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The eco-efficiency of actual production processes is still one dominating research area in engineering. However, neglecting the environmental impacts of production equipment, technical building services and energy supply might lead to sub-optimization or burden-shifting and thus reduced effectiveness. As an established method used in sustainability management, Life Cycle Assessment aims at calculating the environmental impacts from all life cycle stages of a product or system. In order to cope with shortcomings of the static character of life cycle models and data gaps this approach combines Life Cycle Assessment with manufacturing system simulation. Therefore, the two life cycles of product and production system are merged to assess environmental sustainability on product level. Manufacturing simulation covers the production system and Life Cycle Assessment is needed to relate the results to the final product. This combined approach highlights the influences from dynamic effects in manufacturing systems on resulting life cycle impact from both product and production system. Furthermore, the importance of considering indirect peripheral equipment and its effects on the manufacturing system operation in terms of output and energy demands is underlined. The environmental flows are converted into impacts for the five recommended environmental impact categories. Thus, it can be demonstrate that Life Cycle Assessment can enhance the process simulation and help identify hot-spots along the life cycle. The combined methodology is applied for analysing a case study in fourteen scenarios for the integration of volatile energy sources into energy flexible manufacturing control.

Cooling towers are part of industrial technical building services and one of the main drivers for water and energy demands in industry. For planning and operation, the management of cooling tower systems has to focus on several objectives from strategical, tactical and operational perspective. These objectives relate to operational reliability, energy and resource efficiency as well as impacts on health and environment. Due to dynamic interdependencies within connected production systems and with the local environment, cooling tower management can be regarded as a complex task. Thus, a software-based support for decision makers involved in cooling tower management is presented basing upon the new concept of cyber-physical production systems. Enabled by a systematic data acquisition procedure, this approach combines comprehensive methodologies using white-box and black-box models. For a thorough system analysis and performance prediction, three modules have been developed within the cyber system: The system inventory and monitoring module allows to monitor the dynamic cooling tower system behavior and to calculate key performance indicator related to its operational performance. In the simulation module, a white-box model is used to test different alternatives for operational improvements in terms of energy and water demands. By means of the data analytics module, a black-box approach is applied to predict the system behavior with high accuracy basing on historical data. The full potential of the concept comes to bear through a continuous and consistent data basis as well as the synergetic application of the methodologies. As result, the approach provides support for decision makers at every level of cooling tower management. The approach was successfully applied on an industrial cooling tower system in an automotive manufacturing plant located in Germany, proving the advantages of the complementary methodologies. Thereby, the simulation revealed significant reduction potentials of water demands (−7%) and energy demands (−27%) through adapted operational strategies. As a further result, dynamic energy demand profiles could be predicted with a high accuracy (R2 = 0.98) by using multivariate regression analyses.

Energy flexible manufacturing systems offer new opportunities to deal with upcoming challenges of volatile energy supply. In this context, flexibilization of energy intensive system elements is regarded as promising approach. In particular, technical building services (TBS) such as cooling towers exhibit crucial potentials due to their cross-linking within the manufacturing system. Against this background, technical capabilities and operational strategies for energy flexible management of industrial cooling towers are analyzed, basing upon empiric data from a plant located in Germany. A synergetic approach of data-driven analysis and scenario-based simulation is applied to demonstrate benefits of energy flexible TBS management. Further, an approach for practical implementation is proposed within the concept.

One of today’s urging challenges for engineers all over the world is the expansion and integration of renewable energy resources for electricity generation. Enabling production systems to deal with the fluctuating character of renewable energy, the development of novel strategies to flexibilize the energy demand of factories is a crucial task in engineering business. Beside demand side management of production machines, the integration of energy storage technologies in process chains and technical building systems (TBS) has become a promising strategy to foster energy flexibility in production systems. Towards practice-oriented education, learning factories are established as a beneficial concept for research-based learning, not only for universities. However, the urging field of energy flexibility does not yet play a major role for learning factory topics. In order to equip engineers with necessary skills to solve future challenges of increasing renewable energy generation, this paper presents a concept to foster energy flexibility in learning factories focussing the application of energy storage technologies. In this context, two hardware systems featuring energy storage technologies, one based on supercapacitors (SC), second based on lithium-ion battery (LIB), are developed for integration in the electricity supply of the learning factory environment. Furthermore, an energy management system is integrated as centrepiece of the compressed air and electricity supply of a full process chain, whereas the SC is demonstrated as uninterruptible power supply for a single process. The concept and application of energy storage technologies are exemplary applied in the environment of “Die Lernfabrik” at the IWF, TU Braunschweig.

