Extreme variation in apoptosis capacity amongst lymphoid cells of Nijmegen breakage syndrome patients

The human genetic disorder, Nijmegen breakage syndrome (NBS), is characterised by radiosensitivity, immunodeficiency and an increased risk for cancer, particularly lymphoma. The NBS1 gene codes for a protein, nibrin, involved in the processing/repair of DNA double strand breaks and in cell cycle checkpoints. The majority of patients (>90%) are homozygous for a founder mutation. Despite this genetic homogeneity, the syndrome shows considerable clinical variability, for example, in age at development of a malignancy. We hypothesised that one reason for such variation might be individual differences in the clearance of heavily damaged precancerous cells by apoptosis. To test this hypothesis we have examined a set of 30 lymphoblastoid B-cell lines from NBS patients for their capacity to enter into apoptosis after a DNA-damaging treatment. There was a substantial 40-fold variation in apoptosis between cell lines from different patients. NBS patient cell lines could be grouped into a large, apoptosis-deficient group and a smaller group with essentially normal apoptotic response to DNA damage. In both groups, cell lines were proficient in TP53 phosphorylation and stabilisation after the same DNA-damaging treatment. Thus the observed variation in apoptosis capacity is not due to failure to activate TP53. Despite the large variation in apoptosis, no statistically significant correlation between apoptotic capacity of patient cell lines and clinical course of the disease was apparent.     

Indirect genetic effects and sexual conflicts: Partner genotype influences multiple morphological and behavioral reproductive traits in a flatworm

The expression of an individual's phenotypic traits can be influenced by genes expressed in its social partners. Theoretical models predict that such indirect genetic effects (IGEs) on reproductive traits should play an important role in determining the evolutionary outcome of sexual conflict. However, empirical tests of (i) whether reproductive IGEs exist, (ii) how they vary among genotypes, and (iii) whether they are uniform for different types of reproductive traits are largely lacking. We addressed this in a series of experiments in the simultaneously hermaphroditic flatworm Macrostomum lignano. We found strong evidence for IGEs on both morphological and behavioral reproductive traits. Partner genotype had a significant impact on the testis size of focal individuals—varying up to 2.4‐fold—suggesting that IGEs could mediate sexual conflicts that target the male sex function. We also found that time to first copulation was affected by a genotype × genotype interaction between mating partners, and that partner genotype affected the propensity to copulate and perform the postcopulatory suck behavior, which may mediate conflicts over the fate of received ejaculate components. These findings provide clear empirical evidence for IGEs on multiple behavioral and morphological reproductive traits, which suggests that the evolutionary dynamics of these traits could be altered by genes contained in the social environment.     

Multiplexing clonality: combining RGB marking and genetic barcoding

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.     

ATP-dependent remodeling by SWI/SNF and ISWI proteins stimulates V(D)J cleavage of 5 S arrays

Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG proteins and HhaI might have different requirements for maximal accessibility of the substrate.     

Objective quality evaluation of fluorescence images to optimize automatic image acquisition.

BACKGROUND: Because different spectral sensitivities of human eye and image sensor lead to different perception of fluorescence signals, data generation is an important step in image analysis, because following work steps depend on it. METHODS: We developed a method to determine image parameters allowing an objective appraisal of quality of image data as well as a separation of object and background. RESULTS: Calculated parameters can be used for an automated adjustment of camera parameters in image analysis systems. DISCUSSION: Our approach for objective adjusted data generation achieves an improvement of analysis quality.     

High recurrence of rearrangements involving chromosome 14 in an ataxia telangiectasia lymphoblastoid cell line and in its mutagen-treated derivatives

Summary The cytogenetic characterization of CH cell line obtained by Epstein-Barr-virus transformation of the lymphocytes of a patient affected by ataxia telangiectasia is reported. Control CH cells and 2 subcultures treated with the mutagens R7000 or NQO were developed in parallel and studied. A common chromosome anomaly, a der(14) t(11;14) (q13.2;q32), was found in all the studied karyotypes, indicating that it occurred either in vivo or early in vitro. In non-treated cultures, additional anomalies were present in 6 derived subclones. All R-7000 treated cells had the same karyotype corresponding to one of the subclones observed without prior treatment. All NQO-treated cells acquired 2 common anomalies, and could be differentiated into 2 subclones because of the addition of a t(7;14) or a t(11;14). Chromosome 14 was involved in various rearrangements after breakage in band q11.2 or q12 in 6/8 subclones. This was not correlated with tumorigenicity, which was clearly increased in mutagen-treated cells as tested by in vitro growth in semi-solid medium and in vivo by grafts into nude mice or growth on the chorio-allantoic membrane of chick embryos. The CH cell line and its derivatives appear to be a promising in vitro system, showing various stages progressing towards malignancy, and reproducing a number of chromosome anomalies spontaneously occurring in AT patients.     

Distribution of regional lung aeration and perfusion during conventional and noisy pressure support ventilation in experimental lung injury

In acute lung injury (ALI), pressure support ventilation (PSV) may improve oxygenation compared with pressure-controlled ventilation (PCV), and benefit from random variation of pressure support (noisy PSV). We investigated the effects of PCV, PSV, and noisy PSV on gas exchange as well as the distribution of lung aeration and perfusion in 12 pigs with ALI induced by saline lung lavage in supine position. After injury, animals were mechanically ventilated with PCV, PSV, and noisy PSV for 1 h/mode in random sequence. The driving pressure was set to a mean tidal volume of 6 ml/kg and positive end-expiratory pressure to 8 cmH?O in all modes. Functional variables were measured, and the distribution of lung aeration was determined by static and dynamic computed tomography (CT), whereas the distribution of pulmonary blood flow (PBF) was determined by intravenously administered fluorescent microspheres. PSV and noisy PSV improved oxygenation and reduced venous admixture compared with PCV. Mechanical ventilation with PSV and noisy PSV did not decrease nonaerated areas but led to a redistribution of PBF from dorsal to ventral lung regions and reduced tidal reaeration and hyperinflation compared with PCV. Noisy PSV further improved oxygenation and redistributed PBF from caudal to cranial lung regions compared with conventional PSV. We conclude that assisted ventilation with PSV and noisy PSV improves oxygenation compared with PCV through redistribution of PBF from dependent to nondependent zones without lung recruitment. Random variation of pressure support further redistributes PBF and improves oxygenation compared with conventional PSV.     

A novel statistical approach for the evaluation of comet assay data

The present study forms part of a weight-of-evidence framework including genotoxicological studies in the upper Danube River basin, which aim at elucidating the reasons for the decline in fish catch. The major focus of this paper is the assessment of genotoxicity of sediments from the Danube River basin by use of the comet assay with RTL-W1 cells and with embryos of zebrafish (Danio rerio). A frequently discussed question in this type of approach is how to aggregate and compare the data obtained from genotoxicity testing. There is a need to develop mathematical method combining the information from dose-response curves and level of effectiveness (maximum genotoxic effect). For comparison and ranking of the genotoxic potential of samples from different locations along the Danube River, several methods based on EC50, Lowest Observed Effect Concentration (LOEC), and maximum induction factor were compared with respect to their validity. An evaluation system termed the "3-step analysis" was developed to facilitate consideration of a maximum number of aspects of the raw data. The so-called "concentration-dependent induction factor" (CDI) introduces an index for a straightforward, precise and realistic assessment of the genotoxic potential of any kind of field sample or genotoxic agent.     

Molecular recognition of the protein phosphatase 1 glycogen targeting subunit by glycogen phosphorylase

Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (G(L)) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a G(L)-derived peptide. The structure reveals the C terminus of G(L) binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. G(L) mimics interactions that are otherwise employed by the activator AMP. Functional studies show that G(L) binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for G(L) binding potency. Our study validates the G(L)-phosphorylase interface as a novel target for small molecule interaction.