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Stratified flows and the resulting density-driven currents occur in the natural environment and commonly in saline lakes. In the Great Salt Lake, Utah, USA, the northern and southern portions of the lake are divided by an east-to-west railroad causeway that disrupts natural lake currents and significantly increases salt concentrations in the northern section. To support management efforts focused on addressing rising environmental and economic concerns associated with varied saltwater densities throughout the lake, the causeway was recently modified to include a new breach. The purpose of this new breach is to enhance salt exchange between the northern and southern sections of the lake. Since construction, it typically exhibits a strong density-driven bidirectional flow pattern, but estimating flows and salt exchange has proven to be difficult. To obtain much needed insights into the ability of this hydraulic structure to exchange water and salt between the two sections of the lake, a field campaign coupled with CFD modeling was undertaken. Results from this study indicate that the vertical velocity profile in the breach is sensitive to density differences between flow layers along with breach geometry and water surface elevations. The CFD model was able to accurately represent the bidirectional flows through the breach and provides for improved estimates of water and salt exchanges between the north and south sections of the lake.

The focus of this study is to describe the hydraulic effects of stormwater discharge, thus sediment transport occurring as a result of increased discharge from a stormwater detention pond, based on measurements made in a small high-slope Danish stream. In order to extrapolate the findings and predict the result of larger discharge flow rates from the detention pond in this study, 11 traditional threshold equations were tested, and results were compared to the sediment transport experiment with five formulas predicting the threshold based on shear stress and six based on stream power. The sediment transport experiment was constructed as a staircase pattern, step-wise increasing the discharge. During the experiment, measurements of sediment transport in the stream were made in two stations downstream from the point of discharge. Results from those measurements showed that there was no notable correlation between suspended sediment transport and bed sediment transport, and that suspended transport peaked during the periods of low flow conditions. Bed sediment transport peaked before the maximum flow, indicating that the available sediment for transport is a limiting factor. When comparing the calculated threshold of the collected sediment particle sizes to the shear stress and stream power calculated during the experiment, all 11 tested formulas overestimated the sediment transport and particle size moved by a specific flow. This result is in correspondence with results found in other experiments, and here the expected explanation is that the form roughness of the stream bed makes less energy available for sediment transport. This implies that the hydraulic impact from discharge of stormwater into small streams has to be evaluated on a case-by-case basis, rather than relying on general threshold sediment transport models.

Infiltration excess runoff, i.e., runoff as a result of the rainfall intensity exceeding the infiltration capacity of the soil surface, has traditionally been considered the only contributor to the surface runoff from urban pervious areas. However, recent studies show that subsurface throughflow also can be a significant contributor to urban stormwater runoff. Although rainfall-runoff from urban pervious areas can contribute with large quantities of runoff, only little knowledge exists on this topic. In this study, experimental field observations of subsurface throughflow from the literature are used to assess the capability of different models to simulate this type of runoff. It is investigated how well three new modeling approaches in urban drainage engineering (linear reservoir, regression, and shallow neural network models) performs in simulating subsurface throughflow compared to two commonly used models (the time-area and kinematic wave model). The models are compared with the measured runoff rate and evaluated by the root mean square error (RMSE), Nash-Sutcliffe efficiency (NSE), and Bayesian likelihood (L). Generally, a neural network containing 60 neurons and using up to 180 min of data back in time produces the best results (RMSE=0.59 Lmin-1, NSE=0.91, and L=0.92). However, both the kinematic wave (RMSE=1.06 L min-1, NSE=0.71, and L=0.76) and linear reservoir model (RMSE=0.98 L min-1, NSE=0.75, and L=0.78) generate reasonable results despite their significantly simpler modeling approaches.

Rainfall simulators can enhance our understanding of the hydrologic processes affecting the total runoff to urban drainage systems. This knowledge can be used to improve urban drainage designs. In this study, a rainfall simulator is developed to simulate rainfall on urban green surfaces. The rainfall simulator is controlled by a microcomputer programmed to replicate the temporal variations in rainfall intensity of both historical and synthetic rainfall events with constant rainfall intensity on an area of 1 m². The performance of the rainfall simulator is tested under laboratory conditions with regard to spatial uniformity of the rainfall, the kinetic energy of the raindrops, and the ability to replicate historical and synthetic rainfall events with temporally varying intensity. The rainfall simulator is applied in the field to evaluate its functionality under field conditions and the influence of wind on simulated rainfall. Finally, a field study is carried out on the relationship between runoff, soil volumetric water content, and surface slope. Performance and field tests show that the simulated rainfall has a uniform spatial distribution, whereas the kinetic energy of the raindrops is slightly higher than that of other comparable rainfall simulators. The rainfall simulator performs best in low wind speed conditions. The simulator performs well in replicating historical and synthetic rainfall events by matching both intensity variations and accumulated rainfall depth. The field study shows good correlation between rainfall, runoff, infiltration, soil water content, and surface slope.

