Gene targeting in human pluripotent cell-derived neural stem cells for the study and treatment of neurological disorders

Modern stem cell technology has made remarkable progress in recent years with the discovery of induced pluripotency, enabling the production of any cell type from any person in unlimited quantities, as well as improved protocols for the generation of stable neural stem cells to generate all cells of the central nervous system. This forms the base to examine neurons derived from patients of neurodegenerative diseases in vitro, allowing elucidation of disease mechanisms, as well as to regenerative production of neurons and other cells of the CNS. This study investigated two different techniques in order to achieve a specific genetic change in human neural stem cells, enabling the generation of modified neurons. A zinc finger nuclease (ZFN) attempt was used for the targeted destruction of the adenosine kinase gene (ADK) to obtain an adenosine releasing cell population comprising anti-epileptic properties. With recombinant adeno-associated virus (AAV), the Machado-Joseph disease (MJD) causing prolonged allele of ataxin-3 was replaced with the other allele of normal length. The destruction of the ADK gene by frame shifting in course of repairing the ZFN-induced double-strand break was confirmed by sequencing and Western blot analysis and led to a strong increase in adenosine efflux in neural stem cells, which was maintained after differentiation into neurons. Use of this cell population in mouse models of epilepsy to test the anticonvulsant properties have so far not led to a clear result. While due to small animal quantities in a kainate-induced model only a statistically non-significant trend toward reduced seizure frequency in animals with ADK-/- cells compared to wild type cells could be observed, in a model of electrical seizure induction (Kindling) animals showed a strongly increased resistance to the amount of current necessary to induce a seizure. After AAV-mediated exchange of the pathogenic ATXN3-allele against the physiological length, PCR and Western blot analysis confirmed the loss of extended transcript and protein variants in the cells. Following Cre-mediated removal of the selection cassette, a normal protein variant was transcribed from both alleles. MJD patient-derived neurons showed the formation of insoluble microaggregates following glutamate-induced activation. This aggregation was no longer detectable after gene correction. The method used consequently led to neurons, which no longer showed the disease phenotype. Both techniques used were highly efficient and produced very few false positive clones. These results indicate that neural stem cells are an ideal population for genetic manipulation and that the techniques used provide new perspectives for the study of disease mechanisms in the culture dish as well as open the field towards cell therapeutic applications

Wafer bonding process for zero level vacuum packaging of MEMS

It is well known that the packaging of electronic devices is of paramount importance, none more so than in MEMS were fragile mechanical elements are realized. Among the different approaches, wafer to wafer bonding guarantees the advantages of the wafer scaling and provides protection of the devices during the final phase of fabrication. Direct bonding, also known as fusion bonding, is seldom implemented in MEMS fabrication due to the high surface quality required, the high temperature involved and the compulsory wet activation process. In this paper a direct bonding process for MEMS inertial sensor without the need of any wet activation step is presented.acceptedVersio

Wafer bonding process for zero level vacuum packaging of MEMS

It is well known that the packaging of electronic devices is of paramount importance, none more so than in MEMS were fragile mechanical elements are realized. Among the different approaches, wafer to wafer bonding guarantees the advantages of the wafer scaling and provides protection of the devices during the final phase of fabrication. Direct bonding, also known as fusion bonding, is seldom implemented in MEMS fabrication due to the high surface quality required, the high temperature involved and the compulsory wet activation process. In this paper a direct bonding process for MEMS inertial sensor without the need of any wet activation step is presented.acceptedVersio

Update on the role of antipsychotics in the treatment of Tourette syndrome

Tourette syndrome (TS) is a neuropsychiatric disorder with typical onset in childhood and characterized by chronic occurrence of motor and vocal tics. The disorder can lead to serious impairments of both quality of life and psychosocial functioning, particularly for those individuals displaying complex tics. In such patients, drug treatment is recommended. The pathophysiology of TS is thought to involve a dysfunction of basal ganglia-related circuits and hyperactive dopaminergic innervations. Congruently, dopamine receptor antagonism of neuroleptics appears to be the most efficacious approach for pharmacological intervention. To assess the efficacy of the different neuroleptics available, a systematic, keyword-related search in PubMed (National Library of Medicine, Washington, DC) was undertaken. Much information on the use of antipsychotics in the treatment of TS is based on older data. Our objective was to give an update and therefore we focused on papers published in the last decade (between 2001 and 2011). Accordingly, the present review aims to summarize the current and evidence-based knowledge on the risk-benefit ratio of both first and second generation neuroleptics in TS

Estudio conceptual de sistema de liberación de cohetes

El presente trabajo presenta las condiciones de funcionamiento que debe poseer un sistema utilizado para liberar un cohete, y describe el estudio comparativo entre dos alternativas propuestas para dicho sistema. El sistema encargado de liberar un cohete funciona usualmente como el vínculo entre el vehículo y la estructura sujeta al suelo. Esté vínculo debe soportar todas las cargas generadas por el peso propio del cohete, así como las cargas generadas por el viento cuando este incide sobre el fuselaje del vehículo, también las cargas debidas al funcionamiento del motor en el instante previo a su liberación. También debe ser capaz de funcionar en un instante de tiempo muy breve, sin posibilidad de falla y con una mínima dispersión de tiempo entre la liberación de la sujeción más rápida y la más lenta. Como primera instancia se plantean dos alternativas de sistemas de liberación: una consta de una estructura metálica que al momento de liberar es accionada por dos pistones hidráulicos. La segunda es una estructura metálica que posee un mecanismo que es accionado neumáticamente. Como parte del análisis comparativo se presentan análisis de la cinemática de cada sistema y de la capacidad de tomar carga de cada uno. De esta forma se evalúan ambas alternativas para definir cual resulta más viable. Como conclusión se define como más viable la utilización del sistema del mecanismo accionado neumáticamente debido a la simplicidad y resistencia estructural y velocidad de accionamiento.Facultad de Ingenierí

Assessing the societal benefits of river restoration using the ecosystem services approach

This paper is a contribution from the EU seventh framework funded research project REFORM (Grant Agreement 282656).The success of river restoration was estimated using the ecosystem services approach. In eight pairs of restored–unrestored reaches and floodplains across Europe, we quantified provisioning (agricultural products, wood, reed for thatching, infiltrated drinking water), regulating (flooding and drainage, nutrient retention, carbon sequestration) and cultural (recreational hunting and fishing, kayaking, biodiversity conservation, appreciation of scenic landscapes) services for separate habitats within each reach, and summed these to annual economic value normalized per reach area. We used locally available data and literature, did surveys among inhabitants and visitors, and used a range of economic methods (market value, shadow price, replacement cost, avoided damage, willingness-to-pay survey, choice experiment) to provide final monetary service estimates. Total ecosystem service value was significantly increased in the restored reaches (difference 1400 ± 600 € ha−1 year−1; 2500 − 1100, p = 0.03, paired t test). Removal of one extreme case did not affect this outcome. We analysed the relation between services delivered and with floodplain and catchment characteristics after reducing these 23 variables to four principal components explaining 80% of the variance. Cultural and regulating services correlated positively with human population density, cattle density and agricultural N surplus in the catchment, but not with the fraction of arable land or forest, floodplain slope, mean river discharge or GDP. Our interpretation is that landscape appreciation and flood risk alleviation are a function of human population density, but not wealth, in areas where dairy farming is the prime form of agriculture.PostprintPeer reviewe

Absolute Energy Level Positions in CdSe Nanostructures from Potential-Modulated Absorption Spectroscopy (EMAS)

YesSemiconductor nanostructures like CdSe quantum dots and colloidal nanoplatelets exhibit remarkable optical properties, making them interesting for applications in optoelectronics and photocatalysis. For both areas of application a detailed understanding of the electronic structure is essential to achieve highly efficient devices. The electronic structure can be probed using the fact that optical properties of semiconductor nanoparticles are found to be extremely sensitive to the presence of excess charges that can for instance be generated by means of an electrochemical charge transfer via an electrode. Here we present the use of potential modulated absorption spectroscopy (EMAS) as a versatile spectroelectrochemical method to obtain absolute band edge positions of CdSe nanostructures versus a well-defined reference electrode under ambient conditions. In this the spectral properties of the nanoparticles are monitored dependent on an applied electrochemical potential. We developed a bleaching model that yields the lowest electronic state in the conduction band of the nanostructures. A change in the band edge positions caused by quantum confinement is shown both for CdSe quantum dots as well as for colloidal nanoplatelets. In the case of CdSe quantum dots these findings are in good agreement with tight binding calculations. The method presented is not limited to CdSe nanostructures but can be used as a universal tool. Hence, this technique allows the determination of absolute band edge positions of a large variety of materials used in various applications

The emergence of the brain non-CpG methylation system in vertebrates

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.This work was supported by the Australian Research Council (ARC) Centre of Excellence programme in Plant Energy Biology (grant no. CE140100008). R.L. was supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship, ARC Future Fellowship (no. FT120100862) and Howard Hughes Medical Institute International Research Scholarship. A.d.M. was funded by an EMBO long-term fellowship (no. ALTF 144-2014). J.L.G.-S. was supported by the Spanish government (grant no. BFU2016- 74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (no. MDM-2016-0687). B.V. was supported by the Biomedical Research Council of the Agency for Science, Technology and Research of Singapore. F.G. was supported by an ARC Future Fellowship (no. FT160100267). C.W.R. was supported by an NSF grant (no. IOS-1354898). J.R.E. is an investigator of the Howard Hughes Medical Institute. Genomic data was generated at the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics

Non-contrast CT markers of intracerebral hematoma expansion : a reliability study

Objectives: We evaluated whether clinicians agree in the detection of non-contrast CT markers of intracerebral hemorrhage (ICH) expansion. Methods: From our local dataset, we randomly sampled 60 patients diagnosed with spontaneous ICH. Fifteen physicians and trainees (Stroke Neurology, Interventional and Diagnostic Neuroradiology) were trained to identify six density (Barras density, black hole, blend, hypodensity, fluid level, swirl) and three shape (Barras shape, island, satellite) expansion markers, using standardized definitions. Thirteen raters performed a second assessment. Inter and intra-rater agreement were measured using Gwet’s AC1, with a coefficient > 0.60 indicating substantial to almost perfect agreement. Results: Almost perfect inter-rater agreement was observed for the swirl (0.85, 95% CI: 0.78-0.90) and fluid level (0.84, 95% CI: 0.76-0.90) markers, while the hypodensity (0.67, 95% CI: 0.56-0.76) and blend (0.62, 95% CI: 0.51-0.71) markers showed substantial agreement. Inter-rater agreement was otherwise moderate, and comparable between density and shape markers. Inter-rater agreement was lower for the three markers that require the rater to identify one specific axial slice (Barras density, Barras shape, island: 0.46, 95% CI: 0.40-0.52 versus others: 0.60, 95% CI: 0.56-0.63). Inter-observer agreement did not differ when stratified for raters’ experience, hematoma location, volume or anticoagulation status. Intrarater agreement was substantial to almost perfect for all but the black hole marker. Conclusion: In a large sample of raters with different backgrounds and expertise levels, only four of nine non-contrast CT markers of ICH expansion showed substantial to almost perfect inter-rater agreement