A sustainable development towards future production systems requires a holistic understanding of the overall production system. As supplier of cooling medium for processes, machines and air-conditioning, cooling towers play a key role for various production processes and the overall factory. Due to long operating time, significant energy and water demands as well as related environmental impacts, the improvement of cooling tower operations has come into focus of industry. Beyond this background, this paper presents an approach for analysing the energy and water demand as well as the global warming potential of forced-draft cooling towers for industrial purposes. As the energy and water demand of cooling towers is highly influenced by regional conditions, different locations all over the world have been considered in the analysis regarding regional climatic data as well as country-specific electrical energy mixes.

In the last decade, renewable energy supply has gained increasing interest, as it can contribute to the diversification of the energy mix. Energy flexibility offers consumers the opportunity to benefit from fluctuating energy prices, connected to the volatility of wind and solar power. This paper aims at mapping flexibility strategies and their requirements in the process industry environment. The conception of flexibility measures requires a characterization of energy flows, in order to identify hotspots and estimate the flexibility potential. An integrated methodology has been developed with the purpose of supporting investment decisions for a case-study in the paper production sector in Germany.

Die Berucksichtigung von Umweltaspekten uber alle Produktlebenszyklusphasen ist erforderlich, um Problemverschiebungen zu erkennen und ggfs. zu vermeiden. Kunststoffe auf Basis nachwachsender Rohstoffe und biologisch abbaubare Kunststoffe konnen einen positiven Beitrag zu einer nachhaltigen Entwicklung leisten. Vorteile konnen sich insbesondere in der Herstellungs- und End-of-Life-Phase ergeben. Allerdings stellt sich die Frage, ob sich bei der Verarbeitung von Biokunststoffen im Vergleich zu konventionellen Kunststoffprodukten ebenfalls Vorteile ergeben. Da die fur die Verarbeitung erforderliche Energie auch wesentlich zu den potenziellen Umweltwirkungen beitragt, ist es das Ziel, den Energiebedarf zur Verarbeitung von Biokunststoffen besser zu verstehen. Ein wesentliches Verarbeitungsverfahren zur Herstellung von Kunststoffprodukten ist das Kunststoffspritzgiesen. In dieser Studie erfolgt daher eine Analyse und Bewertung der Energieeffizienz von Spritzgiesverfahren einschlieslich der notwendigen Nebenprozesse.

Considering environmental aspects is of growing interest over all life cycle phases of products. Being based on renewable and/or bio-degradable resources, bioplastics enable a positive contribution towards sustainable products in material and end of life phase. However, bioplastics can be more energy intensive during their manufacturing phase than their conventional competitors. As this contributes to the overall environmental sustainability of a product as well, it is necessary to acquire a better understanding about the energy demand of bioplastics in manufacturing. Therefore, this paper presents a study on the energy demand of bioplastic materials in injection moulding, milling, and fused deposition modelling 3D-printing including necessary preparation and drying processes. Based on this, a quantitative energy related assessment of manufacturing bioplastics in comparison to conventional plastics is possible.

Im Dezember 2015 wurde das „Gesetz zur Strafbarkeit der geschaftsmasigen Forderung der Selbsttotung“ beschlossen. Leider verunklart es die bisher eindeutige Rechtslage. Es macht wahrscheinlich, dass Pflegende, Pflege Organisierende, ehrenamtliche Hospizmitarbeiter und Arzte bei Sterbenswunschen die Kommunikation mit Hilfsbedurftigen abbrechen. Das Sterbehilfegesetz (§ 217 StGB) verunsichert alle, die in ihrem Beruf Menschen mit Sterbewunschen begegnen. Der einzige Ratschlag, der sich geben lasst, bevor die Rechtslage nach dem neuen Gesetz eindeutig geklart ist, besteht demnach in folgendem: Wer in die Lage kommt, dass von ihm Hilfe zum Suizid erwartet wird, sollte in jedem Fall alles tun, um zu klaren, ob der Entschluss dazu freiverantwortlich, ohne so genannte Willensmangel, getroffen wurde. Und wenn sich der mogliche Helfer der Freiverantwortlichkeit versichert hat, sollte er das dokumentieren und fur andere erkennbar machen.

