Microarray-based kinetic colorimetric detection for quantitative multiplex protein phosphorylation analysis

Commonly used colorimetric detection applied to protein microarrays with enzymatic signal amplification leads to non‐linear signal production upon increase in analyte concentration, thereby considerably limiting the range and accuracy of quantitative readout interpretation. To extend the detection range, we developed a kinetic colorimetric detection protocol for the analysis of ELISA microarrays designed to measure multiple phosphorylated proteins using the platforms ArrayTube? and ArrayStrip?. With our novel quantification approach, microarrays were calibrated over a broad concentration range spanning four orders of magnitude of analyte concentration with picomolar threshold. We used this design for the simultaneous quantitative measurement of 15 phosphorylated proteins on a single chip.     

Construction of pH‐sensitive Her2‐binding IgG1‐Fc by directed evolution

For most therapeutic proteins, a long serum half‐life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half‐life. In this study, we aimed to investigate whether pH‐dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C‐terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2‐ECD) at pH 6 compared to pH 7.4. A yeast‐displayed Fcab‐library was alternately selected for binding at pH 7.4 and non‐binding at pH 6.0. Selected Fcab variants showed clear pH‐dependent binding to soluble Her2‐ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH‐dependent interactions with Her2‐positive cells whereas their conformational and thermal stability was pH‐independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH‐dependent binding sites in proteins.     

Worker Personality and Its Association with Spatially Structured Division of Labor

Division of labor is a defining characteristic of social insects and fundamental to their ecological success. Many of the numerous tasks essential for the survival of the colony must be performed at a specific location. Consequently, spatial organization is an integral aspect of division of labor. The mechanisms organizing the spatial distribution of workers, separating inside and outside workers without central control, is an essential, but so far neglected aspect of division of labor. In this study, we investigate the behavioral mechanisms governing the spatial distribution of individual workers and its physiological underpinning in the ant Myrmica rubra. By investigating worker personalities we uncover position-associated behavioral syndromes. This context-independent and temporally stable set of correlated behaviors (positive association between movements and attraction towards light) could promote the basic separation between inside (brood tenders) and outside workers (foragers). These position-associated behavior syndromes are coupled with a high probability to perform tasks, located at the defined position, and a characteristic cuticular hydrocarbon profile. We discuss the potentially physiological causes for the observed behavioral syndromes and highlight how the study of animal personalities can provide new insights for the study of division of labor and self-organized processes in general.     

Chemotaxis of mesenchymal stem cells within 3D biomimetic scaffolds--a modeling approach

Bone tissue engineering is a promising strategy to repair local defects by implanting biodegradable scaffolds which undergo remodeling and are replaced completely by autologous bone tissue. Here, we consider a Keller-Segel model to describe the chemotaxis of bone marrow-derived mesenchymal stem cells (BMSCs) into a mineralized collagen scaffold. Following recent experimental results in bone healing, demonstrating that a sub-population of BMSCs can be guided into 3D scaffolds by gradients of signaling molecules such as SDF-1α, we consider a population of BMSCs on the surface of the pore structure of the scaffold and the chemoattractant SDF-1α within the pores. The resulting model is a coupled bulk/surface model which we reformulate following a diffuse-interface approach in which the geometry is implicitly described using a phase-field function. We explain how to obtain such an implicit representation and present numerical results on μCT-data for real scaffolds, assuming a diffusion of SDF-1α being coupled to diffusion and chemotaxis of the cells towards SDF-1α. We observe a slowing-down of BMSC ingrowth after the scaffold becomes saturated with SDF-1α, suggesting that a slow release of SDF-1α avoiding an early saturation is required to enable a complete colonization of the scaffold. The validation of our results is possible via SDF-1α release from injectable carrier materials, and an adaptation of our model to similar coupled bulk/surface problems such as remodeling processes seems attractive.     

