Untersuchung von Membran-DNA Komplexen in äußeren elektrischen Feldern

Untersuchung von Membran-DNA Komplexen in äußeren elektrischen Feldern Die vorliegende Arbeit beschreibt die Entwicklung, Optimierung und Automatisierung einer neuartigen Methode zum Transferieren von DNA-Molekülen in Polyacrylamidgele. Zuerst wird die DNA-Lösung durch Kapillarkräfte in geeignete poröse Membranen absorbiert. Danach wird ein äußeres elektrisches Feld angelegt. Die Bindungskräfte zwischen der Membran und der DNA überlagern sich mit der elektrischen Feldkraft derart, dass eine resultierende Kraft auf die DNA-Moleküle in Richtung des Gels wirkt. Die praktischen Arbeiten wurden auf der am EMBL entwickelten automatischen DNA-Sequenziereinheit Arakis durchgeführt. Über die automatische Detektion und Analyse der DNA-Fragmente hinaus konnte mit dieser Methode das Auftragen der Proben vollständig automatisiert werden. Membrangebundene Proben trugen dazu bei den Durchsatz auf Arakis, bei gleichbleibender Qualität der Sequenzdaten, um mehr als den Faktor 4 zu erhöhen. Die Methode wurde erfolgreich auf das Sequenziersystem ABI PRISM 377 der Firma 'Applied Biosystems' übertragen. Zur Kommerzialisierung wurde eine Lizenz an die Firma 'MWG-Biotech' vergeben

Tow-Photon Polymerization (2PP) enables 3D microsystems for Pharmatechnology

Two-photon polymerization(2PP) is a process for three-dimensional (3D) micro-and Nano structuring based on the locally controlled curing of liquid precursors (light-sensitive resins) by photochemical triggered polymerization. In this decade, will be hearing a lot about this technic being applied to pharmaceutical applications like fabricating 3D microchannels for nanoparticle precipitation, nano-porous membranes and scaffolds for cell culturing, biomimetic organ-on-chip systems. This paper presents 2pp applied microsystems for continuously generating lipid nanoparticles which are one of the most important drug carrier system. The most important advantages of 2pp is manufacturing 3D shapes that is not possible with lithographic micro and nano fabrication technologies. Also, it will be shown how 2pp fabricated microchannel can be integrated with continuous size measurement by flowDLS for the feed-back controlled generation of nanoparticles

On the Geometry of Linking Production to Cost: The Case for Cobb-Douglas

In introductory and intermediate microeconomics courses, students are taught about production functions and cost functions but rarely are they shown a direct link between production and cost. If we assume production is Cobb-Douglas, then the CDProductionToCost.xlsx Excel file shows the link between short-run, SR, and long-run, LR, production and cost in geometric detail. This geometric approach is particularly useful in classes without a calculus prerequisite. The file has three interactive figure sheets and four information sheets. The figure sheets do not require a knowledge of Excel to operate; they can be manipulated simply using sliders and click-boxes. To focus attention on the graphical material presented, the equations that created each figure are hidden and the interactive figure sheets are write-protected. The information sheets are not write-protected so instructors can alter them as they wish. The accompanying paper provides additional graphical and algebraic detail and suggests alternative ways to use the file to deliver material to students. Although the main purpose of this file is to show the geometric link between production and cost, the supplementary sheets allow instructors (and students) to explore the underlying production function and cost- minimizing solutions that allow one to link production to cost

NeuroExaminer: an all-glass microfluidic device for whole-brain in vivo imaging in zebrafish

While microfluidics enables chemical stimuli application with high spatio-temporal precision, light-sheet microscopy allows rapid imaging of entire zebrafish brains with cellular resolution. Both techniques, however, have not been combined to monitor whole-brain neural activity yet. Unlike conventional microfluidics, we report here an all-glass device (NeuroExaminer) that is compatible with whole-brain in vivo imaging using light-sheet microscopy and can thus provide insights into brain function in health and disease

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets.

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization

The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization

The survival motor neuron (SMN) complex fulfils essential functions in the assembly of snRNPs, which are key components in the splicing of pre-mRNAs. Little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here we systematically screened all human phosphatase gene products for a regulatory role in the SMN complex. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a readout for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies, suggesting impaired SMN complex function, among those the catalytically inactive, non–receptor-type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN, which is important for its proper function in snRNP assembly

Identification of a novel Rev-interacting cellular protein

BACKGROUND: Human cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. RESULTS: Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. CONCLUSION: These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors

Stabilized Production of Lipid Nanoparticles of Tunable Size in Taylor Flow Glass Devices with High-Surface-Quality 3D Microchannels.

Nanoparticles as an application platform for active ingredients offer the advantage of efficient absorption and rapid dissolution in the organism, even in cases of poor water solubility. Active substances can either be presented directly as nanoparticles or can be integrated in a colloidal carrier system (e.g., lipid nanoparticles). For bottom-up nanoparticle production minimizing particle contamination, precipitation processes provide an adequate approach. Microfluidic systems ensure a precise control of mixing for the precipitation, which enables a tunable particle size definition. In this work, a gas/liquid Taylor flow micromixer made of chemically inert glass is presented, in which the organic phases are injected through a symmetric inlet structure. The 3D structuring of the glass was performed by femtosecond laser ablation. Rough microchannel walls are typically obtained by laser ablation but were smoothed by a subsequent annealing process resulting in lower hydrophilicity and even rounder channel cross-sections. Only with such smooth channel walls can a substantial reduction of fouling be obtained, allowing for stable operation over longer periods. The ultrafast mixing of the solutions could be adjusted by simply changing the gas volume flow rate. Narrow particle size distributions are obtained for smaller gas bubbles with a low backflow and when the rate of liquid volume flow has a small influence on particle precipitation. Therefore, nanoparticles with adjustable sizes of down to 70 nm could be reliably produced in continuous mode. Particle size distributions could be narrowed to a polydispersity value of 0.12