Improved segmentation of deep brain grey matter structures using magnetization transfer (MT) parameter maps.

Basal ganglia and brain stem nuclei are involved in the pathophysiology of various neurological and neuropsychiatric disorders. Currently available structural T1-weighted (T1w) magnetic resonance images do not provide sufficient contrast for reliable automated segmentation of various subcortical grey matter structures. We use a novel, semi-quantitative magnetization transfer (MT) imaging protocol that overcomes limitations in T1w images, which are mainly due to their sensitivity to the high iron content in subcortical grey matter. We demonstrate improved automated segmentation of putamen, pallidum, pulvinar and substantia nigra using MT images. A comparison with segmentation of high-quality T1w images was performed in 49 healthy subjects. Our results show that MT maps are highly suitable for automated segmentation, and so for multi-subject morphometric studies with a focus on subcortical structures.

Interplanetary transfer of photosynthesis: an experimental demonstration of a selective dispersal filter in planetary island biogeography.

We launched a cryptoendolithic habitat, made of a gneissic impactite inoculated with Chroococcidiopsis sp., into Earth orbit. After orbiting the Earth for 16 days, the rock entered the Earth's atmosphere and was recovered in Kazakhstan. The heat of entry ablated and heated the rock to a temperature well above the upper temperature limit for life to below the depth at which light levels are insufficient for photosynthetic organisms ( approximately 5 mm), thus killing all of its photosynthetic inhabitants. This experiment shows that atmospheric transit acts as a strong biogeographical dispersal filter to the interplanetary transfer of photosynthesis. Following atmospheric entry we found that a transparent, glassy fusion crust had formed on the outside of the rock. Re-inoculated Chroococcidiopsis grew preferentially under the fusion crust in the relatively unaltered gneiss beneath. Organisms under the fusion grew approximately twice as fast as the organisms on the control rock. Thus, the biologically destructive effects of atmospheric transit can generate entirely novel and improved endolithic habitats for organisms on the destination planetary body that survive the dispersal filter. The experiment advances our understanding of how island biogeography works on the interplanetary scale.

Gene transfer into Xenopus hepatocytes: transcriptional regulation by members of the nuclear receptor superfamily.

A procedure to culture Xenopus laevis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid- or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin B1 gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-transfection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics.

Peripheral T cell activation and deletion induced by transfer of lymphocyte subsets expressing endogenous or exogenous mouse mammary tumor virus.

Murine T cell reactivity with products of the minor lymphocyte stimulatory (Mls) locus correlates with the expression of particular variable (V) domains of the T cell receptor (TCR) beta chain. It was recently demonstrated that Mls antigens are encoded by an open reading frame (ORF) in the 3' long terminal repeat of either endogenous or exogenous mouse mammary tumor virus (MMTV). Immature thymocytes expressing reactive TCR-V beta domains are clonally deleted upon exposure to endogenous Mtv's. Mature T cells proliferate vigorously in response to Mls-1a (Mtv-7) in vivo, but induction of specific anergy and deletion after exposure to Mtv-7-expressing cells in the periphery has also been described. We show here that B cells and CD8+ (but not CD4+) T cells from Mtv-7+ mice efficiently induce peripheral deletion of reactive T cells upon transfer to Mtv-7- recipients, whereas only B cells stimulate specific T cell proliferation in vivo. In contrast to endogenous Mtv-7, transfer of B, CD4+, or CD8+ lymphocyte subsets from mice maternally infected with MMTV(SW), an infectious homologue of Mtv-7, results in specific T cell deletion in the absence of a detectable proliferative response. Finally, we show by secondary transfers of infected cells that exogenous MMTV(SW) is transmitted multidirectionally between lymphocyte subsets and ultimately to the mammary gland. Collectively our data demonstrate heterogeneity in the expression and/or presentation of endogenous and exogenous MMTV ORF by lymphocyte subsets and emphasize the low threshold required for induction of peripheral T cell deletion by these gene products.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Evidential relevance in scene to offender transfer cases: development and analysis of a likelihood ratio for offence level propositions

The present paper focuses on the analysis and discussion of a likelihood ratio (LR) development for propositions at a hierarchical level known in the context as 'offence level'. Existing literature on the topic has considered LR developments for so-called offender to scene transfer cases. These settings involve-in their simplest form-a single stain found on a crime scene, but with possible uncertainty about the degree to which that stain is relevant (i.e. that it has been left by the offender). Extensions to multiple stains or multiple offenders have also been reported. The purpose of this paper is to discuss a development of a LR for offence level propositions when case settings involve potential transfer in the opposite direction, i.e. victim/scene to offender transfer. This setting has previously not yet been considered. The rationale behind the proposed LR is illustrated through graphical probability models (i.e. Bayesian networks). The role of various uncertain parameters is investigated through sensitivity analyses as well as simulations.

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation.

BACKGROUND: The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. RESULTS: In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. CONCLUSIONS: This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.

A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.

How can a dual oriT system contribute to efficient transfer of an integrative and conjugative element?

Integrative and conjugative elements (ICEs) are particularly interesting model systems for horizontal gene transfer, because they normally reside in an integrated state in the host chromosome but can excise and self-transfer under particular conditions, typically requiring exquisite regulatory cascades. Despite important advances in our understanding of the transfer mechanisms of a number of ICE, many essential details are lacking. Recently we reported that ICEclc, a 103 kb ICE of Pseudomonas knackmussii B13, has two active origins of transfer (oriTs), which is very much unlike conjugative plasmids that usually employ a single oriT. We discuss here how this dual oriT system could function and how it actually could have presented an evolutionary advantage for ICEclc distribution.