Visualization, analysis, and design of COMBO-FISH probes in the grid-based GLOBE 3D genome platform

The genome architecture in cell nuclei plays an important role in modern microscopy for the monitoring of medical diagnosis and therapy since changes of function and dynamics of genes are interlinked with changing geometrical parameters. The planning of corresponding diagnostic experiments and their imaging is a complex and often interactive IT intensive challenge and thus makes high-performance grids a necessity. To detect genetic changes we recently developed a new form of fluorescence in situ hybridization (FISH) - COMBinatorial Oligonucleotide FISH (COMBO-FISH) - which labels small nucleotide sequences clustering at a desired genomic location. To achieve a unique hybridization spot other side clusters have to be excluded. Therefore, we have designed an interactive pipeline using the grid-based GLOBE 3D Genome Viewer and Platform to design and display different labelling variants of candidate probe sets. Thus, we have created a grid-based virtual "paper" tool for easy interactive calculation, analysis, management, and representation for COMBO-FISH probe design with many an advantage: Since all the calculations and analysis run in a grid, one can instantly and with great visual ease locate duplications of gene subsequences to guide the elimination of side clustering sequences during the probe design process, as well as get at least an impression of the 3D architectural embedding of the respecti

e-Human Grid Ecology: Understanding and approaching the Inverse Tragedy of the Commons in the e-Grid Society.

With ever-new technologies emerging also the amount of information to be stored and processed is growing exponentially and is believed to be always at the limit. In contrast, however, huge resources are available in the IT sector alike e.g. the renewable energy sector, which are often even not at all used. This under-usage bares any rational especially in the IT sector where e.g. virtualisation and grid approaches could be fast implemented due to the great technical and fast turnover opportunities. Here, we describe this obvious paradox for the first time as the Inverse Tragedy of the Commons, in contrast to the Classical Tragedy of the Commons where resources are overexploited. From this perspective the grid IT sector attempting to share resources for better efficiency, reveals two challenges leading to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Thus, the challenges of grid implementation are similar to those of e.g. climate protection. This is well described by the classic Human Ecology triangle and our extension to a rectangle: environment-individual-society-environment. Extension of this classical interdisciplinary field of basic and applied research to an e-Human Grid Ecology rational, allows the Inverse Tragedy of the Commons of the grid sector to be understood and approached better and implies obvious guidelines in the day-to-day management for grid and other (networked) resources, which is of importance for many fields with similar paradoxes as in (e-)society

Large-scale resource sharing at public funded organizations. e-Human "Grid" Ecology.

With ever-new technologies emerging also the amount of information to be stored and processed is growing exponentially and is believed to be always at the limit. In contrast, however, huge resources are available in the IT sector alike e.g. the renewable energy sector, which are often even not at all used. This under-usage bares any rational especially in the IT sector where e.g. virtualisation and grid approaches could be fast implemented due to the great technical and fast turnover opportunities. Here, we describe this obvious paradox for the first time as the Inverse Tragedy of the Commons, in contrast to the Classical Tragedy of the Commons where resources are overexploited. From this perspective the grid IT sector attempting to share resources for better efficiency, reveals two challenges leading to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Thus, the challenges of grid implementation are similar to those of e.g. climate protection. This is well described by the classic Human Ecology triangle and our extension to a rectangle: environment-individual-society-environment. Extension of this classical interdisciplinary field of basic and applied research to an e-Human Grid Ecology rational, allows the Inverse Tragedy of the Commons of the grid sector to be understood and approached better and implies obvious guidelines in the day-to-day management for grid and other (networked) resources, which is of importance for many fields with similar paradoxes as in (e-)society

Large-scale resource sharing at public funded organizations. e-Human "Grid" Ecology.

With ever-new technologies emerging also the amount of information to be stored and processed is growing exponentially and is believed to be always at the limit. In contrast, however, huge resources are available in the IT sector alike e.g. the renewable energy sector, which are often even not at all used. This under-usage bares any rational especially in the IT sector where e.g. virtualisation and grid approaches could be fast implemented due to the great technical and fast turnover opportunities. Here, we describe this obvious paradox for the first time as the Inverse Tragedy of the Commons, in contrast to the Classical Tragedy of the Commons where resources are overexploited. From this perspective the grid IT sector attempting to share resources for better efficiency, reveals two challenges leading to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Thus, the challenges of grid implementation are similar to those of e.g. climate protection. This is well described by the classic Human Ecology triangle and our extension to a rectangle: environment-individual-society-environment. Extension of this classical interdisciplinary field of basic and applied research to an e-Human Grid Ecology rational, allows the Inverse Tragedy of the Commons of the grid sector to be understood and approached better and implies obvious guidelines in the day-to-day management for grid and other (networked) resources, which is of importance for many fields with similar paradoxes as in (e-)society

How to measure diagnosis-associated information in virtual slides

The distribution of diagnosis-associated information in histological slides is often spatial dependent. A reliable selection of the slide areas containing the most significant information to deriving the associated diagnosis is a major task in virtual microscopy. Three different algorithms can be used to select the appropriate fields of view: 1) Object dependent segmentation combined with graph theory; 2) time series associated texture analysis; and 3) geometrical statistics based upon geometrical primitives. These methods can be applied by sliding technique (i.e., field of view selection with fixed frames), and by cluster analysis. The implementation of these methods requires a standardization of images in terms of vignette correction and gray value distribution as well as determination of appropriate magnification (method 1 only). A principle component analysis of the color space can significantly reduce the necessary computation time. Method 3 is based upon gray value dependent segmentation followed by graph theory application using the construction of (associated) minimum spanning tree and Voronoi’s neighbourhood condition. The three methods have been applied on large sets of histological images comprising different organs (colon, lung, pleura, stomach, thyroid) and different magnifications, The trials resulted in a reproducible and correct selection of fields of view in all three methods. The different algorithms can be combined to a basic technique of field of view selection, and a general theory of “image information” can be derived. The advantages and constraints of the applied methods will be discussed

Porting Erasmus Computing Grid (Condor enabled Applications for EDGeS)

Today advances in scientific research as well as clinical diagnostics and treatment are inevitably connected with information solutions concerning computation power and information storage. The needs for information technology are enormous and are in many cases the limiting factor for new scientific results or clinical diagnostics and treatment. At the Hogeschool Rotterdam and the Erasmus MC there is a massive need for computation power on a scale of 10,000 to 15,000 computers equivalent to ~20 to ~30 Tflops (1012 floating point operations per second) for a variety of work areas ranging from e.g. MRI and CT scan and microscopic image anlysis to DNA sequence analysis, protein and other structural simulations and analysis. Both institutions have already 13,000 computers, i.e. ~18 Tflops of computer power, available! To make the needed computer power accessible, we started to build the Erasmus Computing Grid (ECG), which is connecting local computers in each institution via central management systems. The system guaranties security and any privacy rules through the used software as well as through our set-up and a NAN and ISO certification process being under way. Similar systems run already world-wide on entire institutions including secured environments like government institutions or banks. Currently, the ECG has a computational power of ~5 Tflops and is one of or already the largest desktop grid in the world. At the Hogeschool Rotterdam meanwhile all computers were included in the ECG. Currently, 10 departments with ~15 projects at the Erasmus MC depend on using the ECG and are preparing or prepared their analysis programs or are already in production state. The Erasmus Computing Grid office and an advisory and control board were set-up. To sustain the ECG now further infrastructure measures have to be taken. Central hardware and specialist personal needs to be put in place for capacity, security and usability reasons for the application at Erasmus MC. This is also necessary in respect to NAN and ISO certification towards diagnostic and commercial ECG use, for which there is great need and potential. Beyond the link to the Dutch BigGrid Initiative and the German MediGRID should be prepared for and realized due to the great interest for cooperation. There is also big political interest from the government to relieve the pressure on computational needs in The Netherlands and to strengthen the Dutch position in the field of high performance computing. In both fields the ECG should be brought into a leading position by establishing the Erasmus MC a centre of excellence for high-performance computing in the medical field in respect to Europe and world-wide. Consequently, we successfully started to build a super-computer at the Hogeschool Rotterdam and Erasmus MC with great opportunities for scientific research, clinical diagnostics and research as well as student training. This will put both institutions in the position to play a major world-wide role in high-performance computing. This will open entire new possibilities for both institutions in terms of recognition and new funding possibilities and is of major importance for The Netherlands and the EU

