Nach oben pdf Provision of safe donor pigs for xenotransplantation

Provision of safe donor pigs for xenotransplantation

Provision of safe donor pigs for xenotransplantation

which are responsible of taking care of tissue preservation. Then embryonic stem cells from culture of preimplantation embryos, which are seen as pluripotent, as they can differentiate in any cells existing in the human body. And lastly reprogrammed adult cells, referred to as induced PSCs (iPSCs) (reviewed in LANE et al., 2014). The PSCs can be obtained by manipulating the stem cell microenvironment, or “niche”, to facilitate repair by endogenous stem cells (reviewed in LANE et al., 2014). The niche was first described by R. Schofield in 1978 (SCHOFIELD, 1978) as hypothetical interaction of stem cells and their environmental cells. There are niches in many tissues, for example in skin (FUCHS, 2009), intestines (BARKER, 2014; TAN & BARKER, 2014), and the nervous system (CONOVER & NOTTI, 2008). Regarding the heart or the myocardial tissue, it was shown, that PSCs can differentiate into cardiomyocytes in animal models (LAFLAMME et al., 2007; XIONG et al., 2013). Improvement of the function of infarcted hearts, that were transplanted with mouse and guinea pig PSC-derived cardiac progenitors (SHIBA et al., 2012) and even long-term benefit has been reported in the repair of a damaged heart (HARRIS et al., 2007; SCHOLL et al., 2010). Importantly, induced PSCs proved as an innovative and attractive source of PSCs, simply generated from differentiated cells by somatic cell reprogramming (TAKAHASHI & YAMANAKA, 2006). Still, in many cases organs are so severely damaged that cellular regeneration is believed to be of little use and replacement by a “new” heart is the only chance to save patients´ lives (reviewed in MOU et al., 2015). On the other hand, the availability of personalized, autologous iPSC, stimulated also the field of tissue engineering (reviewed in BERTHIAUME et al., 2011). The term tissue engineering comprises living cells, biocompatible materials and suitable biochemical factors for creating tissue-like structures. State-of-the-art approaches use scaffolds made from naturally derived and synthetic polymers, bioresorbable inorganic materials, hybrids, or decellularized tissue scaffolds (reviewed in PINA et al., 2019), which are then cellularized either with iPSCs or donor cells. This was done, for example with rat kidneys and discarded human transplant kidneys, using human inducible pluripotent stem cell–derived endothelial cells for re‐ endothelialization (LEUNING et al., 2019). This may be a possible application in the future, but, as the heart is a very complex organ and consisting of multiple cell types, with various functions, an appropriate multi-cellular composition, which resembles the heart does not exist until now (OWEN & HARDING, 2019).
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(Re)producing transgenic pigs for xenotransplantation – selection of founder animals and establishment of breeding herds

(Re)producing transgenic pigs for xenotransplantation – selection of founder animals and establishment of breeding herds

The importance of pigs in translational biomedical research has been on a constant increase, as their anatomical and physiological suitability as model animals is distinct (Aigner et al. 2010). Furthermore, pigs are considered a feasible source of replacement organs or tissues in the context of xenotransplantation (Petersen et al. 2009). But potential donor animals need to be tailored in their genetic properties as an imperative prerequisite for overcoming detrimental graft rejection processes (Sachs and Galli 2009). Somatic cell nuclear transfer has evolved into the preferential transgenic technology for achieving this (Melo et al. 2007). However, even though it is a successful method for generating novel transgenic pig lines, efficiency in large scale reproduction of already established lines has been disappointingly low (Palmieri et al. 2008). A feasible rectification of this issue can be found in the establishment of breeding herds where transgenic pigs are expanded by means of natural reproduction. By this, substantial numbers of experimental animals can be generated within a viable time frame. The conflicting matters of inbreeding and segregation of multiple transgenes, however, have to be taken into account. Rising inbreeding coefficients have been connected to lower productivity of breeding stock (Charlesworth and Charlesworth 1987; Ralls et al. 1988; Lynch 1989; 1991). While homozygosity of transgene integration sites would rectify the problem of transgene segregation and limit time requirements, it can only be achieved on the expense of inbreeding. Reproduction of already established (multiple) transgenic pigs by breeding can therefore be accomplished if the issues of time, transgene segregation and inbreeding are weighed against each other and a suitable breeding strategy that accommodates all of them is identified. When incorporating novel transgenes into already established breeding herds, selection of transgenic founder animals has to be performed on the basis of careful evaluation of genomic and expression analyses in order to be able to fully exploit cumulative effects of transgenes in multiple transgenic animals.
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Detection of a gammaretrovirus, XMRV, in the human population: Open questions and implications for xenotransplantation

