Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

Adipose-derived stem cells (ASCs) have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342) represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL), with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique

Cation selectivity of the presequence translocase channel Tim23 is crucial for efficient protein import.

Virtually all mitochondrial matrix proteins and a considerable number of inner membrane proteins carry a positively charged, N-terminal presequence and are imported by the TIM23 complex (presequence translocase) located in the inner mitochondrial membrane. The voltage-regulated Tim23 channel constitutes the actual protein-import pore wide enough to allow the passage of polypeptides with a secondary structure. In this study, we identify amino acids important for the cation selectivity of Tim23. Structure based mutants show that selectivity is provided by highly conserved, pore-lining amino acids. Mutations of these amino acid residues lead to reduced selectivity properties, reduced protein import capacity and they render the Tim23 channel insensitive to substrates. We thus show that the cation selectivity of the Tim23 channel is a key feature for substrate recognition and efficient protein import

ODS-materials for high temperature applications in advanced nuclear systems

AbstractA ferritic ODS-alloy (Fe-14Cr-1W-0.25Ti) has been manufactured by application of the powder metallurgical production route involving at first mechanical alloying of ∼10kg pre-alloyed steel powder together with an Y2O3 addition for 12h in a high energy industrial ball mill under hydrogen atmosphere at the company ZOZ GmbH. As a next step, one part of the alloyed powder was hot extruded into rods while another portion was hot isostatically pressed into plates. Both materials were then heat treated. A characterization program on these ODS-alloy production forms included microstructural and mechanical investigations: SANS and TEM assume the existence of Y2Ti2O7 nano clusters and show a bimodal distribution of ODS-particle sizes in both ODS variants. EBSD maps showed a strong 〈110〉 texture corresponding to the α fiber for the hot extruded ODS and a slight 〈001〉 texture for the hipped ODS material. Fracture toughness tests in different specimen orientations (extruded ODS) with mini 0.2T C(T) specimens together with Charpy impact tests revealed anisotropic mechanical properties: Promising (fracture) toughness levels were obtained in the specimen orientation perpendicular to the extrusion direction, while the toughness levels remained low in extrusion direction and generally for the hipped ODS material at all test temperatures. The fracture toughness tests were performed according to ASTM E 1921 and 1820 standards

Mitochondrial protein synthesis adapts to influx of nuclear-encoded protein.

Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits

Mapping protein interactions in the active TOM-TIM23 supercomplex

Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

Unlocking the presequence import pathway.

Translocation of presequence-containing precursor proteins into the inner mitochondrial membrane and matrix is an essential process that is facilitated by the translocase of the outer membrane (TOM) together with the presequence translocase of the inner membrane (TIM23). After initial recognition by receptors of the TOM complex followed by transport across the outer membrane, the precursor emerges into the intermembrane space (IMS). Recognition of the presequence by Tim50 triggers rearrangements of the presequence translocase, priming it for inner membrane translocation. Subsequently, the precursor can be released into the membrane or translocated into the mitochondrial matrix aided by the import motor. This heat-shock protein 70 (Hsp70)-based motor drives precursor unfolding and translocation and is subject to dynamic remodelling. Here, we review recent advances in understanding of the mechanisms underlying protein transport along the presequence pathway

Driving Simulator study for intelligent cooperative intersection safety system (IRIS)

About forty percent of all accidents occur at intersections. The Intelligent Cooperative Intersection Safety system (IRIS), as part of the European research project SAFESPOT, is a roadside application and aims at minimizing the number of accidents at controlled and uncontrolled intersections. IRIS uses vehicle-to-infrastructure communication to track and analyze the movements of all individual vehicles. A key element of IRIS is to identify dangerous situations in time and take the appropriate measure to prevent collisions. This paper presents the concept of IRIS, points out the complexity of intersections and addresses the usefulness and results of a driving simulator study. KEYWORDS Intersection safety, SAFESPOT, IRIS, Cooperative, Vehicle-to-Infrastructure, Roadside application, Driving simulator, Driver behaviour, Human Factor

Humane Fettstammzellen produzieren BDNF und führen zu einer dosisabhängigen Neuritogenese von Spiralganglienzellen

Einleitung: Cochleaimplantate (CI) finden breite Anwendung in der Versorgung von hochgradig schwerhörigen oder ertaubten Patienten. Das Hörergebnis mit dem CI ist interindividuell sehr variabel und in anspruchsvollen Hörsituationen oft eingeschränkt. Ursächlich hierfür ist unter anderem die eingeschränkte Anzahl unabhängiger Frequenzbänder, die mit den derzeitig eingesetzten Geräten an den Hörnerv übertragen werden können. Zur Verbesserung dieser Situation wurde in der Literatur vielfach die Applikation neurotropher Faktoren vorgeschlagen. Humane Fettgewebsstammzellen (adipose-derived stem cells, ASC) sind multipotente Zellen, die neurotrophe Substanzen produzieren. In der vorliegenden Studie wurde die Wirkung von humanen ASC auf Spiralganglienzellen (SGZ) von Ratten untersucht. Methoden: SGZ von Ratten (postnataler Tag 5/6) wurden durch eine mikroskopische Präparation und enzymatische Digestion isoliert. Die ASC wurden aus Fettgewebe von fünf humanen Spendern isoliert. Kokulturen von SGZ und ASC wurden angelegt, wobei jeweils 1x104 SGZ (im Well) mit 5x104, 1x105, 2x105, 4x105 ASC oder ohne ASC (Kontrolle) kultiviert wurden. Die Fixierung erfolgte 2, 4 und 8 Tage nach Versuchsbeginn. Über mikroskopische Aufnahmen erfolgte eine Analyse der Neuritenlängen. Die Produktion von BDNF wurde mit einem ELISA Test untersucht.Ergebnisse: ASC führten zu physiologisch relevanter BDNF-Abgabe in das Medium. Diese war abhängig von der Anzahl der eingesetzten Zellen. Die Kokultivierung mit ASC führte zu einem vermehrten Überleben und einer dosisabhängig gesteigerten Neuritogenese der SGZ. Schlussfolgerungen: ASC scheinen Spinalganglienzellen in ihrem Neuritenwachstum positiv zu beeinflussen. Weitere Untersuchungen sind notwendig um die genaue Interaktion zwischen SGZ und ASC zu analysieren.Der Erstautor gibt keinen Interessenkonflikt an