Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis.

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement.

Changes in lamina structure are followed by spatial reorganization of heterochromatic regions in caspase-8-activated human mesenchymal stem cells.

Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis.

Detection of tumor cells in bone marrow, peripheral blood and lymph nodes by automated imaging devices.

The presence of tumor cells in bone marrow, peripheral blood and lymph nodes has proven its clinical and prognostic value. Since the frequency of these cells in bone marrow and blood is sometimes as low as 1 per million and due to the fact that for the analysis of lymph nodes many sectioning levels have to be analyzed, automated imaging devices have been suggested as an useful alternative to conventional manual screening of specimens. The aim of this paper is to review the performance of current equipment that is commercially available, based on literature published so far.

Array CGH detection of a cryptic deletion in a complex chromosome rearrangement.

Balanced complex chromosome rearrangements (CCR) are extremely rare in humans. They are usually ascertained either by abnormal phenotype or reproductive failure in carriers. These abnormalities are attributed to disruption of genes at the breakpoints, position effect or cryptic imbalances in the genome.

Insights from genomic microarrays into structural chromosome rearrangements.

Array-based comparative genomic hybridization allows high-resolution screening of copy number abnormalities in the genome, and becomes an increasingly important tool to detect deletions and duplications in tumor and post-natal cytogenetics. Here we illustrate that genomic arrays can also provide novel clues regarding the structural basis of chromosome rearrangement, including instability and mechanisms of formation of ring chromosomes. We also showed that array results might impact the recurrence risks for relatives of affected individuals.

Automated analysis of multiple sections for the detection of occult cells in lymph nodes.

Purpose: At present, reverse transcription (RT)-PCR against carcino-embryonic antigen mRNA is one of the few research tools for the detection of occult cells in histopathologically assessed negative lymph nodes from patients with colorectal cancer. The aim of this study was to investigate the suitability of supervised low-resolution image analysis of immunohistochemically stained sections as alternative.

Experimental Design: Multiple sections (n = 50) of regional lymph nodes from patients with colorectal cancer were immunohistochemically stained and analyzed by applying low-resolution image analysis (flatbed scanning) for semiautomated detection of cytokeratin (CK)-positive stained cells.

Automated acquisition of stained tissue microarrays for high-throughput evaluation of molecular targets.

At present, limiting factors in the use of tissue microarrays (TMAs) for high-throughput analysis relate to the visual evaluation of the staining patterns of each of the individual cores in the array and to the subsequent input of the results into a database. Such a database is essential to correlate the data with tumor type and outcome, and to evaluate the performance against other markers achieved in separate experiments. So far, these steps are mostly performed by hand, and consequently are time-consuming and potentially prone to bias and errors, respectively.