Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail

Background  

High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL).     

In situ quantification of aberrant p53 in colorectal neoplasia

Aberrant p53 protein accumulation was measured immunohistologically in 342 colorectal paraffin-embedded tissue sections from 115 patients (24 with adenocarcinoma, 59 with adenoma and 32 'hospital controls'). Subjective scoring was compared with quantitative cell imaging, including dichotomous (p53⁺/p53^-) status, ng p53^mut mg^-1 enterocyte protein, and tumour burden and patient body 'burden' of aberrant p53. A total of 62.5% cancer patients, 23.7% adenoma patients and 3.1% hospital controls were accorded p53⁺ status on the basis of p53 quantification. Quantitative p53⁺/p53^- assignment had a stronger inverse association with survival (χ²=6.17, p=0.013, Kaplan-Meier test) than subjective 'visual estimation' (χ²=0.57, p=0.449). There was a strong inverse relationship between the p53 'body burden' and the months of post-diagnosis survival (hazard ratio=1.42, p=0.0004, Cox proportional hazards). Absolute quantification for inactivated p53 permits objective and reproducible scoring, adjusts for intra-laboratory immunostaining 'batch effects', corrects for fixation artefacts, and standardizes for inter-laboratory differences in fixation, antibody selection and staining method. Clinically, in situ quantification of p53 will permit more accurate survival prognoses and will inform therapy selection and dose. Ultimately, accurate quantitative tissue/blood p53 correlations may provide a minimally invasive and systemic surrogate measure for these same clinical purposes.     

Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper‐preserved archaeological bone using size exclusion chromatography

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Ecdysone receptor expression and activity in adult Drosophila melanogaster

Disrupting components of the ecdysone/EcR/USP signaling pathway in insects leads to morphological defects and developmental arrest. In adult Drosophila melanogaster decreased EcR function affects fertility, lifespan, behavior, learning, and memory; however we lack a clear understanding of how EcR/USP expression and activity impacts these phenotypes. To shed light on this issue, we characterized the wild-type expression patterns and activity of EcR/USP in individual tissues during early adult life. EcR and usp were expressed in numerous adult tissues, but receptor activity varied depending on tissue type and adult age. Receptor activity did not detectably change in response to mating status, environmental stress, ecdysone treatment or gender but is reduced when a constitutively inactive ecdysone receptor is present. Since only a subset of adult tissues expressing EcR and usp contain active receptors, it appears that an important adult function of EcR/USP in some tissues may be repression of genes containing EcRE's.     

Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.     

Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

Regeneration in the auditory system of nymphal and adult bush crickets Tettigonia viridissima

Regeneration and reestablishment of synaptic connections is an important topic in neurobiological research. In the present study, the regeneration of auditory afferents and the accompanying effects in the central nervous system are investigated in nymphs and adults of the bush cricket Tettigonia viridissima L. (Orthoptera: Tettigoniidae). In all animals in which the tympanal nerve is crushed, neuronal tracing shows a regrowth of the afferents into the prothoracic ganglion. This regeneration is seen in both adult and nymphal stages and starts 10–15 days after nerve crushing. Physiological recordings from the leg nerve indicate a recovery of tympanal fibres and a formation of functional connections to interneurones in the same time range. Electrophysiological recordings from the neck connective suggest additional contralateral sprouting of interneurones and the formation of aberrant connections. The regeneration processes of the tympanal nerve in nymphal stages and adults appear to be similar.