Intense rainfall in urban areas can often generate severe flood impacts. Consequently, it is crucial to design systems to minimize potential flood damages. Traditional, simple design of urban drainage systems assumes agreement between rainfall return period and its consequent flood return period; however, this does not always apply. Hydraulic infrastructures found in urban drainage systems can increase system heterogeneity and perturb the impact of severe rainfall response. In this study, a surface flood return period assessment was carried out at Lystrup (Denmark), which has received the impact of flooding in recent years. A 35 years' rainfall dataset together with a coupled 1D/2D surface and network model was used to analyse and assess flood return period response. Results show an ambiguous relation between rainfall and flood return periods indicating that linear rainfall–runoff relationships will, for the analysed case study, be insufficient for flood estimation. Simulation-based mapping of return periods for flood area and volume has been suggested, and moreover, a novel approach has been developed to map local flood response time and relate this to rainfall characteristics. This approach allows to carefully analyse rainfall impacts and flooding response for a correct flood return period assessment in urban areas.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the causative agent of one of the most important porcine diseases with a high impact on animal health, welfare, and production economy. PRRSV exhibits a multitude of immunoevasive strategies that, in combination with a very high mutation rate, has hampered the development of safe and broadly protective vaccines. Aiming at a vaccine inducing an effective cytotoxic T cell response, a bioinformatics approach was taken to identify conserved PRRSV-derived peptides predicted to react broadly with common swine leukocyte antigen (SLA) class I alleles. Briefly, all possible 9- and 10-mer peptides were generated from 104 complete PRRSV type 2 genomes of confirmed high quality, and peptides with high binding affinity to five common SLAs were identified combining the NetMHCpan and positional scanning combinatorial peptide libraries binding predictions. Predicted binders were prioritized according to genomic conservation and SLA coverage using the PopCover algorithm. From this, 53 peptides were acquired for further analysis. Binding affinity and stability of a subset of 101 peptide-SLA combinations were validated in vitro for 4 of the 5 SLAs. Eventually, 23% of the predicted peptide-SLA combinations showed to form complexes with a dissociation half-life ≥30 min. Additionally, combining the two prediction methods proved to be more robust across alleles than either method used alone in terms of predicted-to-observed correlations. In summary, our approach represents a finely tuned epitope prediction pipeline providing a rationally selected ensemble of peptides for future in vivo experiments with pigs expressing the included SLAs. Fil: Welner, Simon. Technical University of Denmark; Dinamarca Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Jungersen, Gregers. Technical University of Denmark; Dinamarca Fil: Larsen, Lars Erik. Technical University of Denmark; Dinamarca

Rainfall-runoff-generating mechanisms in urban green areas are scarcely understood, and limited knowledge and data on rainfall-runoff processes are available. Therefore, a large-scale experimental field station was established to investigate the inherent hydrological processes of a grass-covered 4,300 m2 urban catchment consisting of sandy loam soil. A facility to collect surface runoff from the area was designed. Runoff, soil moisture properties, and rainfall were measured simultaneously by a flow meter, in-ground soil sensors, and rain gauges, respectively. Measured soil volumetric water content was above 0.34 m3 H2O m−3 soil during fall and winter and ranging between 0.13 and 0.34 m3 H2O m−3 soil during late spring and summer. Measured runoff recorded from September 2016 until July 2018 strongly indicates that subsurface throughflow was the dominant runoff type. There was good correlation between the dynamics of soil water content and runoff. Accumulated rainfall and runoff was linearly correlated for soil volumetric water contents above 0.34 m3 H2O m−3 soil. The relationship between runoff and rainfall shows a runoff coefficient of 0.18 for the 4,300 m2 area.

Nutrition has proven to be of great importance for the postoperative clinical outcome. Several studies have shown that infectious complications in the surgical patient , are reduced by pre- or postoperative nourishment. We discuss cellular immunity in relation to both enteral and parenteral nutrition and present an updated literature study of current evidence. The aim of this paper is to give an overview of studies, that compare different immunological parameters in the surgical patient being nourished either enterally or parenterally.

Hydraulic conductivity (Ks) and effective porosity (ϕeff) for saturated water flow are essential hydraulic properties for describing fluid and chemical transport in soil and groundwater systems. Typically, Ks is predicted by pedotransfer functions of soil texture and total porosity or ϕeff. This study shows that a more conceptual approach that uses a volume-weighted ratio of coarser (part of the sand fraction) to finer (clay and organic matter) particles alongside total porosity could explain variations in both Ks and φeff in intact 100-cm3 samples of 20 sandy surface and subsurface soils with

Affinity and stability of peptides bound by major histocompatibility complex (MHC) class I molecules are important factors in presentation of peptides to cytotoxic T lymphocytes (CTLs). In silico prediction methods of peptide-MHC binding followed by experimental analysis of peptide-MHC interactions constitute an attractive protocol to select target peptides from the vast pool of viral proteome peptides. We have earlier reported the peptide binding motif of the porcine MHC-I molecules SLA-1*04:01 and SLA-2*04:01, identified by an ELISA affinity-based positional scanning combinatorial peptide library (PSCPL) approach. Here, we report the peptide binding motif of SLA-3*04:01 and combine two prediction methods and analysis of both peptide binding affinity and stability of peptide-MHC complexes to improve rational peptide selection. Using a peptide prediction strategy combining PSCPL binding matrices and in silico prediction algorithms (NetMHCpan), peptide ligands from a repository of 8900 peptides were predicted for binding to SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01 and validated by affinity and stability assays. From the pool of predicted peptides for SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01, a total of 71, 28, and 38 % were binders with affinities below 500 nM, respectively. Comparison of peptide-SLA binding affinity and complex stability showed that peptides of high affinity generally, but not always, produce complexes of high stability. In conclusion, we demonstrate how state-of-the-art prediction and in vitro immunology tools in combination can be used for accurate selection of peptides for MHC class I binding, hence providing an expansion of the field of peptide-MHC analysis also to include pigs as a livestock experimental model. Fil: Pedersen, Lasse Eggers. Technical University of Denmark; Dinamarca Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Harndahl, Mikkel. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Jungersen, Gregers. Technical University of Denmark; Dinamarca