Detection of a gammaretrovirus, XMRV, in the human population: Open questions and implications for xenotransplantation

XMRV (xenotropic murine leukaemia virus-related virus) is a gammaretrovirus that has been detected in human patients with prostate carcinoma, chronic fatigue syndrome (CFS) and also in a small percentage of clinically healthy individuals. It is not yet clear whether the distribution of this virus is primarily limited to the USA or whether it is causally associated with human disease. If future investigations confirm a broad distribution of XMRV and its association with disease, this would have an impact on xenotransplantation of porcine tissues and organs. Xenotransplantation is currently being developed to compensate for the increasing shortage of human material for the treatment of tissue and organ failure but could result in the transmission of porcine pathogens. Maintenance of pathogen-free donor animals will dramatically reduce this risk, but some of the porcine endogenous retroviruses (PERVs) found in the genome of all pigs, can produce infectious virus and infect cultured human cells. PERVs are closely related to XMRV so it is critical to develop tests that discriminate between them. Since recombination can occur between viruses, and recombinants can exhibit synergism, recipients should be tested for XMRV before xenotransplantation.
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Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation

Distribution of Porcine Cytomegalovirus in Infected Donor Pigs and in Baboon Recipients of Pig Heart Transplantation

a large number of individuals die on the waiting list for the required organ [ 2 ]. Pigs are for several reasons the most suitable donor species. These reasons include the size of the organs, the functional compatibility (for example, pig insulin has been used successfully for the treatment of diabetes over decades), and the ability to create cloned and genetically modified pigs in a short time [ 3 , 4 ]. However, the transplantation of pig cells, tissues, and organs may not only save and prolong human life, it may also be associated with the transmission of potentially zoonotic porcine microorganisms, and the porcine cytomegalovirus (PCMV) among them [ 5 , 6 ]. PCMV is widely distributed among pigs; it is a rather stable DNA virus that can easily be transmitted from pig to pig by milk, urine, and faeces [ 7 , 8 ]. Transplacental transmission of PCMV has also been described [ 8 , 9 ]. When evaluating the risk posed by PCMV, it was found that the survival time of pig kidneys from PCMV-infected pigs transplanted into baboons [ 10 ] or cynomolgus monkeys [ 11 ] was significantly reduced, indicating that PCMV may also pose a risk for clinical xenotransplantation [ 12 – 14 ]. The transmission of PCMV was also observed after pig heart transplantation, which was associated with injury of the transplant, and an increased incidence of consumptive coagulopathy [ 15 ]. Early weaning excluded PCMV, resulted in a prolonged survival of the transplant, and prevented consumptive coagulopathy [ 15 ].
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Xenotransplantation — A special case of One Health

Xenotransplantation — A special case of One Health

The chronic shortage of human transplants to treat tissue and organ failure has led to the development of xeno- transplantation, the transplantation of cells, tissues and organs from another species to human recipients. For a number of reasons, pigs are best suited as donor animals. Successful, routine xenotransplantation would have an enormous impact on the health of the human population, including the young, who sometimes require a re- placement organ or islet cells, but especially the elderly, who more often suffer the consequences of organ failure. The first form of xenotransplantation applied to humans is the use of pig islet cells to treat insulin-dependent di- abetes, a procedure that will have a significant economic impact. However, although xenotransplantation using pig cells, tissues and organs may save and prolong the lives of patients, it may also be associated with the trans- mission of porcine microorganisms to the recipient, eventually resulting in emerging infectious diseases. For this reason, the health of both the donor animals and the human recipients represents a special and sensitive case of the One Health concept. Basic research leading to strategies how to prevent transmission of porcine microorgan- isms by selection of virus-free animals, treatment of donor pigs by antiviral drugs, vaccines, colostrum depriva- tion, early weaning, Caesarean delivery, embryo transfer and/or gene editing should be undertaken to supply an increasing number of potential recipients with urgently required transplants. The methods developed for the detection and elimination of porcine microorganisms in the context of xenotransplantation will also contrib- ute to an improvement in the health of pig populations in general and an increase in the quality of meat products. At present, there is evidence for transmission of porcine viruses to humans eating pork and having contact with pigs, however the impact of these viruses on public health is still unknown.
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Porcine Circoviruses and Xenotransplantation