In order to respond to environmental requirements, a growing number of manufacturing companies operate on-site renewable energy generation as part of their own energy supply. As a vision, plants could be fully energy self-sufficient and independent from electricity suppliers. From perspective of manufacturing systems, this requires the ability to flexibly adapt the energy demand of production machines and processes to current electricity generation and availability from grid. Such energy flexible manufacturing systems can achieve different development levels of energy flexibility: energy demand flexibility, balanced energy self-sufficiency and real energy self-sufficiency. Within this work, scenario-based simulations are used to assess strategies towards energy self-sufficiency for the case of a manufacturing system in a learning factory environment. Several aspects regarding dimensioning of on-site renewable energy generation and resulting oversupply, productivity and demand side management are addressed to show occurring tradeoffs related with energy self-sufficiency. The study shows, that a coupling of demand side management and storage technologies is the most promising combination to achieve a high degree of energy self-sufficiency (88%) for the regarded manufacturing system.

Sustainability and life cycle thinking is becoming part of companies’ “green identity”. For this reason, Life Cycle Assessment (LCA) is a common tool to assess the environmental impacts of manufactured products in order to improve it—starting with raw material selection to efficiency improvements of manufacturing processes and optimization of operation. Focusing the manufacturing stage, usually only the machines and processes directly related to the manufacturing are taken into account for assessment. Peripheral processes e.g. pump and pipe systems and technical building services (TBS) e.g. air conditioning, process water supply, are often neglected due to poor data availability. Supplying cooling water for production machines, cooling towers (CTs) are a central part of the industrial TBS—and therefore part of almost every manufacturing system. Neglecting the environmental impact of CT would result in a major white spot when accomplish a LCA. In order to close this gap, this paper presents the first LCA of industrial CTs showing the hot spots of environmental impacts along the entire CT life cycle.

Industry releases vast amounts of heat energy as dissipative waste heat to the atmosphere. It is therefore necessary to acquire a better understanding of the waste heat potentials in manufacturing. The paper presents an integrated approach for identifying and quantifying waste heat potentials of different production processes. The identification is based on an estimation procedure followed by a simulative assessment of production processes to quantify and allocate waste heat over time. The approach further elaborates on a potential source and demand matching of heat streams. A case study from the automotive industry demonstrates the applicability of the approach.

Scope There is a growing interest in food constituents that could reduce intestinal glucose absorption to prevent overshooting plasma glucose and insulin levels in patients with prediabetes and diabetes mellitus type 2. Methods and results We here demonstrate that an extract and individual polyphenols from apple diminish sodium-coupled glucose transporter 1 (SGLT1) mediated glucose uptake in vitro and in vivo. Inhibition of transport of sugars by SGLT1 was shown in Xenopus oocytes and in mice jejunal segments. Strongest inhibition was observed for phlorizin with IC50 values for transport inhibition of 0.46 ± 0.19 and 4.1 ± 0.6 μM in oocytes and intestinal segments, respectively. An oral glucose tolerance test performed in volunteers with prior administration of the apple extract reduced venous blood glucose and plasma insulin levels, similar to findings obtained in C57BL/6N mice. Analysis of human urine samples revealed that the extract increased modestly renal glucose loss that is most likely a result of inhibition of renal glucose reabsorption by phloretin derivatives found in plasma of the volunteers. Conclusion Although the apple extract substantially decreased intestinal glucose absorption in all test systems, the finding that there are systemic effects that relate to inhibition of glucose transport processes beyond the intestine addresses safety issues that need further exploitation.