Data for adjustment of weather radar rainfall estimations are mostly obtained from rain gauge observations. However, the density of rain gauges is often very low. Yet in many urban catchments, runoff sensors are typically available which can measure the rainfall indirectly. By utilising these sensors, it may be possible to improve the ground rainfall estimate, and thereby improve the quantitative precipitation estimation from weather radars for urban drainage applications. To test the hypothesis, this paper presents a rainfall measurement method based on flow rate measurements from well-defined urban surfaces. This principle was used to design a runoff measurement system in a parking structure in Aalborg, Denmark, where it was evaluated against rain gauges. The measurements show that runoff measurements from well-defined urban surfaces perform just as well as rain gauges. This opens up the possibility of improving the ground rainfall estimate by the use of flow rate measurements.

Weather radar data used for urban drainage applications are traditionally adjusted to point ground references, e.g., rain gauges. However, the available rain gauge density for the adjustment is often low, which may lead to significant representativeness errors. Yet, in many urban catchments, rainfall is often measured indirectly through runoff sensors. This paper presents a method for weather radar adjustment on the basis of runoff observations (Z-Q adjustment) as an alternative to the traditional Z-R adjustment on the basis of rain gauges. Data from a new monitoring station in Aalborg, Denmark, were used to evaluate the flow-based weather radar adjustment method against the traditional rain-gauge adjustment. The evaluation was performed by comparing radar-modeled runoff to observed runoff. The methodology was both tested on an events basis and multiple events combined. The results indicate that this Z-Q adjustment method performs similarly to Z-R adjustment on the basis of well-placed rain gauges. This opens up the possibility of using flow measurements as an alternative to or in combination with rain gauges.

Outbreaks of Yellow Fever occur regularly in endemic areas of Africa and South America frequently leading to mass vaccination campaigns straining the availability of the attenuated Yellow Fever vaccine, YF-17D. The WHO has recently decided to discontinue regular booster-vaccinations since a single vaccination is deemed to confer life-long immune protection. Here, we have examined humoral (neutralizing antibody) and cellular (CD8 and CD4 T cell) immune responses in primary and booster vaccinees (the latter spanning 8 to 36 years after primary vaccination). After primary vaccination, we observed strong cellular immune responses with T cell activation peaking ≈2 weeks and subsiding to background levels ≈ 4 weeks post-vaccination. The number of antigen-specific CD8+ T cells declined over the following years. In >90% of vaccinees, in vitro expandable T cells could still be detected >10 years post-vaccination. Although most vaccinees responded to a booster vaccination, both the humoral and cellular immune responses observed following booster vaccination were strikingly reduced compared to primary responses. This suggests that pre-existing immunity efficiently controls booster inoculums of YF-17D. In a situation with epidemic outbreaks, one could argue that a more efficient use of a limited supply of the vaccine would be to focus on primary vaccinations.

The present study introduces a nowcast scheme that assimilates radar extrapolation data (RED) into a nowcasting version of the high resolution limited area model (HIRLAM) numerical weather prediction (NWP) model covering the area of Denmark. The RED are based on the Co-TREC (tracking radar echoes by correlation) methodology and are generated from cleaned radar mosaics from the Danish weather radar network. The assimilation technique is a newly developed method that increases model precipitation by increasing low-level convergence and decreasing convergence aloft in order to increase the vertical velocity. A selection criterion based on spatial coverage and temporal duration is applied to identify rain events for evaluation. Eight events from August 2010 comply with the criteria and additionally, an extreme event on 2 July 2011 is also evaluated. The RED assimilated NWP model is verified against a non-assimilated NWP reference run using the fractional skill score. In all cases, the predictability of the RED assimilated NWP model has been increased compared with the non-RED assimilated NWP model. After ending the RED assimilation, the positive effects continue until the skill converges towards the reference run. The level of improved predictability relies on the RED quality, which again relies on the type of event.

We investigate the application of rainfall observations and forecasts from rain gauges and weather radar as input to operational urban runoff forecasting models. We apply lumped rainfall runoff models implemented in a stochastic grey-box modelling framework. Different model structures are considered that account for the spatial distribution of rainfall in different degrees of detail.Considering two urban example catchments, we show that statically adjusted radar rainfall input improves the quality of probabilistic runoff forecasts as compared to input based on rain gauge observations, although the characteristics of these radar measurements are rather different from those on the ground. Data driven runoff forecasting models can to some extent adapt to bias of the rainfall input by model parameter calibration and state-updating. More detailed structures in these models provide improved runoff forecasts compared to the structures considering mean areal rainfall only.A time-dynamic adjustment of the radar data to rain gauge data provides improved rainfall forecasts when compared with rainfall observations on the ground. However, dynamic adjustment reduces the potential for creating runoff forecasts and in fact also leads to reduced cross correlation between radar rainfall and runoff measurements. We conclude that evaluating the performance of radar rainfall adjustment against rain gauges may not always be adequate and that adjustment procedure and online runoff forecasting should ideally be considered as one unit.

