Characterisation of a SNARE-complex involved in Golgi-to-ER retrograde transport in mammalian cells

Intrazellulärer Transport vermittelt die Verteilung von Proteinen und Lipiden zwischen verschiedenen Kompartimenten. Dazu werden Vesikel vom Donorkompartiment abgeschnürt und zur Akzeptormembran transportiert, mit der sie fusionieren. Als zusätzlichen Weg betrachtet man den Transport in Form von Membrantubuli.SNARE Proteine erfüllen eine grundlegende Funktion beim Andocken und bei der Fusion zweier Organelle. Sie lagern vier alpha-Helices zu einem stabilen, parallelen Helixbündel zusammen, das die Fusion von Lipiddoppelschichten erleichtert. In der vorliegenden Arbeit wurde ein SNARE-Komplex in Säugerzellen untersucht, dessen homologe Proteine in Hefe nachweislich am retrograden Transport vom Golgi Apparat zum ER beteiligt sind. Der Komplex müsste analog zur Hefe aus folgenden SNAREs bestehen: mSec22b als R-SNARE und Syntaxin18, mUse1 und mSec20 als Q-SNAREs. Obwohl ein solcher Komplex von der 1R-3Q Regel abweicht, die zuvor für andere SNARE-Komplexe beschrieben wurde, konnte er in dieser Arbeit durch Fluoreszenzmessungen an lebenden Zellen (FRET und BiFC) und Koimmunpräzipitation nachgewiesen werden.Am Golgi-Apparat wird der Abtransport bestimmter Moleküle zum ER über den COPI-Adaptorkomplex und dessen Bindungspartner reguliert. Fehlgeleitete Proteine aus dem ER, die ein KDEL-Motiv am C-Terminus tragen, werden im Golgi durch den KDEL-Rezeptor Erd2 gebunden und zum ER zurücksortiert. Der Rezeptor wandert durch COPI-abhängigen Transport zwischen dem Golgi und dem ER. FRET-Messungen ergaben in dieser Arbeit, dass mSec22b, mUse1 und mSec20 jeweils an Erd2 binden. Ausserdem wurden mSec22b und Use1 durch Elektronenmikroskopie in COPI-Vesikeln nachgewiesen, die in einem zellfreien Assay aus dem Golgi entstanden waren. Antikörper gegen mUse1 präzipitierten auch beta´-COP (eine Untereinheit von COPI) und den KDEL-Rezeptor. Schliesslich konnte ich mit Hilfe der Expression von Choleratoxin als exogenem Frachtmolekül für den KDEL-Rezeptor zeigen, dass der retrograde Transport vom Golgi zum ER gestört wird, wenn man die Menge an mSec22b durch siRNA erniedrigt.Meine Ergebnisse weisen insgesamt stark auf eine Beteiligung der SNAREs mSec22b, mUse1, mSec20 und Syntaxin18 an mehreren Schritten des retrograden Transports zwischen Golgi und ER in Säugerzellen hin

Buzz: Face-to-Face Contact and the Urban Economy

This paper argues that existing models of urban concentrations are incomplete unless grounded in the most fundamental aspect of proximity; face-to-face contact. Face-to-face contact has four main features; it is an efficient communication technology; it can help solve incentive problems; it can facilitate socialization and learning; and it provides psychological motivation. We discuss each of these features in turn, and develop formal economic models of two of them. Face-to-face is particularly important in environments where information is imperfect, rapidly changing, and not easily codified, key features of many creative activities.Agglomeration, clustering, urban economics, face-to-face

Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction cells from human adipose tissue

Fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 are key supplements for the culture of stromal vascular fraction (SVF) cells from adipose tissue, both for typical monolayer (2D) expansion and for streamlined generation of osteogenic-vasculogenic grafts in 3D perfusion culture. The present study investigates whether factors present in human platelet lysate (PL) could substitute for FBS and FGF-2 in 2D and 3D culture models of SVF cells from human lipoaspirates. SVF cells were grown in medium supplemented with 10% FBS+FGF-2 or with 5% PL. In 2D cultures, PL initially supported SVF cell proliferation, but resulted in growth arrest shortly after the first passage. Freshly isolated SVF cells cultured with both media under perfusion for 5 days within 3D ceramic scaffolds induced bone formation after subcutaneous implantation in nude mice. However, blood vessels of donor origin were generated only using FBS+FGF-2-cultured cells. This was unexpected, because the proportion of CD34+/CD31+ endothelial lineage cells was significantly higher with PL than that of FBS+FGF-2 (33% vs. 3%, respectively). These results support the use of PL as a substitute of FBS+FGF-2 for short-term culture of human SVF cells, and indicate that more specific serum-free formulations are required to maintain a functionally vasculogenic fraction of SVF cells expanded under 3D perfusion