Introduction: Interprofessional collaboration is crucial to the optimization of patient care. Aim: This paper aims to provide recommendations for implementing an innovative constructivist educational concept with the core element of video-based self-assessment. Methodology: A course for students in medicine, physiotherapy, and nursing was developed through interprofessional, cross-institutional collaboration. The course consisted of drawing on prior knowledge about the work done by each professional group in regard to a specific clinical scenario and an interprofessional treatment situation, filming a role play of this treatment situation, and a structured self-assessment of the role play. We evaluated the preparation and implementation of the three courses conducted thus far. Concrete recommendations for implementation were made based on evaluation sheets (students), open discussions (tutors, instructors, institutions) and recorded meeting minutes (project managers, project participants). Results: Basic recommendations for implementation include: selecting appropriate criteria for self-assessment and a simulated situation that offers members of each professional group an equal opportunity to act in the role play. In terms of administrative implementation we recommend early coordination among the professions and educational institutions regarding the target groups, scheduling and attendance policy to ensure participant recruitment across all professions. Procedural planning should include developing teaching materials, such as the case vignette and treatment scenario, and providing technical equipment that can be operated intuitively in order to ensure efficient recording. Conclusion: These recommendations serve as an aid for implementing an innovative constructivist educational concept with video-based self-assessment at its core., Einleitung: Interprofessionelle Zusammenarbeit ist eine unabdingbare Voraussetzung für die Optimierung der Patientenbehandlung. Zielsetzung: Ziel dieses Erfahrungsberichtes ist es, Empfehlungen zur Implementierung eines innovativen konstruktivistisch geprägten Lehrkonzepts mit dem Kernelement der videobasierten Selbstreflexion zu geben. Methodik: Im interprofessionellen, interinstitutionellen Diskurs wurde eine Lehrveranstaltung (LV) für Auszubildende der Gesundheits- und Krankenpflege und der Physiotherapie sowie für Medizinstudierende entwickelt. Die LV beinhaltete die Wissensreaktivierung zum Tätigkeitsprofil der beteiligten Professionen, dem Krankheitsbild und einer interprofessionellen Behandlungssituation, die Durchführung eines videodokumentierten Rollenspiels zu dieser Behandlungssituation und die strukturierte Selbstreflexion des Rollenspiels. Die Vorbereitung und Durchführung der drei bisher durchgeführten LV wurde evaluiert. Anhand der Evaluationsbögen (Auszubildende, Studierende), offenen Diskussionen (Tutoren, Dozierende, Institutionen) und protokollierten Treffen (Projektverantwortliche, Projektbeteiligte) wurden konkrete Empfehlungen zur Umsetzung abgeleitet. Ergebnis: Wesentliche Empfehlungen zur didaktischen Umsetzung sind: Auswahl einer geeignetem Behandlungssituation mit ausgewogenem Handlungsanteil der beteiligten Berufsgruppen für das videodokumentierte Rollenspiel und Erarbeitung von Kriterien zur Strukturierung der Selbstreflexion; zur administrativen Umsetzung: frühzeitige interprofessionelle/interinstitutionelle Abstimmung bei der Festlegung der Zielgruppen, der Zeitplanung und der Verbindlichkeit der LV zur Sicherstellung der ausgewogenen Teilnehmerrekrutierung; zur Ablaufplanung: Erstellung von Unterrichtsmaterialien, wie Fallvignette und Behandlungsszenario und Bereitstellung intuitiv bedienbarer Präsentationsmedien, um einen effizienten Videodreh zu ermöglichen. Schlussfolgerung: Aus unserer Sicht stellen diese Empfehlungen eine konkrete Hilfestellung zur Implementierung des innovativen konstruktivistische geprägten Lehrkozepts mit dem Kernelement der videobasierten Selbstreflexion dar., GMS Journal for Medical Education; 33(2):Doc33

As inhalation is the major exposure route for nanoparticles, the question if inhaled particles can overcome the respiratory epithelial barrier and hence enter the body is of great interest. Here, we adapted the for soluble substances well established Calu-3 in vitro air-blood barrier model to the use of nanoparticle transport testing. As the usually used filter supports hindered particle transport due to their small pore size, supports with a pore size of 3 μm had to be used. On those filters, barrier and transport characteristics of the cells were tested and culture conditions changed to obtain optimal conditions. Functionality was confirmed with transport experiments with polystyrene model particles prior to testing of industrially relevant engineered metal oxide particles. Except for CeO2 nanoparticles, no transport across the epithelial barrier model could be detected. Paracellular permeability and barrier function was not affected by any of the nanoparticles, except for ZrO2.

Industrial nanoparticles are not developed to be compatible with in vitro cell culture assays which are carried out in isotonic solutions at physiological pH and often in the presence of proteins. ...

Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurfTM and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurfTM promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 2 PEG 2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.