The topic of this paper is temporal interpolation of precipitation observed by weather radars. Precipitation measurements with high spatial and temporal resolution are, in general, desired for urban drainage applications. An advection-based interpolation method is developed which uses methods for vector field estimation already known from short-term weather radar nowcasting. However, instead of forecasting the weather radar rainfall, the proposed interpolation method exploits the advection of the rainfall in the interpolation. The interpolated rainfall fields are validated by measurements at ground level from laser disdrometers. The proposed interpolation method performs better when compared to traditional interpolation of weather radar rainfall where the radar observation is considered constant in time between measurements. It is demonstrated that the advection-based interpolation method reconstructs a significant part of the temporal variations of the rainfall. Moreover, the interpolated precipitation fields estimate accumulated rain depths more accurately.

Summary It is generally acknowledged that in order to apply radar rainfall data for hydrological proposes adjustment against ground observations are crucial. Traditionally, radar reflectivity is transformed into rainfall rates applying a fixed reflectivity – rainfall rate relationship even though this is known to depend on the changing drop size distribution of the specific rain. This creates a transient bias between the radar rainfall and the ground observations due to seasonal changes of the drop size distribution as well as other atmospheric effects and effects related to the radar observational technology. In this study different bias adjustment techniques is investigated, developing a complete 10-year dataset (2002–2012) of high spatio-temporal resolution radar rainfall based on a radar observations from a single C-band radar from Denmark. Results show that hourly adjustment mean field bias adjustment outperform daily mean field bias adjustment with regards to estimation of rainfall totals and peak rain rates. Furthermore, it is demonstrated that radar rainfall estimates can be improved significantly by implementation of a novel advection interpolation technique.

Merging of radar rainfall data with rain gauge measurements is a common approach to overcome problems in deriving rain intensities from radar measurements. We extend an existing approach for adjustment of C-band radar data using state-space models and use the resulting rainfall intensities as input for forecasting outflow from two catchments in the Copenhagen area. Stochastic greybox models are applied to create the runoff forecasts, providing us with not only a point forecast but also a quantification of the forecast uncertainty. Evaluating the results, we can show that using the adjusted radar data improves runoff forecasts compared to using the original radar data and that rain gauge measurements as forecast input are also outperformed. Combining the data merging approach with short term rainfall forecasting algorithms may result in further improved runoff forecasts that can be used in real time control. Merging of radar rainfall data with rain gauge measurements is a common approach to overcome problems in deriving rain intensities from radar measurements. We extend an existing approach for adjustment of C-band radar data using state-space models and use the resulting rainfall intensities as input for forecasting outflow from two catchments in the Copenhagen area. Stochastic greybox models are applied to create the runoff forecasts, providing us with not only a point forecast but also a quantification of the forecast uncertainty. Evaluating the results, we can show that using the adjusted radar data improves runoff forecasts compared to using the original radar data and that rain gauge measurements as forecast input are also outperformed. Combining the data merging approach with short term rainfall forecasting algorithms may result in further improved runoff forecasts that can be used in real time control.

Binding of peptides to MHC class I (MHC-I) molecules is the most selective event in the processing and presentation of Ags to CTL, and insights into the mechanisms that govern peptide-MHC-I binding should facilitate our understanding of CTL biology. Peptide-MHC-I interactions have traditionally been quantified by the strength of the interaction, that is, the binding affinity, yet it has been shown that the stability of the peptide-MHC-I complex is a better correlate of immunogenicity compared with binding affinity. In this study, we have experimentally analyzed peptide-MHC-I complex stability of a large panel of human MHC-I allotypes and generated a body of data sufficient to develop a neural network-based pan-specific predictor of peptide-MHC-I complex stability. Integrating the neural network predictors of peptide-MHC-I complex stability with state-of-the-art predictors of peptide-MHC-I binding is shown to significantly improve the prediction of CTL epitopes. The method is publicly available at http://www.cbs.dtu.dk/services/NetMHCstabpan. Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Fenoy, Luis Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Harndahl, Mikkel. Universidad de Copenhagen; Dinamarca Fil: Kristensen, Anne Bregnballe. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Ida Kallehauge. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Copenhagen; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested., Author Summary Live attenuated yellow fever vaccine (YF-17D) is an efficient and generally safe vaccine. Nevertheless, in recent years the reporting of serious adverse effects together with the given limitations in the use of this live vaccine in certain risk groups has spurred an interest in developing a more generally applicable and safer alternative. Using an adenovector platform and recombinant vaccines targeting both structural and non-structural YF antigens, we now demonstrate that non-replicating adenobased vaccines may be used to induce a state of host immunity, which like YF-17D vaccination encompasses both major arms of the adaptive immune system. Furthermore, in a murine challenge model, adenovector induced protection fully matched that induced by the current vaccine. Taken together our results strongly suggest that adenovectored vaccines targeting structural and non-structural viral antigens represent a viable and safe alternative to the existing live, attenuated YF vaccine.