Lithium-ion battery cell production in Europe: Scenarios for reducing energy consumption and greenhouse gas emissions until 2030

The market for electric vehicles is growing rapidly, and there is a large demand for lithium-ion batteries (LIB). Studies have predicted a growth of 600% in LIB demand by 2030. However, the production of LIBs is energy intensive, thus contradicting the goal set by Europe to reduce greenhouse gas (GHG) emissions and become GHG emission free by 2040. Therefore, in this study, it was analyzed how the energy consumption and corresponding GHG emissions from LIB cell production may develop until 2030. Economic, technological, and political measures were considered and applied to market forecasts and to a model of a state-of-the art LIB cell factory. Notably, different scenarios with trend assumptions and above/below-trend assumptions were considered. It could be deduced that, if no measures are taken and if the status quo is extrapolated to the future, by 2030, ∼5.86 Mt CO₂-eq will be emitted due to energy consumption from European LIB cell production. However, by applying a combination of economic, technological, and political measures, energy consumption and GHG emissions could be decreased by 46% and 56% by 2030, respectively. Furthermore, it was found that political measures, such as improving the electricity mix, are important but less dominant than improving the production technology and infrastructure. In this study, it could be deduced that, by 2030, through industrialization and application of novel production technologies, the energy consumption and GHG emissions from LIB cell production in Europe can be reduced by 24%.     

Differences in baseline lung cancer mortality between the German uranium miners cohort and the population of the former German Democratic Republic (1960–2003)

A previous analysis of the radon-related lung cancer mortality risk, in the German uranium miners cohort, using Poisson modeling techniques, noted internal (spontaneous) rates that were higher on average than the external rates by 16.5% (95% CI: 9%; 24%). The main purpose of the present paper is to investigate the nature of, and possible reasons for, this difference by comparing patterns in spontaneous lung cancer mortality rates in a cohort of male miners involved in uranium extraction at the former Wismut mining company in East Germany with national male rates from the former German Democratic Republic. The analysis is based on miner data for 3,001 lung cancer deaths, 1.76 million person-years for the period 1960–2003, and national rates covering the same calendar-year range. Simple “age–period–cohort” graphical analyses were applied to assess the main qualitative differences between the national and cohort baseline lung cancer rates. Some differences were found to occur mainly at higher attained ages above 70 years. Although many occupational risk factors may have contributed to these observed age differences, only the effects of smoking have been assessed here by applying the Peto–Lopez indirect method for calculating smoking attributability. It is inferred that the observed age differences could be due to the greater prevalence of smoking and more mature smoking epidemic in the Wismut cohort compared to the general population of the former German Democratic Republic. In view of these observed differences between external population-based rates and internal (spontaneous) cohort baseline lung cancer rates, it is strongly recommended to apply only the internal rates in future analyses of uranium miner cohorts.     

Twenty four new microsatellite markers in two invasive pavement ants, <Emphasis Type="Italic">Tetramorium</Emphasis> sp.E and <Emphasis Type="Italic">T. tsushimae</Emphasis> (Hymenoptera: Formicidae)

Invasive species trigger biodiversity losses and alter ecosystem functioning, with life history shaping invasiveness (Sakai et al., Annu Rev Ecol Syst 32:305–332, 2001). However, pinpointing the relation of a specific life history to invasion success is difficult. One approach may be comparing congeners. The two Palearctic pavement ants, Tetramorium sp.E (widely known as T. caespitum, Schlick-Steiner et al., Mol Phylogenet Evol 40:259–273, 2006) and T. tsushimae have invaded North America (Steiner et al., Biol Invasions 8:117–123, 2006). Their life histories differ in that T. sp.E has separate single-queened colonies but T. tsushimae multi-queened colonies scattered over large areas (Sanada-Morimura et al., Insect Soc 53:141–148, 2006; Schlick-Steiner et al., Mol Phylogenet Evol 40:259–273, 2006; Steiner et al., Biol Invasions 8:117–123, 2006). Comparison of the genetic diversity in the entire native and non-native ranges will elucidate the invasion histories. Here, we present 13 and 11 microsatellites, developed for T. sp.E and T. tsushimae, respectively, and characterize all for both species. Florian M. Steiner, Wolfgang Arthofer and Birgit C. Schlick-Steiner contributed equally to this work.     