Study objectives: We performed an open-label multicenter study to evaluate the safety and efficacy of the dual endothelin receptor antagonist bosentan in patients with inoperable chronic thromboembolic pulmonary hypertension (CTEPH). Patients: Nineteen patients with inoperable CTEPH were enrolled. Measurements: The primary end point was a change in pulmonary vascular resistance (PVR). Secondary end points included 6-min walk test, peak oxygen uptake (VO2), New York Heart Association functional class, serum levels of N-terminal-pro brain natriuretic peptide (NT-pro- BNP), and various other hemodynamic parameters. Results: After 3 months of treatment with bosentan, PVR decreased from 914 329 to 611 220 dynescm -5 (p < 0.001). Functional class and peak VO2 remained unchanged, but 6-min walk distance increased from 340 102 to 413 130 m( p 0.009), and serum NT-pro BNP levels improved from 2,895 2,620 to 2,179 2,301 (p 0.027). One patient died, presumably from influenza A infection, and another patient experienced progressive fluid retention despite reduction of PVR. Other than that, treatment was well tolerated by all patients. Conclusions: This open-label pilot trial suggests that bosentan may offer a therapeutic option for patients with inoperable CTEPH. Randomized controlled trials are warranted to confirm these findings. (CHEST 2005; 128:2363-2367) Abbreviations: CI cardiac index; CPET cardiopulmonary exercise testing; CTEPH chronic thromboembolic pulmonary hypertension; INR international normalized ratio; NT-pro-BNP N terminal-pro brain natriuretic peptide; NYHA New York Heart Association; PAH pulmonary arterial hypertension; PAPm mean pulmonary artery pressure; PCWP pulmonary capillary wedge pressure; PEA pulmonary endarterectomy; PVR pulmonary vascular resistance; RAP right atrial pressure; Svo2 mixed venous oxygen saturation; Vo2 oxygen uptake

Exit from mitosis requires Cdk1 inactivation, with the most prominent mechanism of Cdk1 inactivation being proteolysis of mitotic cyclins [1]. In higher eukaryotes this involves sequential destruction of A- and B-type cyclins. CycA is destroyed first, and CycA/Cdk1 inactivation is required for the metaphase-to-anaphase transition [2]. The degradation of CycA is delayed in response to DNA damage but is not prevented when the spindle checkpoint is activated [3, 4]. Cyclin destruction is thought to be mediated by a conserved motif, the destruction box (D box). Like B-type cyclins, A-type cyclins contain putative destruction box sequences in their N termini [5]. However, no detailed in vivo analysis of the sequence requirements for CycA destruction has been described so far. Here we tested several mutations in the CycA coding region for destruction in Drosophila embryos. We show that D box sequences are not essential for mitotic destruction of CycA. Destruction is mediated by at least three different elements that act in an overlapping fashion to mediate its mitotic degradation.

The ductal carcinoma in situ (DCIS) of the mammary gland represents an early, pre-invasive stage in the development of invasive breast carcinoma and is increasingly diagnosed since the introduction of high-quality mammography screening. Uncertainties in the prognosis for patients with DCIS have caused a controversial discussion about adequate treatment, and it is suspected that most patients undergoing mastectomy may be overtreated. In order to improve treatment and treatment decision, it therefore is highly desirable to identify prognostic markers and therapeutic targets for DCIS. We here introduce a set of transgenic mice (WAP-T and WAP-T-NP lines) presenting with various morphological forms of DCIS-like lesions. In these mice the SV40 large tumor antigen is specifically induced in epithelial cells of the terminal duct lobular units (TDLU). As a consequence of continuous expression of the oncogene, the animals develop multifocal DCIS and consequently invasive carcinoma within strain specific periods of latency. DCIS lesions in transgenic mice exhibit distinct architectural and cytological features which closely resemble those commonly present in humans. We therefore propose these transgenic lines as an experimental model to study the underlying molecular events leading to DCIS and its progression to invasive disease.