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and-II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A02:01 and HLA-DRB101:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A02:01/HLADRB101:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development. Fil: Pedersen, Sara Ram. Universidad de Copenhagen; Dinamarca Fil: Christensen, Jan Pravsgaard. Universidad de Copenhagen; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Korsholm, Karen Smith. Statens Serum Institute; Dinamarca Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Claesson, Mogens Helweg. Universidad de Copenhagen; Dinamarca

Minor histocompatibility antigens (mHags) encoded by the Y-chromosome (H-Y-mHags) are known to play a pivotal role in allogeneic haematopoietic cell transplantation (HCT) involving female donors and male recipients. We present a new H-Y-mHag, YYNAFHWAI (UTY139–147), encoded by the UTY gene and presented by HLA-A*24:02. Briefly, short peptide stretches encompassing multiple putative H-Y-mHags were designed using a bioinformatics predictor of peptide-HLA binding, NetMHCpan. These peptides were used to screen for peptide-specific HLA-restricted T cell responses in peripheral blood mononuclear cells obtained post-HCT from male recipients of female donor grafts. In one of these recipients, a CD8+ T cell response was observed against a peptide stretch encoded by the UTY gene. Another bioinformatics tool, HLArestrictor, was used to identify the optimal peptide and HLA-restriction element. Using peptide/HLA tetramers, the specificity of the CD8+ T cell response was successfully validated as being HLA-A*24:02-restricted and directed against the male UTY139–147 peptide. Functional analysis of these T cells demonstrated male UTY139–147 peptide-specific cytokine secretion (IFNγ, TNFα and MIP-1β) and cytotoxic degranulation (CD107a). In contrast, no responses were seen when the T cells were stimulated with patient tumour cells alone. CD8+ T cells specific for this new H-Y-mHag were found in three of five HLA-A*24:02-positive male recipients of female donor HCT grafts available for this study.

Efficient presentation of peptide-MHC class I (pMHC-I) complexes to immune T cells should benefit from a stable peptide-MHC-I interaction. However, it has been difficult to distinguish stability from other requirements for MHC-I binding, for example, affinity. We have recently established a high-throughput assay for pMHC-I stability. Here, we have generated a large database containing stability measurements of pMHC-I complexes, and re-examined a previously reported unbiased analysis of the relative contributions of antigen processing and presentation in defining cytotoxic T lymphocyte (CTL) immunogenicity [Assarsson et al., J. Immunol. 2007. 178: 7890–7901]. Using an affinity-balanced approach, we demonstrated that immunogenic peptides tend to be more stably bound to MHC-I molecules compared with nonimmunogenic peptides. We also developed a bioinformatics method to predict pMHC-I stability, which suggested that 30% of the nonimmunogenic binders hitherto classified as “holes in the T-cell repertoire” can be explained as being unstably bound to MHC-I. Finally, we suggest that nonoptimal anchor residues in position 2 of the peptide are particularly prone to cause unstable interactions with MHC-I. We conclude that the availability of accurate predictors of pMHC-I stability might be helpful in the elucidation of MHC-I restricted antigen presentation, and might be instrumental in future search strategies for MHC-I epitopes.

Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal requirements. To avoid labeling of the highly variable peptide, we labeled the invariant β2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually label-free assay, we showed that rates of peptide-MHC class I dissociation measured in this assay correlated well with rates of dissociation rates measured conventionally with labeled peptides. This assay can be used to measure the stability of any peptide-MHC Class I combination, it is reproducible and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed rates of dissociation that ranged from 0.1 hours to 46 hours depending on the peptide used.

A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable peptide-HLA-I complex formation required for long term presentation. Tapasin is thought to be central to this essential quality control, but the underlying mechanisms remain unknown. Here, we report that the N-terminal region of tapasin, Tpn(1-87), assisted folding of peptide-HLA-A*02:01 complexes according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database and one consisting of medium to high affinity non-SYFPEITHI ligands, were studied in the context of HLA-A*02:01 binding and stability. We show that the SYFPEITHI peptides induced more stable HLA-A*02:01 molecules than the other ligands, although affinities were similar. Remarkably, Tpn(1-87) could functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I- and peptide-specific function, together with the functions exerted by the more C-terminal parts of tapasin, are major features of tapasin-mediated HLA-I quality control. These findings are important for understanding the biogenesis of HLA-I molecules, the selection of presented T-cell epitopes, and the identification of immunogenic targets in both basic research and vaccine design.

Immunotherapy has increased overall survival of metastatic cancer patients, and cancer antigens are promising vaccine targets. To fulfill the promise, appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice, and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart, we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC, both important in human cancer development and progression, were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09, CASAC, Montanide ISA 51 VG, or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01, -1(*)07:02, -2(*)04:01, -2(*)05:02, and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer.

