allergic diseases. Traditionally, pollen forecasts are based on conventional microscopic counting techniques that are labor-intensive and limited in the reliable identification of species. Molecular biological techniques provide an alternative approach that is less labor-intensive and enables identification of any species by its genetic fingerprint. A particularly promising method is quantitative Real-Time polymerase chain reaction (qPCR), which can be used to determine the number of DNA copies and thus pollen grains in air filter samples. During the birch pollination season in 2010 in Mainz, Germany, we collected air filter samples of fine (

We present the first stage of a new online-coupled global to regional-scale modeling framework for the simulation of the spatiotemporal evolution of aerosols and trace gases. The underlying meteorological model is the new nonhydrostatic model system ICON (ICOsahedral Nonhydrostatic) which allows a local grid refinement with two-way interactions between the grids. We develop the extension ART (Aerosol and Reactive Trace gases) with the goal of simulating interactions between trace substances and the state of the atmosphere. Within this paper, we present the basic equations and give an overview of the physical parameterizations as well as numerical methods we use. First applications of the new model system for trace gases, monodisperse particles and polydisperse particles are shown. The simulated distribution of two very short-lived substances (VSLS), bromoform (CHBr3) and dibromomethane (CH2Br2) reflecting the fast upward transport shows a good agreement with observations and previous model studies. Also, the shape of the simulated tropical profiles is well reproduced. As an example for the treatment of monodisperse particles we present the simulated ash plume of the Eyjafjallajökull eruption in April 2010. Here, a novel approach for the source function is applied. The pattern of the simulated distribution of volcanic ash particles shows a good agreement with previous studies. As an example for the treatment of a polydisperse aerosol, where number densities and mass concentrations are accounted for, we simulated the annual emissions of sea salt. We obtain a total emission flux of 26.0 Pg yr−1 and an emission flux of particles with diameter less than 10 μm of 7.36 Pg yr−1.

We applied a parameterization for the emission of mineral dust particles which takes into account the relevant processes such as saltation and combines previous, physically based parameterizations. The size distribution of the soil particles is taken into account to describe the saltation. The emitted particles are described by three log-normal distributions with fixed standard deviations. A comparison of the results of a stand alone version of our parameterization with observations shows that despite tuning of model parameters there are still differences. Finally, we included the parameterization within our three-dimensional mesoscale model system for the area of the Dead Sea. The channelling effect of the Jordan Valley and stable stratification during the day modifies the horizontal distribution of the dust particles. At greater distances the size and the mass distributions of the particles is shifted towards smaller diameters due to sedimentation which is important for radiative feedback mechanisms. Sensitivity runs show the advantage of the parameterization which allows a time dependent ratio of the saltation and the emission flux at each grid point.

To ensure the efficiency of strategies to reduce air pollutants caused by road traffic, it is essential to evaluate the emission data to be used in emission calculation models. This basic approach of the BAB II project (BundesAutoBahn––federal highway) has been reached by simultaneous measurements of gaseous and particulate emissions on the windward and lee side perpendicular to a motorway. The differences between the measurements allows for the calculation of the emissions caused by traffic on the motorway. Measurements of CO, NO, NO x , CO 2 , O 3 , VOC, and particulates of high horizontal and vertical resolution were made. The experimental setup was made symmetric to the motorway, with 52 m high towers on each side. The field phase took place from 1 to 25 May 2001 at the motorway A656 near Heidelberg, Germany. In cases of wind direction perpendicular to the motorway, the height of the plume caused by traffic emissions is detectable and the emissions released on the motorway can be calculated. With traffic census carried out simultaneously, the emission data can be estimated using emission factors given in the literature. Comparison between real-world traffic emissions and calculated emissions allows for an evaluation of the emissions calculated by the models. This paper gives an overview of BAB II, its measuring concept, the experimental setup, and the quality assurance and control program. It is shown in detail that the method of emission estimation by measurements of concentration differences between both sides of a motorway works quite well if the meteorological assumptions for determining real-world traffic emissions have been fulfilled. Detailed results in terms of measured and model-calculated emissions of gaseous and particulate species will be reported in a number of subsequent papers.

The aging of soot is one of the key uncertainties in the estimation of both the direct and indirect climate effect. While freshly emitted soot is initially hydrophobic and externally mixed, it can be transferred into an internal mixture by coagulation, condensation or photochemical processes. These aging processes affect the hygroscopic qualities and hence the growth behaviour, the optical properties and eventually the lifetime of the soot particles. However, due to computational limits the aging of soot in global climate models is often only parameterised by an estimated turnover rate resulting in a lifetime of soot of several days. Hence, the aging process of soot is one of the key uncertainties governing the burden and effect of black carbon. In this study, we discuss the time scale on which diesel soot is transferred from an external to an internal mixture based on the results of our simulations with a comprehensive mesoscale model. For daytime conditions during summer condensation of sulphuric acid is dominant and the aging process occurs on a time scale of τ =8h close to the sources and τ =2h above the source region. During winter comparable time scales are found but ammonium nitrate becomes more important. During night time condensation is not effective. Then coagulation is the most important aging process and our results show time scales between 10h and 40h.

New parameterisations were developed to calculate the emission and resuspension flux of pollen grains. These new parameterisations were included in the comprehensive mesoscale model system KAMM/DRAIS. Two types of simulations were performed. In the first case, horizontally homogeneous meteorology was assumed. In this case, pollen concentration rapidly decreased with the distance from the source. In the second case, where the fully three-dimensional model was applied, atmospheric lifetime of the pollen grains increased remarkably. This was mainly caused by the vertical wind speeds induced by topographic effects. Consequently, the pollen grains could travel much larger distances until they were deposited and finally removed from the atmosphere. This is an important finding with respect to the problem of cross pollination. Due to the lack of measurements, a number of parameters had to be assumed. However, the parameterisation proposed may well serve as a starting point of a daily pollen forecast with numerical models.

Numerical simulations with a comprehensive mesoscale- γ model system were carried out to study the relative importance of individual sources for nitrous acid (HONO) on the daily cycle of the HONO concentration during the course of the day. The sources are the gas phase production, direct emissions of HONO, and heterogeneous reactions at aerosol surfaces and at the ground. The results of the simulations were compared to observations made during 18–19 October 2001 at the 200 m research tower at the Forschungszentrum Karlsruhe, Germany. We found that currently used chemical transport models are using a rate constant for the gas-phase reaction of NO with OH which is a factor of two lower than the recently published IUPAC data. The old rate constant gives 50% lower daytime HONO concentrations than the new one. The simulations showed that the direct emissions and the heterogeneous reactions at the ground are the most important sources of HONO during the night. While the observed nocturnal HONO concentrations close to the surface are reproduced by the simulations the daytime HONO concentrations are underestimated by 30–50%. We introduced an artificial photolytic HONO source which is proportional to the photolysis rate constant of NO 2 . This leads to a much better agreement between observed and simulated HONO concentrations during the day. In addition, we compared the production of the hydroxyl radical by the photolysis of HONO with its production via the photolysis of ozone. During 18 October 2001 when the ozone concentrations were low the photolysis of HONO is the dominant source of the OH radical while during a typical summer day the photolysis of HONO contributes only 20% of the photolysis of ozone.

Simultaneous nighttime HONO, NO, NO 2 and particle surface area density gradients in the altitude range 10–190 m were measured for the first time on the meteorological tower at the Forschungszentrum Karlsruhe/Germany using a new, very sensitive HONO instrument, a commercial NO x monitor and a SMPS system. For all gradient measurements during the campaign it was observed that the [HONO]/[NO 2 ] ratio decreased with increasing altitude below 100 m. In contrast, the particle surface area density was found to be more or less constant. Accordingly, no correlation between the [HONO]/[NO 2 ] ratio and the particle surface area density was observed showing that HONO formation was dominated by processes on ground surfaces and that significant HONO formation on particle surfaces could be excluded for the measurement site. Besides the nighttime gradient measurements, high daytime HONO concentrations were observed at constant altitude, which point to a significant and yet unknown daytime source of HONO. In addition, a HONO/NO x ratio from direct emissions of 0.8% was estimated from the field campaign.

New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects = 25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.

A comprehensive field campaign was carried out to check the quality of modelled on-road emissions of NOx and CO by comparing them with real-world emissions on a motorway in the southwestern part of Germany. Three different sets of emission data were determined for a section of the motorway A 656 that connects Mannheim and Heidelberg. First, emission data were pre-calculated, that means before the field campaign took place, using literature data for traffic volume, types of vehicles and driving speeds. Second, detailed traffic measurements during two intensive measurement phases were used to calculate improved emission data based on the actual traffic situation. Third, real-world emission data were determined by meteorological and chemical measurements. It shows that the differences of the pre-calculated emission data and the emission data improved by the traffic measurements differ by less than 15% in case of CO and approx. by 35% in case of NOx. A comparison of the observed emissions with the calculated ones gives an agreement for NOx within the error bars. For CO, however, a discrepancy by a factor of two was found. The real-world emissions were much higher than calculated.

The O3-NOy relation for a polluted planetary boundary layer is investigated using numerical simulations and observations. Box model runs are carried out to study the sensitivity of the transition value of NOy, which separates the low-NOx. and the high-NOx regimes, to changes in emissions and in ambient conditions. It is shown that the transition value of NOy depends on the hydrocarbon and NOx emissions, on water vapor, on radiation, and on temperature. Increasing hydrocarbon emissions, increasing water vapor, increasing radiation, or increasing temperature shifts the transition value to higher levels. One-dimensional simulations, which include vertical mixing and dry deposition, show that increasing dry deposition reduces the transition value of NOy. Sensitivity studies with a three-dimensional nonhydrostatic model system are carried out to investigate the effects of transport. A smoothing procedure is applied to reduce the large scatter of the O3-NOy relation which is found in these cases. After smoothing, a transition value can be identified. For the southwestern part of Germany it is shown that this transition value indicates areas where NOx reduction leads to the most effective ozone reduction. Runs with various scenarios show the dependence of the transition value on emissions and ambient conditions. A normalization procedure is introduced. The normalization procedure is applied to the model results and to observations. Under high-NOx conditions a large difference between the cases with and without advection is found. It shows that in the three-dimensional case the ozone concentration is less sensitive to changes in the NOx emissions than in the case of pure chemistry.

An intercomparison of nine chemical mechanisms (e.g. ADOM, CBM-IV, EMEP, RADM2) as used by 12 contributing groups was conducted. The results for three scenarios are presented covering remote situations with a net O3 loss of around 2.7 ppb (LAND and FREE) and a moderately polluted situation with O3 formation of around 100 ppb (PLUME1) over a 5 day simulation period. The overall tendencies (i.e. the total net production/loss over 5 days) for O3 show a r.m.s. error of 38, 15 and 16%; for H2O2 the errors are 76, 23 and 30% (for LAND, FREE, PLUME1). In terms of ozone production in PLUME1, the most productive mechanisms are EMEP and IVL, the RADM-type mechanisms lie in the mid-range and the CBM-IV type mechanisms fall at the bottom of the range. The differences in H2O2 can partly be explained by an incorrect use of the HO2 + HO2 rate constant and by differences in the treatment of the peroxy radical interactions. In the PLUME1 case the r.m.s. error of the PAN tendency was found to be 29%. Differences between mechanisms for the HO radical are 10, 15 and 19% and for the NO3 radical 35, 16 and 40% (for LAND, FREE, PLUME1) in terms of the r.m.s. error of the results for a 12 h time period centred around the last noon (HO), respectively, a 8 h time period centred around the last midnight (NO3) of simulation. Especially for NO3 some differences are due to different numerical treatment of photolytic processes in the models. Large differences between mechanisms are observed for higher organic peroxides and higher aldehydes with a r.m.s. error of around 50% for the final concentration in PLUME1. The protocol of the intercomparison is given in the appendix, so that the comparison could be repeated for the purpose of mechanism development and sensitivity studies.

Mineral dust has a significant impact on air quality by reducing visibility and causing adverse health effects on humans such as increased respiratory symptoms and decreased lung function. Ground-based monitoring of PM 10 and PM 2.5 is the common metric used for assessing air quality degradation. Because of vast dust sources in Northern China and Mongolia, a limited number of existing ground-based sites across this region renders air quality monitoring difficult. Information about air quality within these regions can only be gained by the use of models or satellite data. The goal of this study is to determine satellite value-added information from three passive and one active satellite sensors for the assessment and improvement of local air quality modeling.

