Diffusion and transport in the human interphase cell nucleus - FCS experiments compared to simulations.

Despite the succesful linear sequencing of the human genome the three-dimensional arrangement of chromatin, functional, and structural components is still largely unknown. Molecular transport and diffusion are important for processes like gene regulation, replication, or repair and are vitally influenced by the structure. With a comparison between fluorescence correlation spectroscopy (FCS) experiments and simulations we show here an interdisciplinary approach for the understanding of transport and diffusion properties in the human interphase cell nucleus. For a long time the interphase nucleus has been viewed as a 'spaghetti soup' of DNA without much internal structure, except during cell division. Only recently has it become apparent that chromosomes occupy distinct 'territories' also in interphase. Two models for the detailed folding of the 30 nm chromatin fibre within these territories are under debate: In the Random-Walk/Giant-Loop-model big loops of 3 to 5 Mbp are attached to a non-DNA backbone. In the Multi-Loop-Subcompartment (MLS) model loops of around 120 kbp are forming rosettes which are also interconnected by the chromatin fibre. Here we show with a comparison between simulations and experiments an interdisciplinary approach leading to a determination of the three-dimensional organization of the human genome: For the predictions of experiments various models of human interphase chromosomes and the whole cell nucleus were simulated with Monte Carlo and Brownian Dynamics methods. Only the MLS-model leads to the formation of non-overlapping chromosome territories and distinct functional and dynamic subcompartments in agreement with experiments. Fluorescence in situ hybridization is used for the specific marking of chromosome arms and pairs of small chromosomal DNA regions. The labelling is visualized with confocal laser scanning microscopy followed by image reconstruction procedures. Chromosome arms show only small overlap and globular substructures as predicted by the MLS-model. The spatial distances between pairs of genomic markers as function of their genomic separation result in a MLS-model with loop and linker sizes around 126 kbp. With the development of GFP-fusion-proteins it is possible to study the chromatin distribution and dynamics resulting from cell cycle, treatment by chemicals or radiation in vivo. The chromatin distributions are similar to those found in the simulation of whole cell nuclei of the MLS-model. Fractal analysis is especially suited to quantify the unordered and non-euclidean chromatin distribution of the nucleus. The dynamic behaviour of the chromatin structure and the diffusion of particles in the nucleus are also closely connected to the fractal dimension. Fractal analysis of the simulations reveal the multi-fractality of chromosomes. First fractal analysis of chromatin distributions in vivo result in significant differences for different morphologies and might favour a MLS-model-like chromatin distribution. Simulations of fragment distributions based on double strand breakage after carbon-ion irradiation differ in different models. Here again a comparison with experiments favours a MLS-model. FCS in combination with a scanning device is a suitable tool to study the diffusion characteristics of fluorescent proteins in living cell nuclei with high spatial resolution. Computer simulations of the three-dimensional organization of the human interphase nucleus allows a detailed test of theoretical models in comparison to experiments. Diffusion and transport in the nucleus are most appropriately described with the concept of obstructed diffusion. A large volume fraction of the nucleus seems to contain a cytosol-like liquid with an apparent viscosity 5 times higher than in water. The geometry of particles and structure as well as their interactions influence the mobilities in terms of speed and spatial coverage. A considerable amount of genomic sites is accessible for not too large particles. FCS experiments and simulations based on the polymer model are in a good agreement. Using recently developed in vivo chromatin markers, a detailed study of mobility vs. structure is subject of current work

Hsp90 Inhibition Affects Cell Metabolism by Disrupting Mitochondrial Protein Insertion

Hsp90 is a molecular chaperone interacting with hundreds client proteins. Little is known, however, about Hsp90 influence on cancer cell metabolism. Here we show that the inhibition of Hsp90 affects indirectly glycolysis by disrupting mitochondrial insertion of the elements of Translocase of the Outer Membrane (TOM) complex in tumor cells. Improper insertion of Tom40 decreases the abundance of many mitochondrial proteins resulting in reduced mitochondrial activity. This is accompanied by increased production and release of lactate and serine. These results indicate an increased rate of glycolysis with serine synthesis compensating for the loss of energy and mitochondrially derived metabolites, potentially enhancing the metastatic potential of surviving cells. Our results provide novel insights into the effects of Hsp90 inhibition on cancer cell metabolism

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin

Implementation and applications of gas chromatography/ atmospheric pressure chemical ionization time-of-flight mass spectrometry in metabolomics

With the commercial introduction of atmospheric pressure chemical ionization for gas chromatography in 2008, GC-APCI coupled to high-resolution time-of-flight mass spectrometry (GC-APCI-HRTOFMS) became an interesting addition to the metabolomics toolbox. APCI is a soft ionization technique and its application to hyphenate GC to high resolution MS opens up promising new means for the identification of unknown signals in complex matrices. The actual utility of GC-APCI-HRTOFMS in metabolic fingerprinting and profiling applications to biological matrices is the topic of this doctoral thesis. During comparison of GC-APCI-HRTOFMS with GC×GC-EI-TOFMS, GC-EI-TOFMS, GC-CI-qMS, and GC-EI-qMS in my master thesis, it was noticed that reproducibility of APCI was affected greatly by differences in humidity in the laboratory. Therefore, the impact of humidity in the APCI source on ionization efficiency and repeatability was systematically studied in the initial project of this doctoral thesis. Water was continuously infused to ensure a constant humidity during APCI in the analysis of methylchloroformate (MCF)- and methoxime-trimethylsilyl (MeOx-TMS) derivatized metabolites. These two different derivatization strategies are most commonly pursued in GC-MS based metabolome analyses. Several infusion rates were tested and a rate of 0.4 mL/h yielded an average 16.6-fold increase in intensity of the protonated molecules ([M+H]+) of 20 MCF-derivatized metabolites through suppression of in-source fragmentation. Water infusion, however, did not improve efficiency and repeatability of APCI of methoxime-trimethylsilyl (MeOx-TMS) derivatives of metabolite standards. Then, the impact of water infusion on metabolic fingerprinting of biological specimens was investigated. Water infusion led to a marked increase in the number of metabolites identified in MCF-derivatized cancer cell extracts via their [M+H]+ ions and improved repeatability of peak areas, almost doubling the number (N=23) of identified, significantly regulated metabolites (false discovery rate <0.05) between controls and cancer cells treated with the heat-shock protein 90 (Hsp90) inhibitor 17-DMAG. Next, matrix effects caused by co-eluting compounds were investigated that might influence ionization. Strikingly, recovery of three out of seven internal standards used in a spike-in experiment was below 75% in one or several of the three biological matrices tested, namely Escherichia coli extract, serum, and urine. This was due to suppression by their respective endogenous metabolite that was present at a high concentration. Ion suppression caused by a co-eluting compound was further shown for three pairs of co-eluting standards employing standard mixtures with increasing concentrations of the co-eluting compounds. Overall these experiments demonstrated that matrix effects have to be taken into consideration in GC-APCI-MS. In the course of my doctoral thesis, Bruker Daltonics (Bremen, Germany) introduced a redesigned APCI source. To test the capabilities of this source, MeOx-TMS derivatized supernatants of untreated cancer cells were analyzed by GC-APCI-HRTOFMS using both the original APCI I and the redesigned APCI II source. The latter source almost doubled the number of spectral features with signal-to-noise ratios greater than 20 that could be extracted from metabolite fingerprints and increased the absolute number of identified metabolites by 33% from 36 to 48. In addition, the median area RSDs of extracted features decreased from 33% to 24%. These improvements further resulted in a more than fourfold median decrease in lower limits of quantification to 0.002 - 3.91 µM as evidenced for 20 MeOx-TMS derivatized metabolite standards and a concomitant increase in the linear range by 0.5 to almost three orders of magnitude. Finally, GC-APCI(II)-HRTOFMS was applied to the enantioselective quantitative profil-ing of the oncometabolite D-2-hydroxyglutarate (D-2-HG). MCF derivatization and GC analysis on a chiral gamma-cyclodextrin (Rt-rDEXsa) column were used to separate the D from the L enantiomer of 2-HG. Separation was optimized to avoid co-elution of D-2-HG with a highly abundant matrix compound present in cell culture media supplemented with bovine serum albumin. The use of APCI-HRTOFMS yielded highly specific quantifier ions and the infusion of water enhanced lower limits of quantification and repeatability by factors of ten and two, respectively. Analysis of a racemic 2-HG standard after MCF derivatization yielded a total of four peaks instead of the expected two signals for the D- and L-enantiomer. It was then shown that in addition to an open-chain three-fold derivative of 2-HG the methyl ester of the D/L-2-HG lactone is formed during derivatization. Since lactone of D/L-2-HG was found to be naturally present in biological specimens, the developed method was eventually based on the open-chain three-fold derivative of 2-HG yielding LLOQ values of 0.49 µM (D-2-HG) and 0.24 µM (L-2-HG). The GC-APCI(II)-HRTOFMS approach was successfully applied to the determination of D/L-2-HG concentration levels in urine specimens of 23 acute myeloid leukemia (AML) patients and 6 healthy controls, which were validated by HPLC-MS/MS. The yet to be discovered source of 2-HG lactone in sera of AML patients carrying neomorphic isocitrate dehydrogenase mutations promises to shed new light on the pathogenesis and progression of AML. In summary this doctoral thesis demonstrates that GC-APCI-HRTOFMS is a useful addition to the established GC-MS approaches in metabolomics. Studies on factors potentially influencing the ionization, namely water infusion, matrix effects and source type, distinctly widened the applicability of GC-APCI-HRTOFMS for qualitative and quantitative analysis of MeOx-TMS and MCF derivatized metabolites. The ability of APCI along with water infusion to efficiently ionize a broad range of MCF metabolites was proven and in the following applied to comparative metabolic fingerprinting in extracts of treated cancer cells. Finally, the outstanding quantitative capabilities of GC-APCI(II)-HRTOFMS were used in a first enantioselective profiling application for quantitative determination of the oncometabolite D-2-HG. Altogether, this doctoral thesis contributed significantly to the excellent progress GC-APCI-MS has made towards becoming a routine tool in metabolomics

Enhanced metabolite profiling using a redesigned atmospheric pressure chemical ionization source for gas chromatography coupled to high-resolution time-of-flight mass spectrometry.

An improved atmospheric pressure chemical ionization (APCI II) source for gas chromatography-high-resolution time-of-flight mass spectrometry (GC-HRTOFMS) was compared to its first-generation predecessor for the analysis of fatty acid methyl esters, methoxime-trimethylsilyl derivatives of metabolite standards, and cell culture supernatants. Reductions in gas turbulences and chemical background as well as optimized heating of the APCI II source resulted in narrower peaks and higher repeatability in particular for late-eluting compounds. Further, APCI II yielded a more than fourfold median decrease in lower limits of quantification to 0.002-3.91 μM along with an average 20 % increase in linear range to almost three orders of magnitude with R (2) values above 0.99 for all metabolite standards investigated. This renders the overall performance of GC-APCI-HRTOFMS comparable to that of comprehensive two-dimensional gas chromatography (GCþinspace×þinspaceGC)-electron ionization (EI)-TOFMS. Finally, the number of peaks with signal-to-noise ratios greater than 20 that could be extracted from metabolite fingerprints of pancreatic cancer cell supernatants upon switching from the APCI I to the APCI II source was more than doubled. Concomitantly, the number of identified metabolites increased from 36 to 48. In conclusion, the improved APCI II source makes GC-APCI-HRTOFMS a great alternative to EI-based GC-MS techniques in metabolomics and other fields

Virtuelle Werkstatt - Multimodale Interaktion für intelligente virtuelle Konstruktion

Fröhlich C, Wachsmuth I, Latoschik ME. Virtuelle Werkstatt - Multimodale Interaktion für intelligente virtuelle Konstruktion. In: Gausemeier J, Grafe M, eds. 8. Paderborner Workshop Augmented &amp; Virtual Reality in der Produktentstehung. Paderborn: HNI; 2009: 241-255.Das von der Deutschen Forschungsgemeinschaft geförderte Projekt „Virtuelle Werkstatt“ wurde im November 2001 mit dem Ziel begonnen, computergrafisch visualisierte dreidimensionale Modelle realer Konstruktionsteile in der Virtuellen Realität erprobbar zu machen. Das Projekt wurde in zwei Phasen gefördert, wobei die erste von November 2001 bis Dezember 2004 und das anschließende Folgeprojekt von Januar 2006 bis Dezember 2007 durchgeführt wurde. Während dieser zwei Förderungsphasen wurden viele technische Neuerungen auf dem Gebiet der Virtuellen Konstruktion erarbeitet. Zu diesen Neuerungen gehören unter anderem Arbeiten in den Bereichen der Künstlichen Intelligenz, des Virtuellen Prototypings, der multimodalen Mensch-Maschine-Interaktion, sowie der Softwareentwicklung für Simulationssysteme der Virtuellen Realität. In diesem Artikel wird ein Überblick über die durchgeführten Arbeiten gegeben

Continuous water infusion enhances atmospheric pressure chemical ionization of methyl chloroformate derivatives in gas chromatography coupled to time-of-flight mass spectrometry-based metabolomics

The effects of continuous water infusion on efficiency and repeatability of atmospheric pressure chemical ionization of both methyl chloroformate (MCF) and methoxime-trimethylsilyl (MO-TMS) derivatives of metabolites were evaluated using gas chromatography–time-of-flight mass spectrometry. Water infusion at a flow-rate of 0.4 mL/h yielded not only an average 16.6-fold increase in intensity of the quasimolecular ion for 20 MCF-derivatized metabolite standards through suppression of in-source fragmentation but also the most repeatable peak area integrals. The impact of water infusion was the greatest for dicarboxylic acids and the least for (hetero-) aromatic compounds. Water infusion also improved the ability to detect reliably fold changes as small as 1.33-fold for the same 20 MCF-derivatized metabolite standards spiked into a human serum extract. On the other hand, MO-TMS derivatives were not significantly affected by water infusion, neither in their fragmentation patterns nor with regard to the detection of differentially regulated compounds. As a proof of principle, we applied MCF derivatization and GC-APCI-TOFMS to the detection of changes in abundance of metabolites in pancreatic cancer cells upon treatment with 17-DMAG. Water infusion increased not only the number of metabolites identified via their quasimolecular ion but also the reproducibility of peak areas, thereby almost doubling the number of significantly regulated metabolites (false discovery rate < 0.05) to a total of 23

Performance evaluation of gas chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry for metabolic fingerprinting and profiling

Gas chromatography-atmospheric-pressure chemical ionization-time-of-flight mass spectrometry (GC-APCI-TOFMS) was compared to GC × GC-electron ionization (EI)-TOFMS, GC-EI-TOFMS, GC-chemical ionization (CI)-quadrupole mass spectrometry (qMS), and GC-EI-qMS in terms of reproducibility, dynamic range, limit of detection, and quantification using a mix of 43 metabolites and 12 stable isotope-labeled standards. Lower limits of quantification for GC-APCI-TOFMS ranged between 0.06 and 7.81 μM, and relative standard deviations for calibration replicates were between 0.4% and 8.7%. For all compounds and techniques, except in four cases, R(2) values were above 0.99. Regarding limits of quantification, GC-APCI-TOFMS was inferior to only GC × GC-EI-TOFMS, but outperformed all other techniques tested. GC-APCI-TOFMS was further applied to the metabolic fingerprinting of two Escherichia coli strains. Of 45 features that differed significantly (false discovery rate < 0.05) between the strains, 25 metabolites were identified through highly accurate and reproducible (Δm ± SD below 5 mDa over m/z 190-722) mass measurements. Starting from the quasimolecular ion, six additional metabolites were identified that had not been found in a previous study using GC × GC-EI-TOFMS and an EI mass spectral library for identification purposes. Silylation adducts formed in the APCI source assisted the identification of unknown compounds, as their formation is structure-dependent and is not observed for compounds lacking a carboxylic group