Porcine Circoviruses and Xenotransplantation

There is no specific treatment for pigs with PCVD. Anti-inflammatory agents and antimicrobials may help to suppress co-factors and secondary diseases associated with PCVD. All in/all out pig flow, thorough cleaning, and rigid disinfection between batches of pigs are measures that can help control the disease [103]. In the case that the donor animal is PCV-infected, it may be considered to analyze whether the xenotransplantation product (e.g., isolated islet cells) is still negative. Since PCV2 is infecting macrophages, certainly all organs are infected and it will be safer to use only negative animals, especially since no effective antiviral treatment is available. PCV2 infection is associated with an immune response including neutralising antibodies, and these coincide with a decrease in serum virus load. Cell-mediated immunity has also been shown to be necessary to control PCV2 infection (for review see [102]). PCV2 vaccines became commercially available in the summer of 2006 (Table 1) [103]. The vaccines reduced the severity and incidence rate of PCVD on many farms. Vaccination against PCV2 did not only imply a direct beneficial effect on pig productivity, but also contributed to reduction of antimicrobial use [104]. PCV2 vaccines effectively increased average daily weight gain (ADWG) and prevented diseases with a positive result for meat production. In all vaccination trials a lower virus load was registered in the vaccinated animals, however, it remains unclear whether the virus load is reduced to zero. In most reported cases, virus transmission took place despite vaccination [102,105–107]. In a study vaccinating 28 pigs, the virus load was not reduced to zero in any of the animals [108]. In another study, 17 of 32 vaccinated animals still showed PCV in the serum, as measured by PCR [90]. PCV2 vaccination of sows was associated with high antibody responses, but did not prevent fetal infections in utero or soon after birth by infectious colostrum in 29 of 100 cases [107]. When comparing four different vaccines, use of the inactivated chimeric vaccines (Fostera PCV and Circovax) resulted in significantly lower viremia compared with use of the subunit vaccines (Circoflex, Porcilis PCV), however, histopathological lesions and PCV antigens were still detected in all 80 immunized animals [109]. Successful vaccination is mainly associated with induction of neutralizing antibodies, but T cell-mediated immunity also plays a role in the reduction of the virus load and prevention of diseases as mentioned above [102,110].
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Approaching virus safety in xenotransplantation: A search for unrecognized herpesviruses in  pigs

Approaching virus safety in xenotransplantation: A search for unrecognized herpesviruses in pigs

Herpesviruses are known to cause morbidity and mortality in patients who have received allotransplantations. Besides activation of latent infection in the recipient, infections occur particularly through virus transmission from the donor-derived cells or organs. Similarly, in xenotransplantation animal herpesviruses could be transmitted to the xenograft recipient and cause xenozoonotic disease [1]. Beta- and gammaherpesviruses are of particular concern, because they are known to reside latently in lymphocytes and macrophages [2,3,4] which are constituents of all vascularized tissues and organs intended for use in xenotransplantation. Therefore a comprehensive knowledge of the herpesviruses infecting potential donor animals is of great value for safe xenotransplantation. The species currently favoured as a donor of xenografts is the pig [5,6]. Remarkably, until recently only two species, one alphaherpesvirus (pseudorabies virus [PRV], suid herpesvirus 1) [7] and one betaherpesvirus (porcine cytomegalovirus [PCMV], suid herpesvirus 2) [8], were known. This changed with the discovery of two closely related gammaherpesviruses in pigs, the porcine lymphotropic herpesviruses 1 and 2 (PLHV-1, PLHV-2) [9,10,11] and, more recently, of a third porcine gammaherpesvirus (PLHV-3) [12]. Having developed detection methods for these viruses, attempts can be started to raise PLHV-free pigs, and to monitor patients undergoing xenotransplantation for inadvertent virus transmission. However, unrecognized herpesviruses would be excluded from these measures and would therefore represent an unassessable risk potential in xenotransplantation.
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Deleting PERV C infectious potential from donor pigs for xenotransplantation