To investigate if acute sleep deprivation affects food purchasing choices in a mock supermarket.On the morning after one night of total sleep deprivation (TSD) or after one night of sleep, 14 normal-weight men were given a fixed budget (300 SEK-approximately 50 USD). They were instructed to purchase as much as they could out of a possible 40 items, including 20 high-caloric foods (2 kcal/g) and 20 low-caloric foods (2 kcal/g). The prices of the high-caloric foods were then varied (75%, 100% (reference price), and 125%) to determine if TSD affects the flexibility of food purchasing. Before the task, participants received a standardized breakfast, thereby minimizing the potential confound produced by hunger. In addition, morning plasma concentrations of the orexigenic hormone ghrelin were measured under fasting conditions.Independent of both type of food offered and price condition, sleep-deprived men purchased significantly more calories (+9%) and grams (+18%) of food than they did after one night of sleep (both P 0.05). Morning plasma ghrelin concentrations were also higher after TSD (P 0.05). However, this increase did not correlate with the effects of TSD on food purchasing.This experiment demonstrates that acute sleep loss alters food purchasing behavior in men.

Hintergrund/Fragestellung: In der Ruckkreuzungspopulation der NZO-Maus, einem Modell fur das metabolische Syndrom, und der diabetesresistenten C57BL/6J- (B6) Maus wurde der Adipositas-QTL Nob3 auf Chromosom 1 identifiziert und schlieslich mittels positioneller Klonierung eine Mikrodeletion im B6-Genom aufgespurt, die zum Verlust der RNA des Interferon activated gene 202b (Ifi202b) fuhrt und durch Suppression des Gens 11β-Hsd1 vor Adipositas schutzt. Ziel war es nun, den Einfluss des Nob3 und der Ifi202b-Expression auf die Diabetesentstehung und β-Zellfunktion zu untersuchen. Methoden: Langerhans-Inseln von NZO- und B6-ob/ob-Mausen wurden durch ein geeignetes diatetisches Regime aus 15-wochiger Kohlenhydratrestriktion und anschliesen-der 2-tagiger Kohlenhydratgabe glucolipotoxischen Bedingungen ausgesetzt und anschliesend fur eine Microarray-basierte Transkriptomanalyse sowie fur eine RNA-Sequenzierung verwendet. Der Einfluss differenziell exprimierter Gene innerhalb des Nob3 auf die β-Zellfunktion wurde mittels 4 rekombinant kongener Linien (RCS), die auf dem B6-Hintergrund unterschiedliche Fragmente von Nob3 der NZO-Maus tragen, untersucht. Ergebnisse: Glucolipotoxizitat fuhrte in den NZO-Inseln zu einem Verlust von p-AKT, was in einem spateren β-Zelluntergang mundete, wahrend B6-ob/ob-Inseln eine gesteigerte Proliferation aufwiesen. Die Transkriptomanalyse ergab genomweit 272 differenziell exprimierte Gene zwischen NZO und B6-ob/ob-Inseln, von denen sich neben Ifi202b 7 weitere im Nob3 befinden. In vivo-Untersuchungen der RCS zeigten, dass die Anwesenheit von Ifi202b und 6 weiteren Genen im proximalen Bereich von Nob3 zu einer verminderten Induktion von β-Zellproliferation unter glucolipotoxischen Bedingungen fuhrte. Erste Ergebnisse mit isolierten Inseln der RCS deuten darauf hin, dass die Prasens von Ifi202b die Phosphorylierung von AKT hemmt. Schlussfolgerung: Der Verlust des Ifi202b, aber moglicherweise auch die differentielle Expression 6 weiterer Gene fuhrt in der B6-Maus neben der Suppression einer Adipositas zu einer Aufrechterhaltung des AKT-Signalwegs und konnte so eine Glucose-induzierte Proliferation auslosen, wodurch B6-Mause vor einem Diabetes geschutzt sind.