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4+ T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1. Fil: Andreatta, Massimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina Fil: Karosiene, Edita. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Stryhn, Anette. Universidad de Copenhagen; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); Argentina. Technical University of Denmark; Dinamarca

Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network-based predictions (NetMHCpan). The updated NetMHCpan server was used to predict BoLA-I binding peptides within the P1 structural polyprotein sequence of FMDV (strain A24 Cruzeiro) for BoLA-1*01901, BoLA-2*00801, BoLA-2*01201, and BoLA-4*02401. Peptide binding affinity and stability were determined for these BoLA-I molecules using the luminescent oxygen channeling immunoassay (LOCI) and scintillation proximity assay (SPA). The functional diversity of known BoLA alleles was predicted using the MHCcluster tool, and functional predictions for peptide motifs were compared to observed data from this and prior studies. The results of these analyses showed that BoLA alleles cluster into three distinct groups with the potential to define “BoLA supertypes.” This streamlined approach identifies potential T cell epitopes from pathogens, such as FMDV, and provides insight into T cell immunity following infection or vaccination. Fil: Pandya, Mital. University of Vermont; Estados Unidos Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Hansen, Andreas Martin. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Golde, William T.. United States Department of Agriculture; Estados Unidos Fil: Barlow, John. University of Vermont; Estados Unidos

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4+ T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8+ T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8+ T cells from these animals revealed a pivotal role for CD8+ T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8+ T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8+ T cells have been demonstrated to possess antiviral activity in vivo.

The Generalized Likelihood Uncertainty Estimation methodology (GLUE) is investigated for radar rainfall calibration and uncertainty assessment. The method is used to calibrate radar data collected by a Local Area Weather Radar (LAWR).In contrast to other LAWR data calibrations, the method combines calibration with uncertainty estimation. Instead of searching for a single set of calibration parameters, the method uses the observations to construct distributions of the calibration parameters. These parameter sets provide valuable knowledge of parameter sensitivity and the uncertainty.Two approaches are analyzed; the static calibration approach, where the LAWR is calibrated once for a long period and the dynamic approach, where the estimate is continuously adjusted based on ground observations.The analysis illustrates that the static calibration performs insufficiently, whereas the dynamic adjustment improves the performance significantly.It is found that even if the dynamic adjustment method is used the uncertainty of rainfall estimates can still be significant.

The overall objective of this paper is to contribute to the standing debate concerning the advantages of separate sewer systems compared to traditional combined sewer systems. By a case study this investigation reveals that transformation of part of a town from being serviced with combined sewer systems to become serviced with separate sewer systems decreases the volumes of storm water and pollutants diverted to the waste water treatment plant and discharged as combined sewer overflow. This happens at the expense of an increase in volumes of storm water and pollutant loads diverted to local receiving waters when detention ponds are not built-in the new separate sewer systems. It is concluded that consequences can be fatal for receiving waters, if no retention of pollutants is integrated into the system.

The effect of stocking density upon the hydrodynamics of a circular tank, configured in a recirculation system, was investigated. Red drums Sciaenops ocellatus of approximately 140 g wet weight, were stocked at five rates varying from 0 to 12 kg m-3. The impact of the presence of fish upon tank hydrodynamics was established using in-tank-based Rhodamine WT fluorometry at a flow rate of 0.23 l s-1 (tank exchange rate of 1.9 h-1). With increasing numbers of animals, curvilinear relationships were observed for dispersion coefficients and tank mixing times. Stocking densities of 3, 6, 9 and 12 kg m-3 resulted in a 0.2-, 0.5-, 2.4-, and 3.2-fold decrease in mixing time relative to that observed for empty tanks (Pb0.001). Udgivelsesdato: APR 28 The effect of stocking density upon the hydrodynamics of a circular tank, configured in a recirculation system, was investigated. Red drums Sciaenops ocellatus of approximately 140 g wet weight, were stocked at five rates varying from 0 to 12 kg m-3. The impact of the presence of fish upon tank hydrodynamics was established using in-tank-based Rhodamine WT fluorometry at a flow rate of 0.23 l s-1 (tank exchange rate of 1.9 h-1). With increasing numbers of animals, curvilinear relationships were observed for dispersion coefficients and tank mixing times. Stocking densities of 3, 6, 9 and 12 kg m-3 resulted in a 0.2-, 0.5-, 2.4-, and 3.2-fold decrease in mixing time relative to that observed for empty tanks (Pb0.001).

The binding of peptides to classical major histocompatibility complex (MHC) class I proteins is the single most selective step in antigen presentation. However, the peptide-binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Here, we demonstrate how a combination of high-throughput assays using positional scanning combinatorial peptide libraries, peptide dissociation, and peptide-binding affinity binding measurements can be combined with bioinformatics to effectively characterize the functionality of BoLA-I molecules. Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight cases, stable and high affinity binding was confirmed. A set of peptides were tested for binding affinity to the eight BoLA proteins and used to refine the predictors of peptide–MHC binding NetMHC and NetMHCpan. The inclusion of BoLA-specific peptide-binding data led to a significant improvement in prediction accuracy for reported T. parva CTL epitopes. For reported CTL epitopes with weak or no predicted binding, these refined prediction methods suggested presence of nested minimal epitopes with high-predicted binding affinity. The enhanced affinity of the alternative peptides was in all cases confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery in cattle. Fil: Hansen, Andreas M.. Universidad de Copenhagen; Dinamarca Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Svitek,Nicholas. International Livestock Research Institute; Kenia Fil: Harndahl, Mikkel. Universidad de Copenhagen; Dinamarca Fil: Golde, William T.. United States Department of Agriculture. Agriculture Research Service.; Estados Unidos Fil: Barlow, John. University of Vermont; Estados Unidos Fil: Vishvanath, Nene. International Livestock Research Institute; Kenia Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Technical University Of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