High Spatial Resolution Cardiovascular Magnetic Resonance at 7.0 Tesla in Patients with Hypertrophic Cardiomyopathy – First Experiences: Lesson Learned from 7.0 Tesla

Background

Cardiovascular Magnetic Resonance (CMR) provides valuable information in patients with hypertrophic cardiomyopathy (HCM) based on myocardial tissue differentiation and the detection of small morphological details. CMR at 7.0T improves spatial resolution versus today’s clinical protocols. This capability is as yet untapped in HCM patients. We aimed to examine the feasibility of CMR at 7.0T in HCM patients and to demonstrate its capability for the visualization of subtle morphological details.

Methods

We screened 131 patients with HCM. 13 patients (9 males, 56 ±31 years) and 13 healthy age- and gender-matched subjects (9 males, 55 ±31years) underwent CMR at 7.0T and 3.0T (Siemens, Erlangen, Germany). For the assessment of cardiac function and morphology, 2D CINE imaging was performed (voxel size at 7.0T: (1.4x1.4x2.5) mm³ and (1.4x1.4x4.0) mm³; at 3.0T: (1.8x1.8x6.0) mm³). Late gadolinium enhancement (LGE) was performed at 3.0T for detection of fibrosis.

Results

All scans were successful and evaluable. At 3.0T, quantification of the left ventricle (LV) showed similar results in short axis view vs. the biplane approach (LVEDV, LVESV, LVMASS, LVEF) (p = 0.286; p = 0.534; p = 0.155; p = 0.131). The LV-parameters obtained at 7.0T where in accordance with the 3.0T data (p_LVEDV = 0.110; p_LVESV = 0.091; p_LVMASS = 0.131; p_LVEF = 0.182). LGE was detectable in 12/13 (92%) of the HCM patients. High spatial resolution CINE imaging at 7.0T revealed hyperintense regions, identifying myocardial crypts in 7/13 (54%) of the HCM patients. All crypts were located in the LGE-positive regions. The crypts were not detectable at 3.0T using a clinical protocol.

Conclusions

CMR at 7.0T is feasible in patients with HCM. High spatial resolution gradient echo 2D CINE imaging at 7.0T allowed the detection of subtle morphological details in regions of extended hypertrophy and LGE.     

Afamin is a novel human vitamin E-binding glycoprotein characterization and in vitro expression

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.     

Apoptosis paves the detour path for CD8 T cell activation against intracellular bacteria

Intracellular bacteria such as Mycobacterium tuberculosis primarily infect macrophages. Within these host cells, the pathogens are confined to phagosomes and their antigens are secluded from the classical MHC I presentation pathway. Moreover, macrophages fail to express certain antigen presenting molecules like CD1 proteins. As a result of this intracellular lifestyle, the pathways for the induction of MHC I- and CD1-restricted CD8 T cells by such microorganisms remain elusive. Based on recent findings in tuberculosis and salmonellosis, we propose a new detour pathway for CD8 T cell activation against intracellular bacteria through apoptotic blebs from infected macrophages. Pathogen-derived antigens including proteins and lipids are delivered from infected cells to non-infected dendritic cells. Subsequently, these professional antigen presenting cells display microbial antigens through MHC I and CD1 to T cells. Thus, cross-priming mediated by apoptotic vesicles is not just a matter of antigen distribution, but an intrinsic immunological function due to the nature of phagosomally located intracellular bacteria. We consider infection-induced apoptosis the conditio sine qua non for antigen-specific CD8 T cell activation by phagosome-enclosed pathogens. This important new function of cell death in antibacterial immunity requires consideration for rational vaccine design.