Deleting PERV C infectious potential from donor pigs for xenotransplantation

To minimize the infectious risk from PERV for pig xenograft recipients the first thing to do is to select donor pig that have the lowest possible copy number of PERV C in their genome. By using screening methods like PCR, nested PCR, real time PCR (RT- PCR), reverse transcriptase RT-PCR or co-culture pig-human cell assays it was found that the copy number of PERV C in pigs is generally low and even PERV C free pigs have been identified (DENNER et al., 2009; KAULITZ et al., 2011b; FIEBIG et al., 2018). Most existing genetically modified donor pigs for xenotransplantation, however, contain PERV C proviruses. Different PERV inactivating approaches might reduce the risk of transmission. As for other gamma retroviruses, the establishment of vaccines against PERV can protect the human recipient. First PERV vaccines have been developed on the basis of neutralizing antibodies against membrane proximal external region in the N-terminal of the TM envelope protein (FIEBIG et al., 2003; KAULITZ et al., 2011a; WAECHTER & DENNER, 2014). Simultaneous immunization with TM and SU proteins revealed a higher neutralizing activity as with the respective proteins alone (DENNER et al., 2012). Other strategies target PERV at the nucleic acid level. For example, the inhibition of PERV expression might be mediated through short interfering RNA (siRNA), a mechanism which is known to be common in eukaryotic cells to suppress protein synthesis and to act as anti-viral responses (CAPLEN et al., 2001). siRNAs are double strand RNA components with a length of about 22 nucleotides. They are recognized by RNA induced silencing complex (RISC) which induces the degradation of RNA targets that are homologous to the siRNA (YANG et al., 2000). Different studies were conducted by introducing siRNA against conserved regions of PERV into PERV-infected human cell lines (KARLAS et al., 2004), porcine cells (MIYAGAWA et al., 2005) and in vivo in transgenic pigs (DIECKHOFF et al., 2008; RAMSOONDAR et al., 2009). All these attempts worked efficiently and demonstrated that pol siRNA expressed in transgenic pigs reduced PERV expression over 3 years without adverse effects (SEMAAN et al., 2012).
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Is it Safe for Me to Fly?

Is it Safe for Me to Fly?

Do the world statistics on aviation safety paint the full picture? No, they do not. Table 1, using International Air Transport Association (IATA) data, presents statistics on aircraft hull losses in the world’s regions as a rate per million flights. A hull loss means the aircraft was totally destroyed or damaged beyond economic repair, so it is a sensible indicator of aircraft operators’ safety performance in the different regions. (It has limitations as an accident severity metric, as an ageing world fleet and the economics of repairs could tend to mean that less severe accidents become hull losses.) The first point to stress is that these rates derive from a statistically small number of events, the rates for the individual regions typically being based on no more than three or four accidents. Assuming something like a Poisson distribution of the number per year, there will be large variations in the rate for each region from year to year. Second, there are strong indications that the rate for Africa is markedly higher than the average: note the numbers in bold italic font, which are those that are more than twice the world average in that year. Improving the safety of operations in Africa is a high priority for IATA, ICAO and other safety organisations.
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Geofencing requirements for onboard safe operation monitoring

Geofencing requirements for onboard safe operation monitoring

(Fig.  1 ). According to it, the necessary certification effort scales with the actual risks of the operation of interest. The specific category uses a so-called specific operation risk assessment (SORA) for analysis and categorizes the required level of rigour for UAS development and operation [ 4 ]. This approach is not targeted solely on the UAS, but towards the operation of a specific UAS in its entirety, including: the mission, the environment, operation conditions, rigour dur- ing development as well as operator and pilot qualification. The DLR (German Aerospace Center) is currently applying the SORA to a cargo application with the project ALAADy (automated low altitude air delivery) [ 5 ] and investigates different means to exploit the advantages of the new specific category concept. In this project, differ- ent drone configurations are compared, all of which carry approximately one metric ton of payload. The mission is cargo delivery on a range of around 600 km flying over sparsely populated areas. The drones are intended to fly at low altitude to circumvent most of the air traffic. Technologi- cally and economically, different concepts are analyzed and * Christoph Torens
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Designing better donor strategies for inclusive business

