86 research outputs found
Hands-on Testing of Last Mile Concepts
AbstractSince 1995 the freight transport performance within Europe has increased annually by 1.2% and is expected to grow further over the coming decades. Being the sources and sinks of transport activities, Europe's cities are strongly affected by the resulting undesirable effects of transportation. This applies in particular to North West Europe (NWE), one of the most urban regions in Europe. NWE therefore stands in urgent need of solutions for efficient urban freight logistics.Focusing on the last mile of freight deliveries the INTERREG IVB NWE project LaMiLo (Last Mile Logistics) addressed this issue. The project promoted Urban Consolidation Centres (UCCs) as they allow and encourage transport consolidation as well as modal shift. Decoupling the last mile at the city borders, large vehicles are used for bundled, long haul transports outside the city whilst sustainable transport means are used inside.In the knowledge that UCC attempts with huge government subsidies have been made several times before but rarely succeeded, the core of the LaMiLo project was to enable environment for UCC operations that accomplish a sustainable last mile and can as well be transformed in successful business models. Thereto, UCC concepts were operationalised and implemented in a number of pilots in metropolitan areas and cities all around NWE:âąParis: A UCC was field-tested to which freight was brought by truck and afterwards delivered by cargo bikes.âąNetherlands: Central hubs were put into operation to streamline the home delivery of parcels. Thereto, a Freight Circle service was launched in the cities of Nijmegen and Maastricht which used e-bikes and enabled customer-specific delivery times.âąLondon: A UCC was set up to bundle goods for the public sector which were destined for the boroughs of Camden and its partners Enfield and Waltham.âąBrussels: A UCC was launched at the Port of Brussels offering warehouse services and deliveries to retailers in the city.The key results gained from the studies primarily include the demonstration of UCCs as possible last mile solutions. Beyond their economic performance, their environmental effects as well as their social impacts were evaluated. Exemplary results include that all the pilots contributed towards emission reduction and total distance travelled through city centres and residential areas. Gained best practice and lessons learnt about the examined UCC concepts were discussed and shared at transnational level to encourage relating solutions for further cities
Charakterisierung der funktionalen Rolle von Cytohesin-1 in der LFA-1-vermittelten T-Zell-AdhÀsion
Das Leukocyten-spezifische Integrin LFA-1 spielt eine wichtige Rolle bei der Immunantwort,
durch die Vermittlung dynamischer Zell-Zell- bzw. Zell-Matrix-Interaktionen. Die kontrollierte
AdhÀsion bzw. DeadhÀsion von Leukocyten bedarf einer spezifischen Regulation des
LFA-1-Integrins und die AufklÀrung der molekularen Grundlagen dieser VorgÀnge ist von
groĂem Interesse.
Cytohesin-1 war unmittelbar vor Beginn dieser Arbeit als cytoplasmatischer Regulationsfaktor
der durch LFA-1 vermittelten ZelladhÀsion identifiziert worden und seine spezifische Interaktion
mit der cytoplasmatischen DomÀne von CD18 konnte in vitro dokumentiert werden.
Im Rahmen dieser Arbeit gelang es zunÀchst, die Assoziation von Cytohesin-1 und LFA-1
auch endogen, im intakten Zellverband, mittels Kolokalisationsstudien in der lymphoblastoiden
B-Zellinie LCL-721, zu demonstrieren. Ferner konnte mit Hilfe von Mutationsanalysen
die, fĂŒr die Interaktion kritische Region in der cytoplasmatischen DomĂ€ne von CD18 lokalisiert
werden. Sie befindet sich im aminoterminalen Bereich und umfaĂt die AminosĂ€uren
WKA(723 - 725). Die Mutation dieser AminosÀurereste nach TRG resultierte in einem vollstÀndigen
Interaktionsverlust mit Cytohesin-1. Die Inhibition der Cytohesin-1/CD18-Bindung
konnte dabei sowohl durch Protein-Protein-Interaktionsanalysen in Hefe als auch durch biochemische
Bindungsstudien in vitro dokumentiert werden, wobei jeweils Fusionsproteine der
cytoplasmatischen DomÀne von CD18 charakterisiert wurden.
Funktionale Analysen der WKA(723-725)-Region von CD18 ergaben, daĂ die Mutation von
WKA(723-725) nach TRG im intakten LFA-1-MolekĂŒl eine signifikante Reduktion der Integrin-
AktivitÀt zur Folge hatte. Sowohl T-Zellklone als auch nicht hÀmatopoetische Zellen,
wie HeLa, wiesen nach Expression von LFA-1(TRG), mit Hilfe rekombinanter Vaccinia-
Viren, eine stark reduzierte AdhÀsionsfÀhigkeit an immobilisiertes ICAM-1 auf. Ferner ergaben
funktionale Studien mit HeLa-Zellen, die LFA-1 stabil exprimierten, daĂ Cytohesin-1 nur
dann eine gesteigerte AdhÀsion dieser Zellen an ICAM-1 induzierte, wenn sie Wildtyp-LFA-1
exprimierten. HeLa-Zellen, die LFA-1(TRG) exprimierten, lieĂen sich durch Cytohesin-1 zu
keiner verstÀrkten AdhÀsion aktivieren.
Diese Ergebnisse demonstrierten die Bedeutsamkeit der Cytohesin-1/CD18-Interaktion fĂŒr
eine effiziente, durch LFA-1 vermittelte ZelladhÀsion. Unklar war jedoch der Mechanismus,
durch den Cytohesin-1 die Integrin/Liganden-Bindung regulierte. Studien mit dem Reporterantikörper
24 lieĂen darauf schlieĂen, daĂ Cytohesin-1 durch die Bindung an CD18 eine KonformationsĂ€nderung in der extrazellulĂ€ren DomĂ€ne des LFA-1-Integrins induzieren
konnte, die möglicherweise die AffinitÀt des Rezeptors modulierte. Diese Modulation der
LFA-1-Konformation schien jedoch nicht hinreichend fĂŒr eine stabile Bindung an ICAM-1 zu
sein, wie eingehendere Analysen von Dr. W. Kolanus zeigten. Vielmehr erforderte eine effiziente
ZelladhÀsion zusÀtzlich die Guaninnukleotid-Austauschfunktion (GEF-Funktion) von
Cytohesin-1, da die GEF-defekte Punktmutante, Cytohesin-1(E157K), nicht mehr in der Lage
war, die AdhÀsion von Jurkat E6-Zellen an ICAM-1 stabil zu induzieren. Biochemische Interaktionsstudien
konnten dabei zeigen, daà die Mutante weiterhin fÀhig war, die cytoplasmatische
DomÀne von CD18 zu binden.
Diese und weitere Ergebnisse von Dr. W. Nagel, die einen Zusammenhang zwischen der
GEF-Funktion von Cytohesin-1 und dem âSpreadingâ von adhĂ€renten Jurkat E6-Zellen aufzeigten,
legen die Vermutung nahe, daĂ Cytohesin-1 durch einen dualen Mechanismus in die
LFA-1-Regulation involviert ist. Sowohl die direkte Interaktion von Cytohesin-1 und dem
Integrin als auch seine GEF-Funktion stellen essentielle Faktoren fĂŒr eine stabile ZelladhĂ€sion,
die durch LFA-1 vermittelt wird, dar. Welche funktionalen Mechanismen dabei durch den
Guaninnukleotid-Austausch und der damit verbundenen Aktivierung einer GTPase induziert
werden, ist noch unklar. PrimÀr wÀre eine Modulation des Aktin-Cytoskelettes und eine damit
verbundene erhöhte laterale MobilitÀt der Integrine denkbar, die eine verstÀrkte Rezeptormultimerisierung
und dadurch eine AviditÀtsÀnderung des Integrins ermöglicht.
Weitere Studien dieser Arbeit analysierten die Regulation von Cytohesin-1 selbst. Es konnte
gezeigt werden, daĂ PI3-Kinase in die Kontrolle der Cytohesin-1-Funktion involviert war.
Die Ăberexpression einer konstitutiv aktiven Form dieser Kinase (P110*) fĂŒhrte zu einer gesteigerten
AdhÀsion von Jurkat E6-Zellen an ICAM-1. Eine Inkubation dieser Zellen mit dem
PI3-Kinase-spezifischen Inhibitor Wortmannin resultierte dagegen in einer signifikanten Reduktion
der ZelladhÀsion.
Weitere funktionale Analysen, die die ZelladhÀsion von Jurkat E6-Zellen nach Koexpression
von P110* und der PH-DomÀne von Cytohesin-1 untersuchten, sowie eingehendere Studien
von Dr. W. Nagel, ermöglichten die Entwicklung eines Modells zur Regulation von Cytohesin-
1. Demzufolge fĂŒhrt die Aktivierung der PI3-Kinase zu einer verstĂ€rkten Rekrutierung
von Cytohesin-1 an die Plasmamembran. Als Rekrutierungsmodul fungiert dabei die PHDomÀne,
die durch Bindung von PtdIns(3,4,5)P3, einem Produkt der PI3-Kinase, die Assoziation
mit der Membran gewĂ€hrleistet. Die Rekrutierung von Cytohesin-1 an die Plasmamembran fĂŒhrt zur Aktivierung von LFA-1 und der damit verbundenen stabilen ZelladhĂ€sion an ICAM-1
A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma
We present a generic dendritic cell (DC) vaccine strategy for patients with renal cell carcinoma (RCC) based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs. Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs) specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naĂŻve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP)-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease
Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation
<p>Abstract</p> <p>Background</p> <p>Antigen-loaded dendritic cells (DC) are capable of priming naĂŻve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) <it>in vitro</it>. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.</p> <p>Methods</p> <p>In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.</p> <p>Results</p> <p>Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of <it>in vitro </it>transcribed (<it>ivt</it>)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of <it>ivt</it>RNA.</p> <p>Conclusions</p> <p>This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or <it>ivt</it>RNA as sources of specific antigen.</p
Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish
Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines
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Large-scale mapping of mutations affecting zebrafish development
BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the TĂŒbingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations
Photography-based taxonomy is inadequate, unnecessary, and potentially harmful for biological sciences
The question whether taxonomic descriptions naming new animal species without type specimen(s) deposited in collections should be accepted for publication by scientific journals and allowed by the Code has already been discussed in Zootaxa (Dubois & NemĂ©sio 2007; Donegan 2008, 2009; NemĂ©sio 2009aâb; Dubois 2009; Gentile & Snell 2009; Minelli 2009; Cianferoni & Bartolozzi 2016; Amorim et al. 2016). This question was again raised in a letter supported
by 35 signatories published in the journal Nature (Pape et al. 2016) on 15 September 2016. On 25 September 2016, the following rebuttal (strictly limited to 300 words as per the editorial rules of Nature) was submitted to Nature, which on
18 October 2016 refused to publish it. As we think this problem is a very important one for zoological taxonomy, this text is published here exactly as submitted to Nature, followed by the list of the 493 taxonomists and collection-based
researchers who signed it in the short time span from 20 September to 6 October 2016
A cluster of multidrug-resistant Mycobacterium tuberculosis among patients arriving in Europe from the Horn of Africa: a molecular epidemiological study
SummaryBackground The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. Methods On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis. In this molecular epidemiological study, we prospectively defined outbreak cases with 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) profiles; phenotypic resistance to isoniazid, rifampicin, ethambutol, pyrazinamide, and capreomycin; and corresponding drug resistance mutations. We whole-genome sequenced all Mycobacterium tuberculosis isolates and clustered them using a threshold of five single nucleotide polymorphisms (SNPs). We collated epidemiological data from host countries from the European Centre for Disease Prevention and Control. Findings Between Feb 12, 2016, and April 19, 2017, 29 patients were diagnosed with multidrug-resistant tuberculosis in seven European countries. All originated from the Horn of Africa or Sudan, with all isolates two SNPs or fewer apart. 22 (76%) patients reported their travel routes, with clear spatiotemporal overlap between routes. We identified a further 29 MIRU-VNTR-linked cases from the Horn of Africa that predated the outbreak, but all were more than five SNPs from the outbreak. However all 58 isolates shared a capreomycin resistance-associated tlyA mutation. Interpretation Our data suggest that source cases are linked to an M tuberculosis clone circulating in northern Somalia or Djibouti and that transmission probably occurred en route before arrival in Europe. We hypothesise that the shared mutation of tlyA is a drug resistance mutation and phylogenetic marker, the first of its kind in M tuberculosis sensu stricto. Funding The Swiss Federal Office of Public Health, the University of Zurich, the Wellcome Trust, National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC), the Medical Research Council, BELTA-TBnet, the European Union, the German Center for Infection Research, and Leibniz Science Campus Evolutionary Medicine of the Lung (EvoLUNG)
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