Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS alpha- and beta-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.     

Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome – MONET – study

Background

In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.

Methods/Design

The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.

Trial registration

Current Controlled Trials ISRCTN97142521.     

IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes

Following activation through high affinity IgE receptors (FcepsilonRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FcepsilonRI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FcepsilonRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH(2)-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.     

Chromosomal promoter replacement in Saccharomyces cerevisiae: Construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms

Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms. Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement. Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways. All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off.     

Virus PCR assay panels: an alternative to the mouse antibody production test

Antibody production tests have traditionally been used to test biological materials for viral contamination. Now molecular biology techniques have emerged as an alternative. The authors compare MAP testing with PCR-based detection methods, focusing on differences in animal use, laboratory requirements, sample size, and limits of detection.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.     

Fate of polydnavirus DNA of the egg-larval parasitoid Chelonus inanitus in the host Spodoptera littoralis

In situ hybridizations show that 5 min after parasitization, polydnavirus DNA is in close vicinity of the parasitoid egg, but 5 h later also in the yolk and partially in the host embryo. Fifteen hours after parasitization, the viral DNA is seen all over the host embryo and hardly in the yolk. The tissue distribution of the viral DNA was analysed and quantified by dot blots in the fifth instar parasitized larvae. On a per host basis, haemocytes and fat body contained the highest amount of viral DNA, while nervous tissue, intestinal tract and carcass contained less. Of the three viral segments tested, all were found in all tissues. Relative to the quantity of host DNA, viral DNA was most abundant in haemocytes, about five times less abundant in fat body and nervous tissue and about 25 times less abundant in intestinal tract. The total quantity of viral DNA per host was 444+/-145 pg which is similar to the quantity injected by the wasp; thus, the viral DNA persists throughout parasitization. The parasitoid larva contains 820+/-80 pg viral DNA integrated in the genome. This illustrates that the dose of viral DNA injected in virions represents approximately one third of the total viral genomic information present in a host at a late stage of parasitism.     

Cuttine edge: T cells from aged mice are resistant to depletion early during virus infection

Aging is associated with decreased expansion of T cells upon stimulation. In young mice, infection induces a transient T cell depletion followed by the development of an Ag-specific T cell response that controls the infection. We found that T cells were depleted early after infection with E55 + murine leukemia retrovirus in young, but not aged, mice. Adoptive transfer experiments showed donor T cells of young, but not aged, mice were depleted due to apoptosis in various tissues of young recipients. However, T cells of neither young nor aged donors were depleted in aged recipients. These results indicate that both environmental and intrinsic cellular properties limit depletion of T cells of aged mice and suggest a novel explanation for the decreased T cell response associated with aging.