Targeted mutagenesis of the Sap47 gene of Drosophila: Flies lacking the synapse associated protein of 47 kDa are viable and fertile

BACKGROUND: Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila. Sequence comparison identifies homologous proteins in numerous species including C. elegans, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila. RESULTS: Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels. CONCLUSION: The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila

Das Kapitalstockmodell als Basiskonzept für eine nachhaltige Entwicklung

Kapitalien sind die Produktionsfaktoren einer Volkswirtschaft und stellen damit das Potenzial zur Befriedigung jeglicher Art von Bedürfnissen und damit zur Schaffung von Wohlfahrt dar. Es können vier Arten von Kapitalien unterschieden werden, das Naturkapital, das Realkapital, das Humankapital und das Sozialkapital. Diese Kapitalien bzw. Kapitalformen sind die Grundlage zur Schaffung von monetären wie auch nicht-monetären Gütern und Dienstleistungen jetzt und in Zukunft. Im Wirtschaftsprozess findet ein Auf- und Abbau dieser Kapitalien statt. Die Kapitalallokation im Rahmen der Marktprozesse garantiert jedoch keinen nachhaltigen Umgang mit den Kapitalien. Soll die Chancengleichheit gegenwärtiger und zukünftiger Generationen gesichert sein, ist es nötig, die Kapitalien einem partizipativen Managementprozess zuzuführen. Dem Sozialkapital kommt darin eine grosse Bedeutung zu. Sozialkapital verhilft zur Über-windung übergewichteter Eigeninteressen hin zu einem kooperativen Verhalten. Im sogenannten Kapitalstockmodell KSM werden die drei Dimensionen "Kapitalformen", "Partizipation" von Akteuren und Stakeholdern sowie der "Managementprozess" als Issue-Management zusammengeführt, um damit nachhaltige Entwicklung im Sinne von Kapitalaufbau zur Bedürfnisbefriedigung jetziger und zukünftiger Gesellschaften zu ermöglichen

Malicious Cyber Operations, “Hackbacks” and International Law: An Austrian Example as a Basis for Discussion on Permissible Responses

In January 2020, Austria publicly announced that some of its governmental institutions have been hit by a significant malicious cyber operation and that it cannot be denied – at least for the moment – that a state was behind this operation. One month later, the Austrian Foreign Ministry declared the cyber operation to be officially over. While Austria noted that it took “countermeasures” against the operation, it is not entirely clear what it meant by that. This article elaborates the question what response options a state like Austria would have against a malicious cyber operation under the current framework of international law. It, hence, tries to answer when a “hackback” is lawful under international law and when it is not

Partitioning climate projection uncertainty with multiple large ensembles and CMIP5/6

Partitioning uncertainty in projections of future climate change into contributions from internal variability, model response uncertainty and emissions scenarios has historically relied on making assumptions about forced changes in the mean and variability. With the advent of multiple single-model initial-condition large ensembles (SMILEs), these assumptions can be scrutinized, as they allow a more robust separation between sources of uncertainty. Here, the framework from Hawkins and Sutton (2009) for uncertainty partitioning is revisited for temperature and precipitation projections using seven SMILEs and the Coupled Model Intercomparison Project CMIP5 and CMIP6 archives. The original approach is shown to work well at global scales (potential method bias < 20 %), while at local to regional scales such as British Isles temperature or Sahel precipitation, there is a notable potential method bias (up to 50 %), and more accurate partitioning of uncertainty is achieved through the use of SMILEs. Whenever internal variability and forced changes therein are important, the need to evaluate and improve the representation of variability in models is evident. The available SMILEs are shown to be a good representation of the CMIP5 model diversity in many situations, making them a useful tool for interpreting CMIP5. CMIP6 often shows larger absolute and relative model uncertainty than CMIP5, although part of this difference can be reconciled with the higher average transient climate response in CMIP6. This study demonstrates the added value of a collection of SMILEs for quantifying and diagnosing uncertainty in climate projections

Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila

Background The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-β1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure. Results In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth. Conclusions The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm. See minireview at http://jbiol.com/content/9/1/8.ISSN:1478-5854ISSN:1475-492

Enhancing Regional RTD and Innovation Development through Foresight & Mentoring : Scenario Development and Action Plan for RTD and Innovation Promotion up to 2020 in Zurich, Switzerland

