Dissection and optimization of Adeno-associated virus (AAV) DNA family shuffling technology: The journey is the reward

Viral vectors based on Adeno-associated viruses (AAV) have a broad application spectrum including gene therapy and basic research. However, because naturally occurring AAV capsids are rarely sufficiently efficient and/or specific for a given application, techniques were developed to broaden the existing capsid repertoire. A prototype technology is DNA family shuffling where, in a first step, homologous cap genes encoding capsid subunits are fragmented and recombined, yielding a viral library which can then be subjected to selection in order to enrich promising variants. The aim of the present study was to dissect and improve four critical steps along this procedure. Firstly (1), two different methodologies for production of cap gene fragments were compared, resulting in the identification of DNase I based fragmentation as the most robust approach. Interestingly, cap DNA concatamer formation during nested PCR was observed, leading to amendment of the PCR purification protocol. Next (2), we studied the impact of chimerism on the essential assembly-activating protein (AAP) that is encoded in an alternative open reading frame within cap and is recombined as well during DNA family shuffling. Importantly, by performing a battery of complementary experiments, we were able to show that shuffling of AAP is not impairing its function, i.e. the support of particle assembly. Furthermore, no influence on titers was observed for wild-type and most chimeric vector productions, altogether relieving long-standing concerns about a potential rate-limiting role of AAP for AAV vector generation and evolution. Thirdly (3), we established a pioneering in vivo AAV library selection strategy in which, unlike most previously reported schemes, we selected novel capsids in specific cell types within an organ instead of the organ as a whole. Specifically, we were motivated by the facts that liver disease is wide-spread in humans and that hepatic stellate cells (HSC) are known to drive liver fibrosis, thus contributing to disease progression. Alas, tools to genetically manipulate HSCs are limited. Therefore, a library encompassing 10 capsid variants was selected in HSC by AAV injection into mice, HSC isolation and PCR rescue using purified total DNA. Following multiple selection rounds, in vivo bulk validation was performed based on next-generation sequencing. In total, 157 capsid variants were screened in parallel and again, the liver was segregated into the single cell types, i.e. hepatocytes, HSC, Kupffer cells and liver sinusoidal endothelial cells. Notably, this revealed that the selection was successful as hepatocyte-detargeted vectors were identified that showed a strong co-transduction of HSC and Kupffer cells. Intriguingly, we noted differences in vector specificity and efficiency on the DNA versus the RNA level. In order to even further restrict the new vectors to a given cell type, vector cassettes were generated bearing cell-type specific promoters and miRNA binding sites to suppress off-targeting in cells expressing these miRNAs. Testing of these constructs in vitro gave promising results especially for the miRNA-based detargeting strategy. Finally (4), we implemented improvements during the selection and analysis steps, including the use of PacBio/SMRT sequencing technology to monitor AAV sequence enrichments throughout the course of selection. Additionally, we managed to increase the stringency of the PCR rescue of cap genes, by incorporating sample-specific barcodes, i.e., short, unique nucleotide stretches, into the AAV library genomes. By using these barcodes as a primer during sample recovery, we could isolate single libraries out of a complex library mixture, as validated in vitro. In the future, this original strategy could be exploited to track individual libraries in vivo upon injection of a mixture of libraries, which should in turn help to accelerate the identification of top-performing variants for validation studies. In summary, different steps along the powerful methodology of DNA family shuffling were improved advancing future vector development and the lingering concern about AAP impairment upon shuffling was dispersed

Development and validation of a test instrument for the assessment of perceived basic motor competencies in first and second graders: the SEMOK-1-2 instrument

Both actual motor competencies (AMC) and perceived motor competencies (PMC) play an important role in motor development research and children's physical and psychological development. PMC refer to children's perceptions of their motor competencies. To assess the PMC of first and second grade children (aged 6–9 years), the SEMOK-1-2 instrument was developed. The instrument is aligned to the validated MOBAK-1-2 instrument which assesses AMC in the competence areas “object movement” and “self-movement” Accounting for possible reading difficulties in younger children, the motor tasks and answer options were illustrated and explained verbally. The purpose of this study was to test and validate the SEMOK-1-2 instrument and investigate the associations between the constructs AMC, PMC and physical activity (PA), whereby PA was measured by the participation in team and individual sports. Data from N = 404 pupils in the German-speaking part of Switzerland from first and second grades (M = 7.8 years, SD = 0.69, 49% boys) were analyzed. Confirmatory factor analyses were conducted to test the factorial validity of the SEMOK-1-2 instrument. Structural equation models were used to investigate the association between the constructs. The analyses confirmed a two-factor structure with the factors PMC “object movement” and PMC “self-movement”, corresponding to the factors existing in the MOBAK-1-2 instrument. Latent correlations between AMC factors and the corresponding PMC factors were r = 0.79 for “object movement” and r = 0.76 for “self-movement”. Associations with external criteria and covariates, such as sex, were associated with both AMC and PMC. Analyses also revealed that children who participated more often in individual and team sports showed higher levels in both AMC and PMC. The confirmation of the two-factorial structure of the SEMOK-1-2 instrument and the associations between AMC and PMC as well as external criteria indicate construct and criterion validity. The SEMOK-1-2 instrument can be economically utilized for assessing PMC and is also suitable for the monitoring of PMC in the context of Physical Education

Does adult ADHD interact with COMT val 158 met genotype to influence working memory performance?