Designing better donor strategies for inclusive business

Finally, the basis for justifying interventions in inclusive business needs to be strengthened considerably in order to improve and inform future decision making for donors as there is – so far – weak ground for arguments based on inclusive business impacts. There are hardly any impact analyses of inclusivity aspect of business or different donor instruments that adhere to scientific standards. In particular, existing studies focus on case studies with little external validity, lacking counterfactuals and report mainly potentials rather than demonstrated impacts. Hence, there is considerable insecurity on what contribution to inclusivity can be expected from businesses, in addition to the insecurity about what levels of inclusivity in which sectors and contexts are financially viable. Establishing causality in measuring impact on the target group (low-income people) is not easy, and measuring overall development impacts is even more difficult, i.e. time-consuming and costly. What makes this question even more complex is the dynamics with which these factors change over time. Hence donors should invest in empirical studies that try to find suitable counterfactuals and use state of the art methods to establish causality, as well as establishing assessment models and comparable indicators.
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Nonprofit Donor Relationship Management : An Evaluation of Blood Donor Relationship Marketing Strategies

Nonprofit Donor Relationship Management : An Evaluation of Blood Donor Relationship Marketing Strategies

However, differentiated marketing is only a recommendation in the field of nonprofit research, especially in the blood donation context, rather than an applied strategy. Moreover, research focusing on differentiation further stated that differentiated marketing increases the recruitment success of organizations by creating appeals that precisely match the preferences of segments (Dibb and Simkin 2010; Kotler and Levy 1986; Manickam 2014). For example, the German Red Cross Blood Donor Service North-East (GRC) realized that, to date, only a few blood services segment their targets and that commonly used marketing approaches mostly are undifferentiated. However, marketing campaigns, whether differentiated or not, attract attention from potential blood donors. Surprisingly, research focusing on the blood donor market strongly recommended using differentiated marketing to increase blood donations more effectively (e.g. Zhou et al. 2012; Shehu et al. 2015), but do not analyze the effectiveness of differentiated marketing campaigns in a next step. For example, Zhou et al. (2012) examined the social values, lifestyles, and attitudes of Chinese blood donors and identified three major blood donor segments. The authors concluded their research by recommending the usage of specific types of advertising appeals and slogans that blood services might use to address the three identified segments. Another study by Shehu et al. (2015) identified four segments of blood donors and recommend using targeting strategies to address the identified segments. Summing up, these studies only recommend that managers implement different campaigns for each identified segment (Veldhuizen et al. 2013; ***). As mentioned previously, empirical tests of the effectiveness of differentiated marketing, especially in a blood donation context, are lacking. Therefore, our experiment compares a
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Risk management for assuring safe drinking water.

Risk management for assuring safe drinking water.

Water Treatment Plants (WTPs) Groundwater arsenic contamination WTP reliability Shallow groundwater contamination WTP design capability - toxins Reservoir & catchment contamination WTP design capability - alkalinity Reservoir contamination & disinfection failure WTP design capability - colour Groundwater contamination not located WTP design capability - iron & manganese Suspected breaching of mains WTP design capability - industrial chemicals Distribution system contamination WTP design capability - pathogens Unidentified contamination WTP design capability - taste & odour Overdose of fluoride
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Type-safe Programming for the Semantic Web

Type-safe Programming for the Semantic Web

Graph-based data models allow for a flexible representation of data that is useful for capturing knowl- edge. In particular, data based on semantic data models, where conceptualizations and schemata are stored inside the data as part of the graph, have grown considerably and fuel many different appli- cations. Most prominently, the knowledge graphs by Google and Microsoft enhance Internet search. Another example is Wikidata [91], an open-source knowledge graph storing several billion semantic statements which are, among others, used in Wikipedia. Schema.org 1 provides schematic information for data which is being used both to enhance search as well as in personal assistants such as Google Now and Cortana. Based on Schema.org, Google stores more than three trillion facts that are extracted from of the Web [73]. A key factor in the popularity of knowledge graphs is their ability to deal with a large variety in the captured knowledge that arises, for example, when integrating different data sources. Figure 1.1 shows an abstracted version of such a knowledge graph.
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Metallic iron for safe drinking water worldwide