Rapid, template-directed ligation reactions between a phosphate-terminated oligonucleotide and an unphosphorylated reaction partner may be induced by cyanogen bromide (BrCN). Frequently, however, the reaction is low yielding, and even a large excess of the condensing agent can fail to induce quantitative conversions. In this study, we used BrCN to induce chemical primer extension reactions. Here, we report that buffers containing hydroxyl groups react with short oligodeoxynucleotides in the presence of BrCN. One stable adduct between HEPBS buffer and cytosine was characterized by mass spectrometry and NMR after HPLC purification, indicating that a side reaction occurred at this nucleobase. Further, a first example of a primer extension reaction between an unmodified oligodeoxynucleotide as primer and dGMP is reported. Together, our results shed light on the potency, as well as the drawbacks of BrCN as a highly reactive condensing reagent for the ligation of unmodified nucleic acids.

In several rodent strains such as the New Zealand Obese (NZO) mouse, the incidence of obesity-associated diabetes mellitus is much higher in males than in females. In the present study, we investigated the effects of ovariectomy on glucose homeostasis in female NZO mice in order to elucidate the mechanism of their diabetes resistance. NZO females were ovariectomized at the age of 4 weeks, received a high-fat diet and body weight, body fat, glucose and insulin tolerance were investigated in comparison to sham-operated mice. In a second experiment, operated mice were fed a carbohydrate-free diet up to the age of 19 weeks before they received the high-fat diet. In comparison with a sham-operated control group, ovariectomized female NZO mice exhibited similar body weights, a reduced glucose tolerance, developed significantly higher blood glucose levels, lost insulin producing β-cells, which finally resulted in a diabetes prevalence of 73% at the age of 16 weeks vs. 25% in controls. Similar to male NZO mice, ovariectomized females presented a more severe insulin resistance in the insulin tolerance test than sham-operated controls. Furthermore, the more severe insulin resistance in ovariectomized mice preceded the development of diabetes and pancreatic insulin depletion that was caused by a dietary regimen of carbohydrate restriction and subsequent re-exposure. In summary our data demonstrate that estrogen protects NZO females from β-cell loss and obesity-associated diabetes mellitus. This effect is due to a reduced insulin resistance and possibly also to a reduced sensitivity of β-cells to glucolipotoxic conditions.

Based on laboratory studies it has been suggested that the most important atmospheric aerosol particles acting as ice nuclei for heterogeneous freezing of cloud droplets at temperatures above −15°C could be from biological sources. Here we present simulations with a regional atmospheric model to investigate their impact on mixed-phase clouds based on recent experimental studies with different biological ice nuclei.

Air pollution is one of the most significant environmental concerns in China today, particularly in the highly industrialized Eastern Chinese mega cities. During the Olympic Summer Games in 2008, the air pollution in China’s capital city of Beijing became an issue of international interest. Air pollution in Beijing has a number of key constituent components, including anthropogenic gaseous pollutants, and small airborne particles from natural and anthropogenic sources. These often exceed WHO recommended levels. To investigate the influence of anthropogenic nitrogen oxides (NOx) and ozone (O3) on the air quality in Beijing and its vicinity, the fully online coupled model system COSMO-ART is applied for the first time to Northern China. For this purpose a five-day simulation period from August 4th to 8th in 2008 was conducted. The simulated NOx and O3 concentrations are compared to observations from a measurement station located in Beijing. Backward trajectories are calculated to get more information about the source areas of the air pollutants. Comparisons of measured and modeled NOx and O3 showed that the daily course of the concentrations can be reproduced by the model very well.

Acute sleep loss increases food intake in adults. However, little is known about the influence of acute sleep loss on portion size choice, and whether this depends on both hunger state and the type of food (snack or meal item) offered to an individual. The aim of the current study was to compare portion size choice after a night of sleep and a period of nocturnal wakefulness (a condition experienced by night-shift workers, e.g. physicians and nurses). Sixteen men (age: 23 ± 0.9 years, BMI: 23.6 ± 0.6 kg/m2) participated in a randomized within-subject design with two conditions, 8-h of sleep and total sleep deprivation (TSD). In the morning following sleep interventions, portion size, comprising meal and snack items, was measured using a computer-based task, in both fasted and sated state. In addition, hunger as well as plasma levels of ghrelin were measured. In the morning after TSD, subjects had increased plasma ghrelin levels (13%, p = 0.04), and chose larger portions (14%, p = 0.02), irrespective of the type of food, as compared to the sleep condition. Self-reported hunger was also enhanced (p