Debate is currently taking place over minimum case numbers for the care of premature infants and neonates in Germany. As a result of the Federal Joint Committee (Gemeinsamer Bundesauschuss, G-BA) guidelines for the quality of structures, processes, and results, requiring high levels of staffing resources, Level I perinatal centers are increasingly becoming the focus for health-economics questions, specifically, debating whether Level I structures are financially viable.Using a multistep contribution margin analysis, the operating results for the Obstetrics Section at the University Perinatal Center of Franconia (Universitäts-Perinatalzentrum Franken) were calculated for the year 2009. Costs arising per diagnosis-related group (DRG) (separated into variable costs and fixed costs) and the corresponding revenue generated were compared for 4,194 in-patients and neonates, as well as for 3,126 patients in the outpatient ultrasound and pregnancy clinics.With a positive operating result of € 374,874.81, a Level I perinatal center on the whole initially appears to be financially viable, from the obstetrics point of view (excluding neonatology), with a high bed occupancy rate and a profitable case mix. By contrast, the costs of prenatal diagnostics, with a negative contribution margin II of € 50,313, cannot be covered. A total of 79.4% of DRG case numbers were distributed to five DRGs, all of which were associated with pregnancies and neonates with the lowest risk profiles.A Level I perinatal center is currently capable of covering its costs. However, the cost-revenue ratio is fragile due to the high requirements for staffing resources and numerous economic, social, and regional influencing factors.

Carrier-mediated transport of small solutes across plasma membranes is central to all living cells, allowing compartmentalization and coordinated metabolic fluxes. Common laboratory techniques for characterizing these transport processes either employ radiolabelled substrates or, in the case of rheogenic carriers, measure transport currents. However, many transport proteins do not cause net charge movement because they carry uncharged substrates or act as electrically silent exchangers. The limited availability of radiotracer substrates might also explain why there are still numerous orphan transporters that await identification of their substrates. High-resolution H NMR spectroscopy might overcome some of the limitations of current methodologies because it allows the detection and quantification of any organic molecule as long as it contains protons or other nuclei detectable with NMR spectroscopy. The aim of our study was to test the suitability of high-resolution NMR spectroscopy for analysis of plasma membrane transport processes. In-cell NMR spectroscopy was pioneered by Serber and Dctsch and has attracted a lot of attention in structural biology. This method opens pathways to the study of the structure and dynamics of biological macromolecules inside a living cell. More recent studies have demonstrated that protein modifications such as phosphorylation, acetylation and deacetylation, as well as protein–protein interactions (STINT-NMR), 8] can be characterized by in-cell NMR spectroscopy. Here we have applied in-cell NMR spectroscopy to the analysis of plasma membrane transport processes. Oocytes of the South African clawed frog Xenopus laevis are a well-established and robust expression system for membrane transport proteins from various sources. 10] Xenopus oocytes are unusually large cells, with a mean diameter of 1.2 mm and internal volume of 1 mL. Upon injection with transporter cRNA, transport processes can be studied 2–4 days later, when carrier proteins have been synthesized. We employed these cells for developing a new transport assay that detects intracellular substrates by NMR analysis. For establishing the NMR-based assay we used the mammalian peptide transporter PEPT1 and the amino acid transporter PAT1. Both are rheogenic transporters that couple substrate uptake to proton movement down an electrochemical proton gradient. Both proteins have previously been expressed and functionally characterized in Xenopus oocytes. To confirm protein production, total protein extracts from cRNA-injected oocytes were separated by SDS-PAGE, transferred to nitrocellulose membranes and labelled with specific antisera. The sera detected PAT1 and PEPT1 with the expected molecular masses but failed to detect these proteins in uninjected cells (see Figure S1 in the Supporting Information). We also recorded substrate-induced currents in oocytes expressing PEPT1 and PAT1 by the two-electrode voltage-clamp technique. Figure S2 demonstrates that oocytes expressing PEPT1 produce significant inward currents when perfused with glycyl-sarcosine (Gly-Sar). Similarly, PAT1-expressing cells display transport currents with the substrate sarcosine (N-methylglycine). In summary, both proteins are functionally expressed in the oocytes. The first series of H NMR measurements were performed with oocyte extracts obtained from 70–80 uninjected oocytes. These extracts were measured either straight (Figure S3A) or after addition of Gly-Sar to a final concentration of 1 mm (Figure S3B). These measurements were performed at 4 8C and demonstrated that Gly-Sar produced distinct NMR signals that were easily detectable against the background of all other cellular metabolites. We next prepared extracts from cells expressing PEPT1. Three days after the injection of the cRNA, the cells were incubated with 20 mm Gly-Sar overnight and then washed to remove extracellular substrate. The extracts prepared from these cells (Figure S3C) displayed the same additional signals that were observed after spiking oocyte extracts with Gly-Sar. This confirms that the NMR approach has the necessary sensitivity and resolution to detect a substrate that was imported through PEPT1. For the next series of experiments, oocytes expressing PEPT1 were exposed to substrate solution as above and then embedded between two symmetric layers of agarose in a 2 mm NMR tube (Figure 1). All measurements involving intact cells were performed at 20 8C (at 4 8C the intracellular signals of Gly-Sar were very broad and this prevented their unambiguous assignment; data not shown). We first determined how many cells were necessary for reproducible measurements. Contrary to our expectations, the H NMR signals became sharper when fewer cells were measured and this improved the detection of Gly-Sar (Figure S4). This observation can prob[a] Prof. Dr. W. Kremer, Prof. Dr. H. R. Kalbitzer Institut f r Biophysik und Physikalische Biochemie und Zentrum f r Magnetische Resonanz in Chemie und Biomedizin Universit t Regensburg Universit tstrasse 31, 93053 Regensburg (Germany) E-mail : werner.kremer@ur.de [b] Dr. C. Schreier, Dr. F. Huber LipoFIT Analytic GmbH Josef-Engert-Str. 9, 93053 Regensburg (Germany) [c] C. Schulze, Prof. Dr. H. Daniel, Dr. J. Stolz Lehrstuhl f r Ern hrungsphysiologie Zentralinstitut f r Ern hrungsund Lebensmittelforschung (ZIEL)— Abteilung Biochemie Wissenschaftszentrum Weihenstephan, Technische Universit t M nchen Gregor-Mendel-Strasse 2, 85350 Freising (Germany) E-mail : stolz@tum.de Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201200514.