Metallic iron for safe drinking water worldwide

It has already been demonstrated that the oxide film on Fe 0 is beneficial for the process of contaminant removal [51-54,65]. In fact, the oxide film acts as contaminant scavenger and the contaminant may be further chemically transformed (oxidized or reduced if applicable). The presentation above clearly disprove the validity of Assumption 1 and corroborates results from other branches of science that iron corrosion is always coupled to oxide film formation at pH > 4.5. Iron corrosion continues under the film because the film is porous and permeable to water and other oxidizing agents [51,52,65-68]. More importantly, using Fe 0 to assist sand filtration does not aim at inducing reductive transformations of contaminants, but to use the process of iron corrosion and the adsorptive properties of in situ generated iron oxides to sustain the filtration process. In other words, in Fe 0 filters, Fe 0 is oxidized by H 2 O and corrosion products are used as trap for contaminants which could be chemically transformed. Depending on their nature and concentration, selected contaminants may influence (inhibit or sustain) the process of iron corrosion. For example, it is well-established that the incorporation of a cation into the structure of iron (oxyhydr)oxides alters the nucleation, crystal growth, and transformation [69]. This impact of the alteration on the further iron corrosion should be carefully characterized in laboratory and field investigations.
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Minimum participation rules for the provision of public goods

Minimum participation rules for the provision of public goods

This paper considers the endogenous formation of an institution to provide a public good. If the institution governs only its members, players have an incentive to free ride on the institution formation of others and the social dilemma is simply shifted to a higher level. Addressing this second-order social dilemma, we study the effectiveness of three different minimum participation requirements: 1. full participation / unanimity rule; 2. partial participation; 3. unanimity first and in case of failure partial participation. While unanimity is most effective once established, one might suspect that a weaker minimum participation rule is preferable in practice as it might facilitate the formation of the institution. The data of our laboratory experiment do not support this latter view, though. In fact, weakening the participation requirement does not increase the number of implemented institutions. Thus, we conclude that the most effective participation requirement is the unanimity rule which leaves no room for free riding on either level of the social dilemma.
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Metallic Iron for Safe Drinking Water Production

Metallic Iron for Safe Drinking Water Production

the failure to treat properly the combined problem of mass transport and chemical reaction in these complex systems. Well-mixed batch experiments that have been undertaken in order to circumvent the mass-transport problem associated with bulk solutions have not always adequately addressed these key issues. Mixing intensity may not only affect the hydrodynamic but also the chemical dynamics, in particular the formation of the oxide-film. A critical review on the process of oxide scale formation and its impact on the process of mass-transport to the Fe 0 surface, have demonstrated that well-mixed batch systems are not an effective tool for investigating the mechanism of aqueous contaminant removal by Fe 0 . In fact, any mixing operation (e.g. stirring, shaking) increases corrosion rate, delays the formation of the oxide scale, or provokes its abrasion. The critical review has let to the conclusions that quantitative contaminant removal occur within the oxide scale on Fe 0 . Consequently: (i) non-shaken batch experiments are proposed as a simple tool to investigate mass-transport limitation through oxide- films at laboratory scale, (ii) experiments characterizing interactions in Fe 0 /H 2 O systems should
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Safe Cooperation of Automated Vehicles