Hepatic steatosis is recognized as hepatic presentation of the metabolic syndrome. Hyperinsulinaemia, which shifts fatty acid oxidation to de novo lipogenesis and lipid storage in the liver, appears to be a principal elicitor particularly in the early stages of disease development. The impact of PGE₂, which has previously been shown to attenuate insulin signaling and hence might reduce insulin-dependent lipid accumulation, on insulin-induced steatosis of hepatocytes was studied. The PGE₂-generating capacity was enhanced in various obese mouse models by the induction of cyclooxygenase 2 and microsomal prostaglandin E-synthases (mPGES1, mPGES2). PGE₂ attenuated the insulin-dependent induction of SREBP-1c and its target genes glucokinase and fatty acid synthase. Nevertheless, PGE₂ enhanced incorporation of glucose into hepatic triglycerides synergistically with insulin. This was most likely due to a combination of a PGE₂-dependent repression of (1) the key lipolytic enzyme adipose triglyceride lipase, (2) carnitine-palmitoyltransferase 1, a key regulator of mitochondrial β-oxidation, and (3) microsomal transfer protein, as well as (4) apolipoprotein B, key components of the VLDL synthesis. Repression of PGC1α, a common upstream regulator of these genes, was identified as a possible cause. In support of this hypothesis, overexpression of PGC1α completely blunted the PGE₂-dependent fat accumulation. PGE₂ enhanced lipid accumulation synergistically with insulin, despite attenuating insulin signaling and might thus contribute to the development of hepatic steatosis. Induction of enzymes involved in PGE₂ synthesis in in vivo models of obesity imply a potential role of prostanoids in the development of NAFLD and NASH.

Mechanisms to simulate the formation of nitrous acid (HONO), an important OH radical source in the atmosphere, have been incorporated into numerical models. The results from the model simulations of HONO formation are compared with field measurements of HONO and conclusions are drawn regarding HONO formation mechanisms in the atmosphere and their representation in models.

Nob3 is a major obesity quantitative trait locus (QTL) identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6), and by introgression of its 38 Mbp peak region into B6 (B6.NZO-Nob3.38). B6.NZO-Nob3.38 mice carrying the NZO allele exhibited markedly increased body weight, fat mass, lean mass and a lower energy expenditure, than the corresponding B6 allele carriers. For positional cloning of the responsible obesity gene, five additional congenic lines (RCS) were generated and characterized, allowing to define a critical genomic interval comprising 43 genes. mRNA profiling and western blotting indicated that Ifi202b, a member of the Ifi200 family of interferon inducible transcriptional modulators, was expressed in NZO-allele carriers but was undetectable in tissues of homozygous B6-allele carriers due to a microdeletion, including the first exon and the 5'-flanking region of Ifi202b in B6. Transcriptome analysis of adipose tissue of RCS revealed a marked induction of 11β-hydroxysteroid dehydrogenase type 1 (11β-Hsd1) expression in mice expressing Ifi202b. Furthermore, siRNA-mediated Ifi202b suppression in 3T3-L1 adipocytes resulted in a significant inhibition of 11β-Hsd1 expression, whereas an adenoviral-mediated overexpression of Ifi202b increased 11β-Hsd1 mRNA levels. Expression of human IFI orthologues was significantly increased in visceral adipose tissue of obese subjects. We suggest that the disruption of Ifi202b in B6 is responsible for the effects of the obesity QTL Nob3, and that Ifi202b modulates fat accumulation through expression of adipogenic genes such as 11β-Hsd1.

COSMO-ART is a recently developed regional model that couples meteorological and air quality processes online. It allows for a consistent chemical forecasting since it avoids temporal and spatial interpolation steps. Additionally, it enables the simulation of feedbacks between chemistry, aerosols and meteorology. Towards this direction, COSMO-ART has already incorporated the direct radiative forcing of aerosol processes. In the framework of this study, COSMO-ART is modified to include the volatility basis set (VBS) treatment of secondary organic aerosol (SOA) chemistry. The VBS approach assumes gas-to-particle partitioning of all (thousands) organics grouped by their saturation concentration and calculates their formation and chemical evolution (aging) into the atmosphere. COSMO-ART simulations are performed for May 2008 covering the greater European area with a horizontal resolution of 14 km and a vertical extend up to 20 km. Results are compared to PMCAMx predictions and are evaluated against EUCAARI measurements. Sensitivity simulations reveal the effectiveness of the VBS approach against a traditional SOA module and the fraction of anthropogenic SOA. Additional simulations excluding organic chemistry, aim at identifying the SOA effect on radiation and atmospheric temperature. An average radiative reduction is predicted over Europe, linked to a moderate temperature decrease.

Hintergrund: Die Pathophysiologie des Typ-2-Diabetes schliest eine progressive Einschrankung und im spateren Verlauf den Untergang von Insulin-produzierenden β-Zellen ein, wenn eine genetische Pradisposition vorliegt. Die NZO (New Zealand Obese)-Maus ist ein Modell, das neben Adipositas und Insulinresistenz auch einen Typ-2-Diabetes mit β-Zellverlust entwickelt. Mittels Kopplungsanalysen wurden mehr als 15 Quantitative Trait Loci (QTL) fur das Metabolische Syndrom und Hyperglykamie auf verschiedenen Chromosomen lokalisiert. Zur Identifizierung von Diabetesgenen sollten die Transkripte der Langerhans-Inseln der NZO mit denen der diabetesresistenten B6.V-Lepob-(ob/ob) Maus verglichen und die Kandidaten auf die QTL-Karten projiziert werden. Methoden: Die RNA aus Langerhans-Inseln von je sechs ob/ob- und NZO-Mausen wurde isoliert und zur Microarray-basierten Expressionsanalyse verwendet. Die Ergebnisse wurden mit den QTL-Analysen abgeglichen. Ein Kandidatengen (Synonym: Diab1) sowie sein putativer Promotor wurden vollstandig in beiden Mauslinien sequenziert, die SNP-Positionen in silico auf Veranderungen der Transkriptionsfaktor (TF)-Bindungsstellen untersucht und die Diab1-Expression mittels quantitativer RT-PCR untersucht. Ergebnisse:Diab1 ist auf Mauschromosom 1 im QTL Nob3 lokalisiert, der mit einem erhohten Korpergewicht und einer Hyperglykamie assoziiert ist. Durch quantitative RT-PCR wurde bestatigt, dass Diab1 in den Langerhans-Inseln der ob/ob-Maus exprimiert wird, wahrend die NZO-Inseln fast keine Expression aufzeigen. Sequenzanalysen des Diab1-Gens identifizierten 10 SNPs (Single Nucleotide Polymorphisms) in Exons, von denen 4 zu einem Aminosaureaustausch fuhren, 30 intronische SNPs und 8 SNPs im UTR sowie 36 SNPs im konservierten putativen Promotor der NZO-Maus. Die in silico-Analyse der TF-Bindungsstellen ergab, dass 14 putative TF-Bindungsstellen nur im ob/ob-Genom liegen (z.B. fur den Peroxisome Proliferator-Activated Receptor) und 9mogliche TF-Bindungsstellen NZO-spezifisch sind (z.B. fur E-Box Binding Factors). Schlussfolgerung: Durch den Abgleich von Expressionsprofilen der Langerhans-Inseln mit Ergebnissen aus Kopplungsanalysen (QTL) wurde ein neues Diabetesgen identifiziert, das im Diabetes-sensitiven NZO-Stamm sowohl im Promotor als auch im kodierenden Bereich zahlreiche Polymorphismen aufweist. Weiterfuhrende Untersuchungen zur Rolle von Diab1 fur die Langerhans-Inseln mussen sich anschliesen.

Significant amounts of mineral dust were transported into the vicinity of the developing tropical storm Helene between 9 to 14 September 2006. A number of different dynamical features contributed to the emission and the transport of dust towards Helene. The aim of this study is to investigate the transport of mineral dust towards Hurricane Helene. A particular focus is placed on the role of the dust radiation feedback. We use the model system COSMO-ART (Aerosols and Reactive Trace gases) in which the dust emission and dust transport as well as the radiation feedback are implemented. With the help of COSMO-ART we analyse the radiative effect of the dust on the convective systems and their environment. Model runs with a horizontal resolution of 28 km were conducted with and without the radiation feedback of the dust. Based on these model runs, we were able to distinguish between dry, dry and dusty, as well as dusty and moist air. The transport of mineral dust is analysed by calculating trajectories. We aim to assess how the dust influences the environment of the developing tropical storm Helene.

Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia ( Donor PBMC chimerisms at d+7, d+14 and d+28 were median 22% (range: 8-34%), 50% (range: 29-53%) and 67% (range: 47-73%) in IBM10. The results of PBMC chimerism for IBM60 were 11% (range: 5-34%), 42% (range: 20-42%) and 59% (range: 44-66%) at these time points (p = n.s.). Donor granulocyte chimerisms of median 33% (range: 11-83%), 100% (range: 58-100%) and 100% (range: 82-100%) were detected at d+7, d+14 and d+28 after HSCT in IBM10, respectively. The granulocyte chimerism in IBM60 amounted to 34% (range: 3-87%), 96% (range: 94-100%) and 98% (range: 96-100%) at the above mentioned time points p=n.s. for all time points). Conclusion: Our data suggest that early granulocyte and PBMC engraftment is not influenced by modification of the HSC infusion rate. However, the period of thrombocytopenia seems to be prolonged following a 60 minutes application. Therefore, longer infusion times in an IBMT setting seem not to be beneficial following toxicity reduced conditioning regimen. Disclosures No relevant conflicts of interest to declare.

Everolimus (RAD001) is an mTOR inhibitor that has been successfully used as an immunosuppressant in solid-organ transplantation. Data in allogeneic hematopoietic stem cell transplantation (HSCT) is limited. This study aimed to investigate pharmacokinetics, safety, and efficacy of RAD001 in a canine allogeneic HSCT model. First, pharmacokinetics of RAD001 were performed in healthy dogs in order to determine the appropriate dosing. Doses of 0.25 mg RAD001 twice daily in combination with 15 mg/kg cyclosporin A (CsA) twice daily were identified as appropriate starting doses to achieve the targeted range of RAD001 (3-8 μg/L) when orally administered. Subsequently, 10 dogs were transplanted using 2 Gy total body irradiation (TBI) for conditioning and 0.25 mg RAD001 twice daily plus 15 mg/kg CsA twice daily for pre- and posttransplantation immunosuppression. Seven of the 10 transplanted dogs were maintained at the starting RAD001 dose throughout the study. For the remaining 3 dogs, dose adjustments were necessary. RAD001 accumulation over time did not occur. All dogs initially engrafted. Five dogs eventually rejected the graft (weeks 10, 10, 13, 27, and 56). Two dogs died of pneumonia (weeks 8 and 72) but were chimeric until then. Total cholesterol rose from median 4.1 mmol/L (3.5-5.7 mmol/L) before HSCT to 6.0 mmol/l (5.0-8.5 mmol/l) at day 21 after HSCT, but remained always within normal range. Changes in creatinine and triglyceride values were not observed. Long-term engraftment rates were inferior to sirolimus/CsA and mycophenolate mofetil (MMF)/CsA regimen, respectively. RAD001/CsA caused a more pronounced reduction of platelet counts to median 2 × 10(9)/L (range: 0-21 × 10(9)/L) and longer time to platelet recovery of 21 days (range: 14-24 days) compared with MMF/CsA. CsA c(2h) levels were significantly enhanced in the RAD001/CsA regimen, but c(0h) and area under the curve from 0 to 12 hours (AUC(0-12h)) values did not differ compared with an MMF/CsA immunosuppression. In summary, immunosuppression consisting of RAD001 and CsA is well tolerated but not as efficient as with other established immunosuppressants in a canine nonmyeloablative HSCT regimen. Hence, our study does not support the application of RAD001/CsA as standard practice in this setting.