Peer reviewedPostprin

Прогнозирование и определение температуры детали в процессе ИПА

Для повышения темпов внедрения и улучшения уровня контроля процесса ионно-плазменного азотирования разработана математическая модель влияния режимов азотирования на температуру подложки

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Werkstoff bei dem in eine Siliciumnitrid-Matrix-Diamant- oder cBN-Partikel als Hartstoff eingebettet sind, und Verfahren zu seiner Herstellung

Die Erfindung betrifft einen Werkstoff, bei dem Diamant- oder cBN-Partikel als Hartstoff in eine Siliciumnitrid-Matrix eingebettet sind, und ein Verfahren zu dessen Herstellung. Der Werkstoff wird dabei mit 10 Vol.-%-60 Vol.-% Diamantpartikel, die eine Beschichtung aus SiC oder TiCxN1-x (mit 0 ≤ x ≤ 1) aufweisen, oder 10 Vol.-%-60 Vol.-% cBN-Partikel, wobei die cBN-Partikel bevorzugt eine Beschichtung aus TiN aufweisen, gebildet. Ein solcher Werkstoff kann als abrasionsbeständiger Verschleißkörper, beispielsweise in der Metallindustrie, eingesetzt werden

On the stability of c-BN-reinforcing particles in ceramic matrix materials

Cubic boron nitride (c-BN) composites produced at high pressures and temperatures are widely used as cutting tool materials. The advent of new, effective pressure-assisted densification methods, such as spark plasma sintering (SPS), has stimulated attempts to produce these composites at low pressures. Under low-pressure conditions, however, transformation of c-BN to the soft hexagonal BN (h-BN) phase can occur, with a strong deterioration in hardness and wear. In the present work, the influence of secondary phases (B2O3, Si3N4, and oxide glasses) on the transformation of c-BN was studied in the temperature range between 1100 °C and 1575 °C. The different heat treated c-BN particles and c-BN composites were analyzed by SEM, X-ray diffraction, and Raman spectroscopy. The transformation mechanism was found to be kinetically controlled solution–diffusion–precipitation. Given a sufficiently low liquid phase viscosity, the transformation could be observed at temperatures as low as 1200 °C for the c-BN–glass composites. In contrast, no transformation was found at temperatures up to 1575 °C when no liquid oxide phase is present in the composite. The results were compared with previous studies concerning the c-BN stability and the c-BN phase diagram

Thermal Stability of TiN Coated Cubic Boron Nitride Powder

Wear-resistant, super hard ceramic composites based on cubic boron nitride (cBN) are of great interest to industry. However, cBN is metastable under sintering conditions at normal pressure and converts into the soft hexagonal BN (hBN). Therefore, efforts are being made to avoid this process. Besides short sintering times, the use of coated cBN-particles is a way to minimize this process. Therefore, the thermal stability of TiN coated cBN powders in high purity argon and nitrogen atmospheres up to temperatures of 1600 °C was investigated by thermogravimetry, X-ray phase analysis, scanning electron microscopy and Raman spectroscopy. The TiN coating was prepared by the atomic layer deposition (ALD)-method. The investigations showed that the TiN layer reacts in Ar at T ≥ 1200 °C with the cBN and forms a porous TiB2 layer. No reaction takes place in nitrogen up to temperatures of 1600 °C. Nevertheless, the 20 and 50 nm thin coatings also undergo a recrystallization process during heat treatment up to temperatures of 1600 °C

Bauteil, das mit ß-Siliciumnitridwerkstoff gebildet ist und ein Verfahren zu seiner Herstellung

Das Bauteil, das mit β-Siliciumnitridwerkstoff gebildet ist weist eine Wärmeleitfähigkeit > 80 W/mK sowie eine dielektrische Durchlagsfestigkeit > 35 KV/mm auf. Ei der Herstellung wird pulverförmiges SiNzu einem blockförmigen Element geformt und bei einer Wärmebehandlung in einer Stickstoffatmosphäre bei einer Temperatur von mindestens 1700 °C verdichtet. Im Anschluss an eine nachfolgende Abkühlung aus dem blockförmigen Element werden Trägerelemente als scheibenförmige Elemente mit einer Dicke der einzelnen Trägerelemente im Bereich 100 µm bis 3 mm mittels eines Drahtsägeprozesses erhalten

Wear behavior of diamond-silicon nitride composites sintered with FAST/SPS

Diamond-silicon nitride composite materials with 40 vol.% of diamond with two different grain sizes (50 µm and 100 µm) were produced using the field-assisted sintering technique/spark plasma sintering (FAST/SPS). A diamond powder with a SiC coating was also used. For the pure matrix material and the composites, the microstructure and the wear behavior under dry oscillating sliding conditions were investigated with use of steel and silicon nitride balls as counter-body materials. The composite materials showed coefficients of friction (COFs) of as low as 0.1, substantially lower than the COFs (0.8–1.2) obtained for the matrix. The highest wear resistance (wear rate: 8*10-8 mm³/Nm) was observed for the composite material with the SiC-coated diamond particles due to the stronger bonding of the diamond particles to the matrix in this material