Changes in dairy cattle breeding goals and selection methods

Over the last decades, the dairy stock has undergone major changes, and the selection of dairy cattle has become an internal business.With current prospects of milk quota removal, falling prices, increasing herd sizes, changes in the farmers’ way of working and diversification of demand, the ideal cow of the future will have to demonstrate autonomy, robustness and feed efficiency, and will have to produce a rich milk whose composition meets the requirements of humans. Selection methods will change radically with the ongoing development of genomic selection. Animal identification, pedigree and performance recording will remain, but progeny testing of bulls will diminish. Resources will be pooled between research and supervision organisations, breeding companies and other operators, and there will be an increased need for training.Dans les dernières décennies, le cheptel a évolué en profondeur et la sélection des bovins laitiers s'est internationalisée. Dans un contexte prévisible de suppression des quotas, de baisse du prix du lait, d'accroissement de la taille des structures, d'évolution du rapport des éleveurs au travail et de diversification de la demande, la vache idéale de demain devra faire preuve d'autonomie, de robustesse et de sobriété, produisant un lait riche dont la composition répondra aux besoins de l'homme. Les méthodes de sélection vont radicalement changer avec le développement en cours de la sélection génomique. L'identification, l'état civil et le contrôle des performances se maintiendront mais l'épreuve de la descendance des taureaux d'insémination va régresser. La mutualisation des moyens sera renforcée entre organismes de recherche et d'encadrement, entreprises de sélection et opérateurs de base, et les besoins de formation vont s'amplifier

Phenotypic characterization of bone marrow mononuclear cells and derived stromal cell populations from human lliac crest, vertebral body and femoral head

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14-91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45-CD34-CD73+, CD45-CD34-CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head

Platelet-Rich Plasma as an Autologous and Proangiogenic Cell Delivery System

Angiogenesis is a key factor in early stages of wound healing and is crucial for the repair of vascularized tissues such as the bone. However, supporting timely revascularization of the defect site still presents a clinical challenge. Tissue engineering approaches delivering endothelial cells or prevascularized constructs may overcome this problem. In the current study, we investigated platelet-rich plasma (PRP) gels as autologous, injectable cell delivery systems for prevascularized constructs. PRP was produced from human thrombocyte concentrates. GFP-expressing human umbilical vein endothelial cells (HUVECs) and human bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in PRP gels in different proportions. The formation of cellular networks was assessed over 14 days by time-lapse microscopy, gene expression analysis, and immunohistology. PRP gels presented a favorable environment for the formation of a three-dimensional (3D) cellular network. The formation of these networks was apparent as early as 3 days after seeding. Networks increased in complexity and branching over time but were only stable in HUVEC-MSC cocultures. The high cell viability together with the 3D capillary-like networks observed at early time points suggests that PRP can be used as an autologous and proangiogenic cell delivery system for the repair of vascularized tissues such as the bone

Brachytic2/ZmABCB1 functions in IAA export from intercalary meristems

Dwarfism traits in Zea mays are regulated by multiple factors including the hormone auxin. Dwarf brachytic2 (br2) mutants harbour lesions in the gene encoding an orthologue of Arabidopsis thaliana ABCB1 which functions in auxin efflux out of meristematic regions in the shoot and root. br2 mesocotyls and coleoptiles exhibit reduced auxin transport. However, the dwarf stature of br2 derives from shortened lower internodes whilst the upper portion of the plant is completely normal. As such, it is counter-intuitive to attribute br2 dwarfism exclusively to reduced auxin export out of the shoot apex. Arabidopsis abcb1 mutants exhibit only minor reductions in auxin transport and plant height unless combined with mutations in the ABCB19 auxin transporter. Phylogenetic modelling analysis excludes the possibility that BR2 is more closely related to ABCB19 which has three more closely related orthologues in maize. BR2 is expressed in nodal meristems, and analyses of auxin transport and content indicate that BR2 function in these grass-specific tissues is analogous to ABCB1 function in the shoot and root apex of Arabidopsis. These results indicate that ABCB1/BR2 function is conserved between dicots and monocots, but also suggests that this function must be understood in the context of the segmental organization of grass plants

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14-91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45-CD34-CD73+, CD45-CD34-CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head