The release of insulin hexamer (39 kD) and amyloglucosidase (AMG, 97 kD), entrapped in spherical Ca-alginate beads, was investigated. While the release of insulin could be described solely by diffusion this was not the case for the 1.6 (rm/rm) larger AMG protein, where rm is the Stokes-Einstein effective molecular radius. Because the alginate bead size was not constant during the release experiments, it was hypothesised that in addition to the diffusional mass transfer, a non-negligible convective flow of liquid in or out of the beads was present due to swelling or shrinkage, respectively. Although it should be expected that the effective diffusion coefficient of AMG is only slightly lower than that of insulin, the results show that the effective diffusions coefficient of AMG was found to be much smaller. In the case of AMG, it was shown that including bead size changes and the resulting convective flow in the numerical model, release could be described more accurately. The numerical model was able to describe the release characteristics from both shrinking, swelling, and non-swelling alginate beads. To evaluate the effect of bead swelling on the protein release rate, a swelling modulus and a release index was defined, describing the different effects on release of smaller and larger proteins.

MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide–HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C–specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8+ T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database. Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Harndahl, Mikkel. Universidad de Copenhagen; Dinamarca Fil: Stryhn, Anette. Universidad de Copenhagen; Dinamarca Fil: Boucherma, Rachid. Inserm; Francia. Groupe Hospitalier Cochin-Port-Royal; Francia Fil: Nielsen, Lise Lotte. Universidad de Copenhagen; Dinamarca Fil: Lemonnier, François A.. Inserm; Francia. Groupe Hospitalier Cochin-Port-Royal; Francia Fil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Technical University of Denmark; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca

Adductor canal catheters preserve quadriceps strength better than femoral nerve catheters and may facilitate postoperative ambulation following total knee arthroplasty. However, the effect of this newer technique on provider workload, if any, is unknown. We conducted a retrospective provider workload analysis comparing these two catheter techniques; all other aspects of the clinical pathway remained the same. The primary outcome was number of interventions recorded per patient postopera- tively. Secondary outcomes included infusion duration, ambulation distance, opioid consumption, and hospital length of stay. Adductor canal patients required a median (10-90th percentiles) of 0.0 (0.0-2.6) interventions com- pared to 1.0 (0.3-3.0) interventions for femoral patients (p \ 0.001); 18/23 adductor canal patients (78 %) com- pared to 2/22 femoral patients (9 %) required no inter- ventions (p \ 0.001). Adductor canal catheter infusions lasted 2.0 (1.4-2.0) days compared to 1.5 (1.0-2.7) days in the femoral group (p = 0.016). Adductor canal patients ambulated further (mean (SD)) than femoral patients on postoperative day 1 (24.5 (21.7) vs. 11.9 (14.6) meters, respectively; p = 0.030) and day 2 (44.9 (26.3) vs. 22.0 (22.2) meters, respectively; p = 0.003). Postoperative opioid consumption and length of stay were similar between groups. We conclude that adductor canal catheters offer both patient and provider benefits when compared to femoral nerve catheters.

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy. Fil: Braendstrup, Peter. Universidad de Copenhagen; Dinamarca Fil: Mortensen, Bo Kok. Universidad de Copenhagen; Dinamarca Fil: Justesen, Sune. Universidad de Copenhagen; Dinamarca Fil: Østerby, Thomas. Universidad de Copenhagen; Dinamarca Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Hansen, Andreas Martin. Universidad de Copenhagen; Dinamarca Fil: Christiansen, Claus Bohn. Copenhagen University Hospital; Dinamarca Fil: Hansen, Morten Bagge. Copenhagen University Hospital; Dinamarca Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Vindeløv, Lars. Copenhagen University Hospital; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Stryhn, Anette. Universidad de Copenhagen; Dinamarca

Secondary settling tanks (SSTs) are the most hydraulically sensitive unit operations in biological wastewater treatment plants. The maximum permissible inflow to the plant depends on the efficiency of SSTs in separating and thickening the activated sludge. The flow conditions and solids distribution in SSTs can be predicted using computational fluid dynamics (CFD) tools. Despite extensive studies on the compression settling behaviour of activated sludge and the development of advanced settling velocity models for use in SST simulations, these models are not often used, due to the challenges associated with their calibration. In this study, we developed a new settling velocity model, including hindered, transient and compression settling, and showed that it can be calibrated to data from a simple, novel settling column experimental set-up using the Bayesian optimization method DREAM(ZS). In addition, correlations between the Herschel-Bulkley rheological model parameters and sludge concentration were identified with data from batch rheological experiments. A 2-D axisymmetric CFD model of a circular SST containing the new settling velocity and rheological model was validated with full-scale measurements. Finally, it was shown that the representation of compression settling in the CFD model can significantly influence the prediction of sludge distribution in the SSTs under dry- and wet-weather flow conditions.

