Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy

Peer reviewe

RNA-basierte Regulation der Genexpression : künstliche Genschalter

RNA-based gene control mechanisms pose an elegant and straightforward way to switch on, off, or fine-tune transgene expression without the need for expressing regulatory proteins. A small molecule effector binds directly to a ligand-binding aptamer RNA structure and thereby modulates expression of an associated target gene. We established genetic switches based on regulation of self-cleaving ribozymes and polyadenylation that allow for control of transgene expression in bacteria, yeast, human cell lines and Caenorhabditis elegans in a robust and dose-dependent manner.publishe

Deletion Mutagenesis of the Amino−Terminal Head Domain of Vimentin Reveals Dispensability of Large Internal Regions for Intermediate Filament Assembly and Stability

Previous studies have shown that the non−−helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino−terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25−38, 44−95, or 40−95 polymerized into wild−type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44−69 or 25−63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25− 68, 44−103, or 88−103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non−−helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non−−helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non−−helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C−terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as−yet−unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild−type vimenti

A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans

The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3'-UTR converts any gene of interest into a tetracycline-inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington's disease models exhibiting ligand-controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans.publishe

Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5'-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.publishe

Maßnahmen des RoboCup zum Wissenschaftsjahr 2006

Die „Maßnahmen des RoboCup zum Wissenschaftsjahr 2006“ hatten zum Ziel, Kinder und Jugendliche für Technik, speziell für Robotik zu begeistern und so auch technische und ingenieurwissenschaftliche Berufe mehr in den Fokus der Aufmerksamkeit zu rücken. Der Rahmen des Informatikjahrs 2006 eignete sich dabei besonders, da durch die verstärkte mediale Aufmerksamkeit und Berichterstattung Schulen, ihre Lehrkräfte und SchülerInnen gut erreicht und begeistert werden konnten. Dafür wurden deutschlandweit gezielt Bildungsstätten und lokale Medien angesprochen und mit Informations- und Werbematerial für eine Informationsveranstaltung in der Stadt versorgt. Durch die Veranstaltung wurden neue Kontakte sowohl innerhalb interessierter Teilnehmer als auch zwischen dem RoboCupJunior-Team und den Interessenten generiert. Die Webseite www.robocupunior.de diente dafür als zentraler Anlaufpunkt. Hier finden sich über das Projektende hinaus didaktisches Material und Kontaktmöglichkeiten zu anderen Teams. Die Webseite konnte innerhalb der Projektlaufzeit insgesamt fast 28.000 Besuche verzeichnen. Als weiterer Schwerpunkt wurden drei bundesweite Qualifikationen und die internationale Weltmeisterschaft erfolgreich organisiert und durchgeführt. Insgesamt nahmen 2006 aus Deutschland 786 SchülerInnen in 258 Teams daran teil. Der vorliegende Schlussbericht stellt alle Projektphasen detailliert dar. Das ebenfalls geförderte „Outdoor-Rescue“-Event erregte zusätzlich große mediale Aufmerksamkeit. Aufbauend auf diesen Erfolg wird es 2007 und 2008 wieder Qualifikationsturniere sowie die Deutschen Meisterschaften „GermanOpen“ im Rahmen der „Hannover Messe“ geben.The “Activities of the RoboCup for the Science Year 2006” was aimed to inspire children and young people for technology, especially for robotics. Technical and engineering professions were to be moved more into the focus of attention. The frame of the Informatics Year 2006 was especially suited because of the increased media attention. Schools, teachers, and pupils could be reached well for becoming enthusiastic. Educational institutions and local media were addressed nationwide, and they were provided with information and promotional material for information meetings at the local sites. These events generated new contacts between interested participants as well as between the RoboCup Junior teams and the interested people. The web site www.robocupunior.de served as the central information point. Still after the end of the project, one can find there the didactic material and the contact data to the teams. The site had a total of almost 28,000 visits within the project period. As a further focal point of the project, the three nationwide competitions for RoboCup Junior qualification and finally the International Junior competitions at the RoboCup World Championship 2006 in Bremen were organized and successfully carried out. A total of 786 students in 258 teams from Germany participated in the competitions. This final report describes all phases of the project in detail. The also funded Outdoor Rescue Event at the RoboCup 2006 in Bremen attracted major media attention. Building on the success of the project, there will be again nationwide qualifying tournaments and the final German competition at the championship "RoboCup German Open" in the context of "Hannover Fair" in 2007 and the following years.Peer Reviewe

Influence of Personalized Exercise Recommendations During Rehabilitation on the Sustainability of Objectively Measured Physical Activity Levels, Fatigue, and Fatigue-Related Biomarkers in Patients With Breast Cancer

Purpose. Only one-third of patients with breast cancer reach the recommended activity level of 15 to 25 MET h/wk. The aim of this study was to determine the influence of personalized exercise recommendations during rehabilitation on patients' physical activity level, fatigue, and self-perceived cognitive function as well as on side effect-associated biomarkers. Methods. Total metabolic rate, physical activity level, mean MET and steps, fatigue, self-perceived cognitive functioning , and biomarkers (C-reactive protein [CRP], interleukin 6, macrophage migration inhibiting factor [MIF], tumor necrosis factor [TNF]-, brain-derived neurotrophic factor [BDNF], insulin-like growth factor 1 [IGF1]) were assessed in 60 patients with breast cancer in the aftercare phase before (t(0)) and 8 months after (t(1)) the intervention. The rehabilitation program consisted of an initial 3-week period and a 1-week stay after 4 months. Results. Paired t-test indicated a statistically significant increase in all activity outcomes from t(0) to t(1). Patients' mean activity level significantly increased from 14.89 to 17.88 MET h/wk. Fatigue and self-perceived cognitive functioning significantly improved from t(0) to t(1). CRP levels significantly decreased, and BDNF as well as IGF1 levels significantly increased over time. Correlation analysis revealed statistically significant negative associations between fatigue, physical activity, and markers of inflammation (TNF- and MIF). Furthermore, significant positive correlations between subjective cognitive functioning and all dimensions of fatigue were observed. Conclusions. The results support the importance of personalized exercise recommendations to increase physical activity levels in patients with breast cancer. Furthermore, the results highlighti an association between physical activity, fatigue, and inflammation

T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca²⁺ Influx

<div><p>CD4 T cells in human infants and adults differ in the initiation and strength of their responses. The molecular basis for these differences is not yet understood. To address this the principle key molecular events of TCR- and CD28-induced signaling in naive CD4 T cells, such as Ca²⁺ influx, NFAT expression, phosphorylation and translocation into the nucleus, ERK activation and IL-2 response, were analyzed over at least the first 3 years of life. We report dramatically reduced IL-2 and TNFα responses in naive CD31⁺ T cells during infancy. Looking at the obligatory Ca²⁺ influx required to induce T cell activation and proliferation, we demonstrate characteristic patterns of impairment for each stage of infancy that are partly due to the differential usage of Ca²⁺ stores. Consistent with those findings, translocation of NFATc2 is limited, but still dependent on Ca²⁺ influx as demonstrated by sensitivity to cyclosporin A (CsA) treatment. Thus weak Ca²⁺ influx functions as a catalyst for the implementation of restricted IL-2 response in T cells during infancy. Our studies also define limited mobilization of Ca²⁺ ions as a characteristic property of T cells during infancy. This work adds to our understanding of infants’ poor T cell responsiveness against pathogens.</p></div