Upon contact with the human body, nanomaterials are known to interact with the physiological surroundings, especially with proteins. In this context, we explored analytical methods to provide biologically relevant information, in particular for manufactured nanomaterials as produced by the chemical industry. For this purpose, we selected two batches of SiO(2) nanoparticles as well as four batches of CeO(2) nanoparticles, each of comparably high chemical purity and similar physicochemical properties. Adsorption of serum proteins and bovine serum albumin (BSA) was quantified by SDS-PAGE in combination with densitometry and further investigated by atomic force microscopy (AFM) and analytical ultracentrifugation (AUC). The protein adsorption to SiO(2) nanoparticles was below the limit of detection, regardless of adjusting pH or osmolality to physiological conditions. In contrast, the four CeO(2) nanomaterials could be classified in two groups according to half-maximal protein adsorption. Measuring the work of adhesion and indention by AFM for the BSA-binding CeO(2) nanomaterials revealed the same classification, pointing to alterations in shape of the adsorbed protein. The same trend was also reflected in the agglomeration behavior/dispersibility of the four CeO(2) nanomaterials as revealed by AUC. We conclude that even small differences in physicochemical particle properties may nevertheless lead to differences in protein adsorption, possibly implicating a different disposition and other biological responses in the human body. Advanced analytical methods such as AFM and AUC may provide valuable additional information in this context.

The alveolar lining fluid (ALF) covering the respiratory epithelium of the deep lung is the first biological barrier encountered by nanoparticles after inhalation. We here report for the first time significant differences for metal oxide nanoparticles to the binding of surfactant protein A (SP-A), the predominant protein component of ALF. SP-A is a physiologically most relevant protein and provides important biological signals. Also, it is involved in the lung’s immune defence, controlling e.g. particle binding, uptake or transcytosis by epithelial cells and macrophages. In our study, we could prove different particle–protein interaction for eight different nanoparticles, whereas particles of the same bulk material revealed different adsorption patterns. In contrast to other proteins as bovine serum albumin (BSA), SP-A does not seem to significantly deagglomerate large agglomerates of particles, indicating different adsorption mechanisms as in the well-investigated model protein BSA. These findings may have important consequences for biological fate and toxicological effects of inhaled nanomaterials.

Background Glial cells such as retinal Muller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Muller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Muller cells. Methodology/Principal Findings The aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Muller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Muller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice. Conclusion These findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Muller cells.

Background: DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2. Methodology/Principal Findings: In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC50-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM. Conclusions/Significance: These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8+and CD4+T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4+and CD8+cells both were equally important in establishing and maintaining a protective immune response. CD4+cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4+lymphocytes provide help to CD8+cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4+cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8+lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8+cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8+T cells. Moreover, this helper effect of CD4+T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8+CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4+T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.