The 3D chromatin structure of the mouse β-haemoglobin gene cluster

Here we show a 3D DNA-FISH method to visualizes the 3D structure of the β-globin locus. Geometric size and shape measurements of the 3D rendered signals (128Kb) show that the volume of the β-globin locus decreases almost two fold upon gene activation. A decrease in length and a distinctive change in shape and surface structure of the locus are also observed. Adding 5’ and 3’end regions to the probe (175Kb) showed a less prominent change in length, shape and structure. It was shown (data not on this poster) that the physical distance between the two flanking regions shift in a similar limited manner, indicating that the flanking regions do not participate in ACH formation and thus active chromatin folding is occurring only within the locus proper

COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci

With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15–30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)—synthetic DNA analogues with a neutral backbone—which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488® showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10–20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome

Chromatin Dynamics of the mouse β-globin locus

Lately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors (TF) to TF binding sites also becomes an important factor to the gene’s activation status. Both such changes have been ascribed to the mouse ?- haemoglobin gene cluster as a trigger to activate globin expression in the erythroid cell lineage. Early models speculated a scanning, random activation or a looping mechanism to activate globin transcription. The chromatin conformation capture (3C) technique has shown that there is a molecular interaction between various DNAse I hypersensitive (HS) sites that are located up- and downstream of the ?-globin gene cluster, the HS sites of the Locus Control Region (LCR) and the promoter by means of a dynamic looping mechanism. The clustering of the HS sites of the LCR and the up- and down- stream HS sites results in the formation of a so called Active Chromatin Hub (ACH) which is depending on at least two erythoid TF: EKLF and GATA-1. The long range interactions between the outlying HS -84/-85, -62/-60 and the 3’HS1 are depending on the presence of CTCF, a TF that is thought to play an important role in long range chromatin interactions across the whole genome. Prior to gene activation, cells of the early erythroid lineage (progenitors) already show a presence of an ACH, which is not found in non-erythroid cells. The final chromatin 3D structure consist of four major loops sizing 25- 38Kb and two minor loops within the LCR sizing 4.5 and 12Kb. To confirm this looping hypothesis (based on 3C technology) we used an in situ hybridization approach to visualize and, after image restoration, quantitatively measure the 3D conformational changes that take place within the locus in erythroid cells before and after differentiation. Globin gene activation is depending on long distance looping of the up- and downstream HS sites and the ?-major promotor to the LCR, resulting in a complex 3D chromatin structure. By staining the m?- globin loci with fluorescence labeled sequence specific probes followed by high-resolution 3D imaging and 3D volume rendering of the deconvolved images, the loci reveal changes in the geometric size and shape properties when cells are differentiated into a active globin transcribing cell. An almost 2x decrease in volume was measured, which was mostly due to a reduction of the longest length measured. This can be explained by a change in loop formation. The almost 70Kb loop between the LCR and the 3’HS1 is folded into two loops of 34 and 35Kb upon interaction of the promotor to the ACH to activate transcription. The limited decrease in volume and length when the locus was probed with an additional 5’ and 3’ end region is surprising. The 5’ end is actively participating in the looping process that stabilizes the ACH. However, the 3’ end has (until now) not been seen to be participate in ACH formation or any complex looping mechanism for globin gene activation. As this part of the locus seems to be the most un-dynamic, it could be the dominating factor that influences the fluorescent signal emitting from the probed DNA region and therefore cloud subtle changes in the chromatin folding mechanism of gene activation. Next to the dynamic chromatin folding process that is occurring between the HS-85/84 and the 3’HS1, a stretch of “rigid” DNA can prevent a DNA region containing activated genes to stay at the edge of a chromosome territory and possibly prevent a close proximity to the silencing effect of (spreading) heterochromatin. An increase in lateral and axial resolution like the 3D Structural Illumination Microscope (SIM) provides, could help solve the problem of detecting subtle 3D changes in chromatin structure. And in the near future will reveal many more details of 3D chromatin organization of not only the m?-globin locus but of many other intra-cellular processes