Safe Cooperation of Automated Vehicles

of a Request message have the option to ignore the Request, or to answer it with a Promise message. A request message is not answered, if the recipient cannot or does not want to guarantee integrity of the reservation. E.g. vehicle 𝑣𝑣 3 in fig. 9 could prefer to be uncooperative, in order to maintain a steep emergency maneuver. A Promise message is realized as a data structure containing the three IDs (𝑗𝑗, 𝑟𝑟, 𝑖𝑖). If a recipient of a Request decides to reply with a Promise message, it has to test first, whether the continued existence of at least one emergency maneuver is guaranteed under the tightened constraints. The question can be decided by re- planning the emergency maneuver under the tightened constraints, yet this could incur unwanted computational demands if many Requests are received. In our approach we opt to test for intersection between the current emergency maneuver and the reservation to determine whether they are compatible. The test is carried out by the Contract Validation module, see fig. 2. This simpler test is guaranteeing correctness, but is more conservative and less cooperative than re-planning. Before a Promise message is issued, the Supervisor module assures that an entry is made in the Occupied set. In this way, eq. (5.15) and therefore safety of the cooperation is guaranteed, irrespective of the performance of the communication layer. Due to the possibility of message loss, eq. (5.15) is not an equivalence relationship. The proposed protocol is guaranteeing safety and is also resilient to misuse, as reservations are limited in space and are only relevant with respect to the reserving vehicle’s actual state.
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PERV und Xenotransplantation:  Impfstoffe, RNAi und genotypische Selektion

PERV und Xenotransplantation: Impfstoffe, RNAi und genotypische Selektion

Der Neutralisationsassay wurde mit virushaltigen Überständen PERV/5°-infizierter HEK 293- Zellen durchgeführt. 100 µl uninfizierte Zellen wurden in einer 96 Well Mikrotiterplatte eingesät und für 4 Stunden im CO 2 -Inkubator MCO-20AIC (Sanyo) bebrütet. 20 µl der dekomplementierten Seren wurden mit 80 µl verdünntem PERV/5° für 30 min bei 37 °C inkubiert und zu den 293-Zellen gegeben. Die Zellen wurden anschließend für 72 h bei 37 °C inkubiert, die Vitalität wurde lichtmikroskopisch am Inversmikroskop (Carl Zeiss AG) überprüft und das Medium wurde anschließend entfernt. Die Zellen wurden bei 95 °C für 30 min inaktiviert (Inkubator Heraeus instruments) und anschließend bei -20 °C gelagert. Die Lyse der Zellen erfolgte durch Zugabe von 100 µl Lysepuffer (nukleasefreies Wasser mit 0.2 mg/ml Proteinase K und 10 % (v/v) 10 × PCR-Puffer) bei 60 °C für mindestens 3 Stunden. Die Proteinase K wurde bei 95 °C für 30 min hitzeinaktiviert. Jeweils 3 µl des Lysates wurden in der PERV-Duplex Real- time PCR pro Reaktionsansatz eingesetzt Für die Quantifizierung wurden die Primer/Sonden Kombinationen gag-for/gag-rev/gag-Sonde und huGAPDH-for/huGAPDH-re/huGAPDH-Sonde verwendet (siehe Tabelle 9). Die optimale Virusverdünnung wurde durch Titration des virushaltigen Überstandes auf 293-Zellen ermittelt. Verdünnungen, die in der Real-time PCR zu stabilen Ct-Werten zwischen 25 und 27 führen, sind geeignet.
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OECD Xenotransplantation Policies and Public Participation

OECD Xenotransplantation Policies and Public Participation

Abdallah Daar reiterated previous clinical xenotransplantation experiments and facts that contributed to public awareness. He referred to the public via opinion polls as a survey public. However, for methodological reasons he was not uncritical of existing studies that had used questionnaires and focus groups. How do you address problems, which are not known? What do respondents know about the details of xenotransplantation? He described several scenarios, which would negatively affect public opinion. These included “ill-planned” transplant from non-human primates, discovery of new dangerous viruses, death of a patient from a pig virus infection, discrediting of an oversight authority to fulfill its tasks and creation of “widespread negative publicity” (ibid. 228). Daar concluded his presentation with lessons learned and set out a strategic map for gaining public acceptance, which included the role of the public (“publicity cuts both ways”), media (“can be fickle”), lobbying (“it works”), the importance of primacy, the imponderability of research, the uncertainty about infection risk, the easiness to find patients for clinical trials, the role of animal groups in shaping public attitude, the role of patients’ advocacy groups (balancing the negative publicity of animal rights groups), and differences between the United States and Europe with regards to a moratorium (Europe being more in favor of a moratorium than the US). He also agreed to the necessity of some sort of “community consent” but said that “at present we have no sensible idea how to obtain such consent” (ibid. 230).
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