The dynamic software updating system JRebel from Zeroturnaround has proven to be an efficient mean to improve developer productivity, as it allows developers to change the code of their applications while developing and testing them. Hence, developers no longer have to go through the tedious cycle of serializing application state, halting execution, redeploy the binary, restarting, and de-serializing state before they can test the effect of a code change. However, the current version of JRebel has its limits, as it does not support all kinds of code changes. In this paper, we compare the three most comprehensive dynamic updating systems developed for Java to date. Together, these systems provide comprehensive support for changing class definitions of live objects, including adding, removing and moving fields, methods, classes and interfaces anywhere in the inheritance hierarchy. We then investigate the effects of dynamic updating by performing a dynamic updating experiment on five consecutive revisions of the classical arcade game Breakout using the dynamic updating system Gosh! (Prior to the acquisition by zeroturnaround.com known as Javeleon.). Based on the result of this experiment we show that dynamic updating of class definitions for live objects may under some circumstances result in different run-time behavior than would be observed after a cold restart of the upgraded application. Finally, we conclude by discussing the implication of integrating the dynamic updating model of Gosh! with JRebel. The successful integration of these two systems will set a new standard for dynamic software updating in Java.

This paper presents a demonstration of how Local Area Weather Radar (LAWR) X-band measurements can be combined with meteorological C-band measurements into a single radar product. For this purpose, a blending method has been developed which combines the strengths of the two radar systems. Combining the two radar types achieves a radar product with both long range and high temporal resolution. It is validated that the blended radar product performs better than the individual radars based on ground observations from laser disdrometers. However, the data combination is challenged by lower performance of the LAWR. Although both radars benefit from the data combination, it is also found that advection based temporal interpolation is a more favourable method for increasing the temporal resolution of meteorological C-band measurements.

Mitochondrial DNA (mtDNA) is maternally inherited without recombination events and has a high copy number, which makes mtDNA analysis feasible even when genomic DNA is sparse or degraded. Here, we present a SNP typing assay with 33 previously described mtDNA coding region SNPs for haplogroup assignment of Caucasians. Samples may be ascribed into eight major Caucasian haplogroups and 28 of the most frequent Caucasian subhaplogroups. The assay consists of a multiplex PCR reaction followed by two single base extension (SBE) reactions. The assay was tested on 49 unrelated Danish individuals previously typed by sequencing of the mitochondrial HV1 and HV2 regions. Haplogroup assignments based on mtDNA coding region SNPs and sequencing of HV1 and HV2 regions gave identical results for 27% of the samples, and except for one sample, differences in haplogroup assignments were at the subhaplogroup level.

An on-line settling column for measuring the dynamic variations of settling velocity of activated sludge has been developed. The settling column is automatic and self-cleansing insuring continuous and reliable measurements. The settling column was tested on sludge from a batch reactor where sucrose was added as an impulse to activated sludge. The continuous measurement of settling velocity revealed a highly dynamic response after the sucrose was added. The results were verified with simultaneous measurement of the initial settling rate. A 200 hour experiment showed variations in settling velocity, which was not apparent in the DSVI (Diluted Sludge Volume Index).

This paper presents a method for estimating runoff coefficients of urban drainage subcatchments based on a combination of high resolution weather radar data and flow measurements from a downstream runoff sensor. By utilising the spatial variability of the precipitation it is possible to estimate the runoff coefficients of the separate subcatchments. The method is demonstrated through a case study of an urban drainage catchment (678 ha) located in the city of Aarhus, Denmark. The study has proven that it is possible to use corresponding measurements of the relative rainfall distribution over the catchment and downstream runoff measurements to identify the runoff coefficients at subcatchment level.

Major histocompatibility complex class I (MHC-I) molecules play an essential role in the cellular immune response, presenting peptides to cytotoxic T lymphocytes (CTLs) allowing the immune system to scrutinize ongoing intracellular production of proteins. In the early 1990s, immunogenicity and stability of the peptide–MHC-I (pMHC-I) complex were shown to be correlated. At that time, measuring stability was cumbersome and time consuming and only small data sets were analysed. Here, we investigate this fairly unexplored area on a large scale compared with earlier studies. A recent small-scale study demonstrated that pMHC-I complex stability was a better correlate of CTL immunogenicity than peptide–MHC-I affinity. We here extended this study and analysed a total of 5509 distinct peptide stability measurements covering 10 different HLA class I molecules. Artificial neural networks were used to construct stability predictors capable of predicting the half-life of the pMHC-I complex. These predictors were shown to predict T-cell epitopes and MHC ligands from SYFPEITHI and IEDB to form significantly more stable MHC-I complexes compared with affinity-matched non-epitopes. Combining the stability predictions with a state-of-the-art affinity predictions NetMHCcons significantly improved the performance for identification of T-cell epitopes and ligands. For the HLA alleles included in the study, we could identify distinct sub-motifs that differentiate between stable and unstable peptide binders and demonstrate that anchor positions in the N-terminal of the binding motif (primarily P2 and P3) play a critical role for the formation of stable pMHC-I complexes. A webserver implementing the method is available at www.cbs.dtu.dk/services/NetMHCstab. Fil: Jørgensen, Kasper W.. Technical University of Denmark; Dinamarca Fil: Rasmussen, Michael. Universidad de Copenhagen; Dinamarca Fil: Buus, Søren. Universidad de Copenhagen; Dinamarca Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human β2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8+ T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ–producing CD8+ T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8+ T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell–based vaccines.