The Regional Economic RTD Policy through Foresight & Mentoring (REFORM) project was one of 18 projects funded under the Sixth Framework Programme within the ‘Regions of Knowledge 2’ call, which were launched in 2006 with the aim to promote increased and improved regional investment in research and development through mutual learning, coordination and collaboration between regional policy developers and regional initiatives. It is accepted that it is essential to enhance the integration and coordination between private and public R&D investments, and to provide appropriate support through R&D policy and infrastructure, to promote economic progress in Europe. The REFORM project developed, supported and implemented a variety of measures and activities which will enable regions to understand their particular needs to enable increased RTD activity and investment in the future. Through mechanisms such as Foresight and a new Mentoring Programme, the project developed a number of individual action plans for partners, which will provide the infrastructure for new RTD policy and its implementation, and thus increase the economic growth of the EU as a whole. Additional actions, including study visits, workshops and a conference supported the transfer of good practice and knowledge to those regions in the developmental stages of increasing RTD investment, increasing the rate of transfer through hands on support, promotion and participation.The Regional Economic RTD Policy through Foresight & Mentoring (REFORM) project was one of 18 projects funded under the Sixth Framework Programme within the ‘Regions of Knowledge 2’ call, which were launched in 2006 with the aim to promote increased and improved regional investment in research and development through mutual learning, coordination and collaboration between regional policy developers and regional initiatives. It is accepted that it is essential to enhance the integration and coordination between private and public R&D investments, and to provide appropriate support through R&D policy and infrastructure, to promote economic progress in Europe. The REFORM project developed, supported and implemented a variety of measures and activities which will enable regions to understand their particular needs to enable increased RTD activity and investment in the future. Through mechanisms such as Foresight and a new Mentoring Programme, the project developed a number of individual action plans for partners, which will provide the infrastructure for new RTD policy and its implementation, and thus increase the economic growth of the EU as a whole. Additional actions, including study visits, workshops and a conference supported the transfer of good practice and knowledge to those regions in the developmental stages of increasing RTD investment, increasing the rate of transfer through hands on support, promotion and participation

CRISPR-induced double-strand breaks trigger recombination between homologous chromosome arms

CRISPR–Cas9–based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms—often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention

The Non-Affected Muscle Volume Compensates for the Partial Loss of Strength after Injection of Botulinum Toxin A

Local botulinum toxin (BTX-A, Botox®) injection in overactive muscles is a standard treatment in patients with cerebral palsy. The effect is markedly reduced in children above the age of 6 to 7. One possible reason for this is the muscle volume affected by the drug. Nine patients (aged 11.5; 8.7–14.5 years) with cerebral palsy GMFCS I were treated with BTX-A for equinus gait at the gastrocnemii and soleus muscles. BTX-A was administered at one or two injection sites per muscle belly and with a maximum of 50 U per injection site. Physical examination, instrumented gait analysis, and musculoskeletal modelling were used to assess standard muscle parameters, kinematics, and kinetics during gait. Magnetic resonance imaging (MRI) was used to detect the affected muscle volume. All the measurements were carried out pre-, 6 weeks post-, and 12 weeks post-BTX-A. Between 9 and 15% of the muscle volume was affected by BTX-A. There was no effect on gait kinematics and kinetics after BTX-A injection, indicating that the overall kinetic demand placed on the plantar flexor muscles remained unchanged. BTX-A is an effective drug for inducing muscle weakness. However, in our patient cohort, the volume of the affected muscle section was limited, and the remaining non-affected parts were able to compensate for the weakened part of the muscle by taking over the kinetic demands associated with gait, thus not enabling a net functional effect in older children. We recommend distributing the drug over the whole muscle belly through multiple injection sites

Erratum to: Poor outcome at 7.5years after Stanisavljevic quadriceps transposition for patello-femoral instability

Introduction: Congenital dislocation of the patella and recurrent symptomatic dislocation in adolescents are difficult pathologies to treat. Stanisavljevic described an extensive release procedure essentially involving medializing the entire lateral quadriceps and medial soft tissue stabilization. There are no significant series reporting the success of this method. This procedure has been performed in our institution over several years and we report our results. Method: Retrospective case series. Between 1990 and 2007, 20 knees in 13 children and adolescents (mean age 12.8years; 4-17, 7 female) with recurrent or congenital dislocation of the patella (8 knees) underwent this procedure after failed conservative treatment (mean follow-up 7.5years; 4-16). All were immobilized in a long leg cast for 6weeks. Results: Five knees in five patients (20%, 1 congenital dislocation) reported their knees as improved without further dislocations. Out of the 15 knees with failures (80%) 12 in six patients (60%) were revised due to redislocation. Three knees in two patients (15%) still had dislocations or subluxations, but any revision was refused. Three knees in three patients caused pain and discomfort during daily activity. Redislocation first developed after a mean of 21.3months (4-72) postoperatively. Only one patient had returned to sport at the 12-month follow-up. Discussion: The Stanisavljevic procedure produces a mediocre success rate with our long-term follow-up series showing a failure rate up to 80%. We therefore recommend more specific procedures dealing with the anatomical deformity such as trochleaplasty to produce superior success rates

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

A new study reveals a functional rule for N-terminal acetylation in higher eukaryotes called the (X)PX rule and describes a generic method that prevents this modification to allow the study of N-terminal acetylation in any given protein