PubMed Health⌕ Search

Biomedical subjects

Alan Forster

Publications and source records attributed to Alan Forster.

15 recordsLinked to original sources
PubMed2006

Interrogation of genomes by molecular copy-number counting (MCC).

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations.

Carcinoma, Renal Cell↗
PubMed2006

Singularity characteristics of needle EMG IP signals.

Clinical electromyography (EMG) interference pattern (IP) signals can reveal more diagnostic information than their constituents, the motor unit action potentials (MUAPs). Singularities and irregular structures typically characterize the mathematically defined content of information in signals. In this paper, a wavelet transform method is used to detect and quantify the singularity characteristics of EMG IP signals using the Lipschitz exponent (LE) and measures derived from it. The performance of the method is assessed in terms of its ability to discriminate healthy, myopathic and neuropathic subjects and how it compares with traditionally used Turns Analysis (TA) methods and a method recently developed by the authors, interscale wavelet maximum (ISWM). Highly significant intergroup differences were found using the LE method. Most of the singularity measures have a performance similar to that of ISWM and considerably better than that of TA. Some measures such as the ratio of the mean LE value to the number of singular points in the signal have considerably superior performance to both methods. These findings add weight to the view that wavelet analysis methods offer an effective way forward in the quantitative analysis of EMG IP signal to assist the clinician in the diagnosis of neuromuscular disorders.

Action Potentials↗
PubMed2005

Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis.

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.

Animals↗
PubMed2005

The Ews-ERG fusion protein can initiate neoplasia from lineage-committed haematopoietic cells.

The EWS-ERG fusion protein is found in human sarcomas with the chromosomal translocation t(21;22)(q22;q12), where the translocation is considered to be an initiating event in sarcoma formation within uncommitted mesenchymal cells, probably long-lived progenitors capable of self renewal. The fusion protein may not therefore have an oncogenic capability beyond these progenitors. To assess whether EWS-ERG can be a tumour initiator in cells other than mesenchymal cells, we have analysed Ews-ERG fusion protein function in a cellular environment not typical of that found in human cancers, namely, committed lymphoid cells. We have used Ews-ERG invertor mice having an inverted ERG cDNA cassette flanked by loxP sites knocked in the Ews intron 8, crossed with mice expressing Cre recombinase under the control of the Rag1 gene to give conditional, lymphoid-specific expression of the fusion protein. Clonal T cell neoplasias arose in these mice. This conditional Ews gene fusion model of tumourigenesis shows that Ews-ERG can cause haematopoietic tumours and the precursor cells are committed cells. Thus, Ews-ERG can function in cells that do not have to be pluripotent progenitors or mesenchymal cells.

Animals↗
PubMed2005

Sensory recovery in the sensate free transverse rectus abdominis myocutaneous flap.

BACKGROUND: Preservation and restoration of sensation to improve the quality of the reconstruction have not always been considered a priority in efforts to reconstruct the breast. Studies have documented spontaneous recovery of sensation in reconstructed breasts, but this recovery was variable and unpredictable. The authors conducted a pilot study to compare sensory recovery in innervated versus noninnervated microsurgical breast reconstruction patients. METHODS: The authors investigated a group of 14 patients who had undergone microsurgical breast reconstruction with a muscle-sparing free transverse rectus abdominis myocutaneous flap. Seven patients had reinnervation of their flaps, and seven did not. The two patient groups were similar in age (mean age, 46 years for the innervated group and 51 years for the noninnervated group) and length of follow-up (mean duration of follow-up, 39 months for the innervated group and 40 months for the noninnervated group). Sensitivity to fine touch was assessed using Semmes-Weinstein monofilaments. By testing over a defined grid and over the entire reconstructed breast, it was possible to develop color maps, or sensory topograms, of the areas sensing different levels of pressure for each patient. The patient's ability to differentiate between hot (60 degrees C) and cold (5 degrees C) was recorded. RESULTS: In a comparison of sensation in flap skin (skin paddle of rectus abdominis myocutaneous flap) with nonflap skin (native breast skin), sensitivity to fine touch was statistically better in (1) innervated flap skin compared with noninnervated flap skin (p = 0.003), (2) innervated nonflap skin compared with noninnervated flap skin (p = 0.006), (3) innervated nonflap skin compared with noninnervated nonflap skin (p = 0.037), and (4) innervated flap skin compared with noninnervated nonflap skin (p = 0.006). Temperature differentiation was significantly better in patients with innervated flaps than in patients with noninnervated flaps in all zones (p = 0.02). These differences persisted beyond a follow-up period of 3 years. CONCLUSIONS: The authors' findings confirm that reinnervation of microsurgically reconstructed breasts achieves sensory recovery that is superior to that in breasts reconstructed without reinnervation. If sensation is important and the anatomical factors are favorable, reinnervation may represent a worthwhile refinement in attempts to achieve normality for patients undergoing breast reconstruction.

Adult↗
PubMed2005

The invertor knock-in conditional chromosomal translocation mimic.

Knock-in models of tumor-specific chromosomal translocations can generate lethal mutations. To circumvent this, a new conditional gene fusion model has been developed (invertor mice) and exemplified with the Ews-ERG fusion oncogene. An ERG segment, flanked by loxP sites, was knocked in to an intron of the Ews gene but in an inverted transcription orientation and lineage-specific Ews-ERG fusion created by Cre-mediated inversion. This invertor method is a completely conditional approach, applicable to any gene fusion, to emulate effects of translocations found in human cancers.

Alleles↗
PubMed2004

Cannabis-induced propriospinal myoclonus.

We report on a case of a 25-year-old woman with clusters of myoclonus induced by a single exposure to inhaled cannabis. Investigations excluded a structural abnormality of the spine. Multi-channel surface EMG with parallel frontal EEG recording confirmed the diagnosis of propriospinal myoclonus.

Adult↗
PubMed2003

The hepatitis C virus internal ribosome entry site facilitates efficient protein synthesis in blood vessel endothelium during tumour angiogenesis.

The development of gene delivery systems for therapeutic use involves vectors (often retrovirus or adenovirus) which typically encode one target protein, but the use of internal ribosome entry sites (IRES) can confer the ability to express more than one protein from bi- or polycistronic mRNAs. IRES elements can display tissue-specific expression, so it is necessary to determine suitable IRES for specific clinical applicability. Blood vessel endothelial cells are important clinically since many different conditions involve neo-vascularisation (angiogenesis). We have demonstrated that the viral hepatitis C IRES element is a powerful mediator of protein synthesis in angiogenesis, such as found in solid tumours. Homologous recombination was used to introduce IRES-lacZ sequences into the Lmo2 gene, which is expressed in endothelial cells. beta-Galactosidase expression was determined during vascular remodelling in mouse embryos and in sprouting endothelium during growth of solid tumours, and showed that the hepatitis C IRES is used efficiently for protein synthesis in endothelial cells. This IRES element can provide the means to express two or more therapeutic genes in blood vessel endothelium in clinical conditions, such as cancer, which depend on angiogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

De novo production of diverse intracellular antibody libraries.

Many therapeutic targets are intracellular proteins and molecules designed to interact with them must effectively bind to their target inside the cell. Intracellular antibodies (intrabodies) recognise and bind to proteins in cells and various methods have been developed to produce such molecules. Intracellular antibody capture (IAC) is based on a genetic screening approach and is a facile methodology with which effective intracellular antibodies can be obtained. During the development of the IAC technology, consensus immunoglobulin variable frameworks were identified which can form the basis of intrabody libraries for direct screening. In this paper, we describe the de novo synthesis of intrabody libraries based on the IAC consensus sequence. The procedure comprises in vitro production of a single antibody gene fragment from oligonucleotides and diversification of CDRs of the immunoglobulin variable domain by mutagenic PCR. Completely de novo intrabody libraries can be rapidly generated in vitro by these approaches. As an example, a single immunoglobulin VH domain intrabody library was screened directly in yeast with an oncogenic BCR-ABL antigen bait and distinct antigen binders were isolated illustrating the functional utility of the library. This second generation IAC approach (IAC2) has many practical advantages, in particular the ability to isolate intrabodies by direct genetic selection, which obviates the need for in vitro production of antigen for pre-selection of antibody fragments.

Amino Acid Sequence↗
PubMed2003

Engineering de novo reciprocal chromosomal translocations associated with Mll to replicate primary events of human cancer.

The etiology of human tumors often involves chromosomal translocations. Models that emulate translocations are essential to understanding the determinants of frank malignancy, those dictating the restriction of translocations to specific lineages, and as a basis for development of rational therapeutic methods. We demonstrate that developmentally regulated Cre-loxP-mediated interchromosomal recombination between the Mll gene, whose human counterpart is involved in a spectrum of leukemias, and the Enl gene creates reciprocal chromosomal translocations that cause myeloid tumors. There is a rapid onset and high penetrance of leukemogenesis in these translocator mice, and high proportions of cells carrying chromosomal translocations can be found in bone marrow as early as 12 days after birth. This de novo strategy is a direct recapitulation of naturally occurring human cancer-associated translocations.

Alleles↗
PubMed2003

The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development.

The LMO2 gene encodes a LIM-only protein and is a target of chromosomal translocations in human T-cell leukemia. Recently, two X-SCID patients treated by gene therapy to rescue T-cell lymphopoiesis developed T-cell leukemias with retroviral insertion into the LMO2 gene causing clonal T-cell proliferation. In view of the specificity of LMO2 in T-cell tumorigenesis, we investigated a possible role for Lmo2 in T-lymphopoiesis, using conditional knockout of mouse Lmo2 with loxP-flanked Lmo2 and Cre recombinase alleles driven by the promoters of the lymphoid-specific genes Rag1, CD19, and Lck. While efficient deletion of Lmo2 was observed, even in the earliest detectable lymphoid cell progenitors of the bone marrow, there was no disturbance of lymphopoiesis in either T- or B-cell lineages, and in contrast to Lmo2 transgenic mice, there were normal distributions of CD4- CD- thymocytes. We conclude that there is no mandatory role for LMO2 in lymphoid development, implying that its specific role in T-cell tumorigenesis results from a reprogramming of gene expression after enforced expression in T-cell precursors.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein.

The expression of antibodies inside cells to ablate protein function has the potential for disease therapy and for target validation in functional genomics. However, due to inefficient expression or folding, only a few antibodies or antibody fragments, usually as single-chain Fv antibody fragments (scFv), bind their antigens in an intracellular environment. We have established a genetic-selection technology (intracellular antibody capture, IAC) to facilitate the isolation of functional intracellular scFv from a diverse repertoire. This approach comprises an in vitro library screen with scFv-expressing bacteriophage, employing bacterially expressed antigen, followed by a yeast in vivo antibody-antigen interaction screen of the sub-library of in vitro scFv antigen-binders. Accordingly, we have isolated panels of scFv that bind intracellularly to the BCR or the ABL parts of the BCR-ABL oncogenic protein. Sequence analysis of the intracellular antibody scFv panels revealed a sequence conservation indicating an intracellular antibody consensus for both VH and VL, which could form the basis for the de novo synthesis of intracellular antibody libraries to be used with intracellular antibody-capture technology.

Amino Acid Sequence↗
PubMed2002

The LIM-domain protein Lmo2 is a key regulator of tumour angiogenesis: a new anti-angiogenesis drug target.

The growth of solid tumours requires a blood supply provided by re-modeling of existing blood vessel endothelium (angiogenesis). Little is known about transcription regulators which are specific for the control of tumour angiogenesis. The proto-oncogene LMO2 encodes a LIM domain transcription regulator which controls angiogenesis during mouse embryogenesis where it regulates remodelling of the capillary network into mature vessels. We now show that Lmo2 expression is augmented in tumour endothelium such as mouse thymomas and human lung tumours. The functional significance of this Lmo2 expression was assessed in teratocarcinomas induced in nude mice by subcutaneous implantation of Lmo2-lacZ targeted ES cells. CD31-positive, sprouting endothelium of ES-cell origin occurred in teratocarcinomas from heterozygous Lmo2-lacZ ES cells but none occurred from null Lmo2-lacZ ES cells. Therefore, in this model Lmo2 is an obligatory regulator of neo-vascularization of tumours. These data suggest that LMO2 function may be a drug target in cancer and other conditions characterized by neo-vascularization.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Interscale wavelet maximum-a fine to coarse algorithm for wavelet analysis of the EMG interference pattern.

A method has been developed, interscale wavelet maximum (ISWM), for characterising the electromyogram (EMG) interference pattern to assist in the diagnosis of neuromuscular disease. EMG signals are decomposed with the redundant dyadic wavelet transform and wavelet maxima (WM) are found. Thresholding methods are applied to remove WM due to noise and background activity. An efficient fine-to-coarse algorithm identifies the WM tree structure for the motor unit action potential rising edges. The WM for each tree are summed at each scale; the largest value is the ISWM. Highly significant differences in ISWM values have been found between healthy, myopathic, and neuropathic subjects that could make the technique a useful diagnostic tool.

Action Potentials↗