The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2- mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 ( BRCA2 ΔE5+7 ), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the ‘ BRCAness’ profile.

The SatNOGS, or Satellite Network Open Ground Stations, project promotes and supports free and open space applications. It seeks to solve the problem of connecting many satellite users/observers to many ground station operators. Modern open software, web, and hardware techniques are used in implementing the Network, Database, Client, and Ground Station subprojects. Modularity in all the systems promotes the dual-use of ground stations by not interfering with local operation while utilizing the great amount of time a civilian, non-commercial ground station would otherwise sit idle.

The EVI1 (ecotropic viral integration site 1) gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML) is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA) derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196) in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D), which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

NATURE METhOdS | VOL.9 NO.7 | JULY 2012 | 683 BioImageXd puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXd has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.

BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.

Presented is an overview of the image-analysis software platform Fiji, a distribution of ImageJ that updates the underlying ImageJ architecture and adds modern software design elements to expand the capabilities of the platform and facilitate collaboration between biologists and computer scientists. Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.

A lipophilic dye based on the Bodipy fluorophore, LD540, was developed for microscopic imaging of lipid droplets. In contrast to previous lipid droplet dyes, it can spectrally be resolved from both green and red fluorophores allowing multicolor imaging in both fixed and living cells. Its improved specificity, brightness and photostability support live cell imaging, which was used to demonstrate by two-color imaging lipid droplet motility along microtubules.

Biological matter is one of the most diverse and important classes of materials. Products of living organisms (wood, bone, cotton, wool, leather, coal, oil, drugs, etc.) are vital to humanity as foodstuffs, energy sources, engineering and construction materials, and chemicals; and by the way, they shape the environment of the biosphere.

The field of High Content Screening (HCS) has evolved from a technology used exclusively by the pharmaceutical industry for secondary drug screening, to a technology used for primary drug screening and basic research in academia. The size and the complexity of the screens have been steadily increasing. This is reflected in the fact that the major challenges facing the field at the present are data mining and data storage due to the large amount of data generated during HCS. On the one hand, technological progress of fully automated image acquisition platforms, and on the other hand advances in the field of automated image analysis have made this technology more powerful and more accessible to less specialized users. Image analysis solutions for many biological problems exist and more are being developed to increase both the quality and the quantity of data extracted from the images acquired during the screens. We highlight in this review some of the major challenges facing automatic high throughput image analysis and present some of the software solutions available on the market or from academic open source solutions.

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Fanconi anemia (FA) is an inherited disease with congenital abnormalities and an extreme risk of acute myeloid leukemia (AML). Genetic events occurring during malignant transformation in FA and the biology of FA‐associated AML are poorly understood, but are often preceded by the development of chromosomal aberrations involving 3q26‐29 in bone marrow of FA patients. We report here the molecular cytogenetic characterization of FA‐derived AML cell lines SB1685CB and SB1690CB by conventional and array comparative genomic hybridization, fluorescence in situ hybridization, and SKY. We identified gains of a 3.7 MB chromosomal region on 3q26.2‐26.31, which preceded transformation to overt leukemia. This region harbors the oncogenic transcription factor EVI1. A third FA‐derived cell line, FA‐AML1, carried a translocation with ectopic localization of 3q26 including EVI1 . Rearrangements of 3q, which are rare in childhood AML, commonly result in overexpression of EVI1, which determines specific gene expression patterns and confers poor prognosis. We detected overexpression of EVI1 in all three FA‐derived AML. Our results suggest a link between the FA defect, chromosomal aberrations involving 3q and overexpression of EVI1 . We hypothesize that constitutional or acquired FA defects might be a common factor for the development of 3q abnormalities in AML. In addition, cryptic imbalances as detected here might account for overexpression of EVI1 in AML without overt 3q26 rearrangements. © 2007 Wiley‐Liss, Inc.

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.

Fanconi anemia (FA) is an inherited disease with congenital abnormalities and an extreme risk of acute myeloid leukemia (AML). Genetic events occurring during malignant transformation in FA and the biology of FA-associated AML are poorly understood, but are often preceded by the development of chromosomal aberrations involving 3q26-29 in bone marrow of FA patients. We report here the molecular cytogenetic characterization of FA-derived AML cell lines SB1685CB and SB1690CB by conventional and array comparative genomic hybridization, fluorescence in situ hybridization, and SKY. We identified gains of a 3.7 MB chromosomal region on 3q26.2-26.31, which preceded transformation to overt leukemia. This region harbors the oncogenic transcription factor EVII. A third FA-derived cell line, EA-AMLI, carried a translocation with ectopic localization of 3q26 including EVII. Rearrangements of 3q, which are rare in childhood AML, commonly result in overexpression of EVII, which determines specific gene expression patterns and confers poor prognosis. We detected overexpression of EVII in all three FA-derived AML. Our results suggest a link between the FA defect, chromosomal aberrations involving 3q and overexpression of EVII. We hypothesize that constitutional or acquired FA defects might be a common factor for the development of 3q abnormalities in AML. In addition, cryptic imbalances as detected here might account for overexpression of EVII in AML without overt 3q26 rearrangements. (c) 2007 Wiley-Liss, Inc.

Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) carry a high risk of haematological cancer. Affected cellular pathways may be modulated in sporadic malignancies and silencing of FANCF through methylation has been shown to cause somatic disruption of the FA pathway. Combined bisulphite restriction analysis for methylation of FANCF, FANCB and NBS1 was used to investigate 81 sporadic acute childhood leukaemias. No methylation was detected at any associated CpG sites analysed. This does not exclude very low levels of FANCF, FANCB or NBS1 methylation, but suggests other factors are responsible for chemo-sensitivity and chromosomal instability in sporadic childhood leukaemia.

With the recent success of the human genome project, researchers must now discover biological functions of the tens of thousands of proteins encoded in the genome. Molecular cell biology and structural biology methods are required for this work. Modern multidimensional bioimaging methods, such as laser scanning confocal microscopy and other light microscopic techniques, have emerged as a major research methodology in this new era of informational biology. These methods all produce vast amounts of digital information and require specialized computer software to process and analyze it. Several programs intended for this purpose have become available in recent years, but microscopists around the world frequently discover them unsuitable for many of their required tasks. The reasons for this are varied, and include: extensive bugs that take too long to fix, prohibitively high prices (also for updates and bug fixes), algorithms that are undisclosed and as such unacceptable for accurate scientific work, limited sets of features dispersed into separate “modules”, complex and difficult user interfaces, and lack of flexibility in adapting to specific research situations.

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

ABSTRACT Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy.