Oligonucleotide-mediated gene editing of Apolipoprotein A-I.

Apolipoprotein A-I (ApoA-I) is the major protein constituent of high density lipoprotein (HDL) and controls reverse cholesterol transport, an important process in preventing atherosclerosis. A natural point mutation, ApoA-lMiiano (ApoA-Im) enhances the atheroprotective potential of HDL. Here, I attempt to introduce this specific modification into the genome of mammalian cells using the gene therapy strategy of oligonucleotide-mediated gene editing. I showed successful APOA-I gene editing in recombinant Chinese hamster ovary (CHO-AI) and human hepatocellular carcinoma (HepG2) cells by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing. However, I was unable to isolate gene-edited cell clones or quantify gene editing. Therefore, I established a recombinant CHO cell line expressing mutated non-fluorescent enhanced green fluorescent protein (mEGFP) that demonstrates successful gene editing through restoration of fluorescence. Using flow cytometry, I studied the influence of transfection reagents and oligonucleotide design on gene editing efficiency and viability of gene-edited cells. I found that Lipofectamine 2000 generated higher initial editing efficiencies with phosphorothioate (PTO) or locked nucleic acid (LNA) modifications, but unmodified oligonucleotides produced significantly more gene-edited clones. I also investigated ways of increasing editing efficiencies or selecting for gene-edited cells independent of the target gene and demonstrated that I had introduced three specific nucleotide alterations by Southern blotting of genomic DNA from gene-edited cell clones. I established that the phenotype in these clones was unstable due to epigenetic down- regulation which was not specific to the gene-edited allele. Variegated gene expression in three mEGFP cell lines demonstrated that gene editing was positively associated with target gene expression. Isolation of ApoA-lM gene-edited cells using flow cytometry failed because an ApoA-I i homodimer antibody was not specific. In summary, I demonstrated that oligonucleotide-mediated gene editing produces stable gene-edited cells in a reporter gene system. However, further research is needed before this method can be applied to non-selectable genes

The presence of valine at residue 129 in human prion protein accelerates amyloid formation

The polymorphism at residue 129 of the human PRNP gene modulates disease susceptibility and the clinicopathological phenotypes in human transmissible spongiform encephalopathies. The molecular mechanisms by which the effect of this polymorphism are mediated remain unclear. It has been shown that the folding, dynamics and stability of the physiological, alpha-helix-rich form of recombinant PrP are not affected by codon 129 polymorphism. Consistent with this, we have recently shown that the kinetics of amyloid formation do not differ between protein containing methionine at codon 129 and valine at codon 129 when the reaction is initiated from the a-monomeric PrPC-like state. In contrast, we have shown that the misfolding pathway leading to the formation of beta-sheet-rich, soluble oligomer waS favoured by the presence of methionine, compared with valine, at position 129. In the present work, we examine the effect of this polymorphism on the kinetics of an alternative misfolding pathway, that of amyloid formation using partially folded PrP allelomorphs. We show that the valine 129 allelomorph forms amyloids with a considerably shorter lag phase than the methionine 129 allelomorph both under spontaneous conditions and when seeded with pre-formed amyloid fibres. Taken together, our studies demonstrate that the effect of the codon 129 polymorphism depends on the specific misfolding pathway and on the initial conformation of the protein. The inverse propensities of the two allelomorphs to misfold in vitro through the alternative oligomeric and amyloidogenic pathways could explain some aspects of prion diseases linked to this polymorphism such as age at onset and disease incubation time. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Development of therapeutic splice-switching oligonucleotides.

Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne Muscular Dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase I clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exonic-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Lastly, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case

Pathogenetic mechanisms of amyloid A amyloidosis

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid

Knowledge discovery from post-project reviews

This article was published in the journal, Construction Management and Economics [© Taylor & Francis (Routledge)] and the definitive version is available at: http://dx.doi.org/10.1080/01446193.2011.588953Many construction companies conduct reviews on project completion to enhance learning and to fulfil quality management procedures. Often these reports are filed away never to be seen again. This means that potentially important knowledge that may assist other project teams is not exploited. In order to ascertain whether useful knowledge can be gleaned from such reports, Knowledge Discovery from Text (KDT) and text mining (TM) are applied. Text mining avoids the need for a manual search through a vast number of reports, potentially of different formats and foci, to seek trends that may be useful for current and future projects. Pilot tests were used to analyse 48 post-project review reports. The reports were first reviewed manually to identify key themes. They were then analysed using text mining software to investigate whether text mining could identify trends and uncover useful knowledge from the reports. Pilot tests succeeded in finding common occurrences across different projects that were previously unknown. Text mining could provide a potential solution and would aid project teams to learn from previous projects. However, a lot of work is currently required before the text mining tests are conducted and the results need to be examined carefully by those with domain knowledge to validate the results obtained

Whole body vibration exercise : are vibrations good for you ?

Heparan sulphate proteoglycans (HSPGs) exist in pancreatic beta cells, and HS seems to modulate important interactions in the islet microenvironment. However, the intra-islet structures of HS in health or altered glucose homeostasis are currently unknown. Here we show that distinct spatial distribution of HS motifs is present in islets in the adult, that intra-islet HS motifs are mostly conserved between rodents and humans, and that HS is abundant in glucagon producing islet alpha cells. In beta cells HS is characterised by 2-O, 6-O and N-sulphated moieties, whereas HS in alpha cells is N-acetylated, N-, and 2-O sulphated and low in 6-O groups. Differential expression of three HS modifying genes in alpha and beta cells was observed and may account for the different HS patterns. Furthermore, we found that FGF1 and FGF2 were present in alpha cells, whereas functional FGFRs exist in beta cells, but not in the alpha cell line aTC1-6, or in primary alpha cells in islets. FGF1 induced signalling was dependent on 2-O, and 6-O HS sulphation in beta cells, and HS desulphation reduced beta cell proliferation and potentiated oxidant induced apoptosis. In leptin resistant animals and in islets from streptozotocin treated rats there was a reduction in alpha cell HS expression. These data demonstrate the distinct HS expression patterns in alpha and beta islet cells and propose a novel role for alpha cells as a source of paracrine FGF ligands to neighbouring beta cells with specific cell-associated HS domains mediating the activation and diffusion of paracrine ligands

Pathogenetic mechanisms of amyloid A amyloidosis.

Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid