Publications by authors named "Susanna Chan"

28 Publications

  • Page 1 of 1

Glycolipid-peptide vaccination induces liver-resident memory CD8 T cells that protect against rodent malaria.

Sci Immunol 2020 06;5(48)

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.

Liver resident-memory CD8 T cells (T cells) can kill liver-stage -infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver T cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate -specific CD8 T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8 T cells expressing canonical markers of liver T cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver T cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver T cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver T cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciimmunol.aaz8035DOI Listing
June 2020

The Chemical Synthesis, Stability, and Activity of MAIT Cell Prodrug Agonists That Access MR1 in Recycling Endosomes.

ACS Chem Biol 2020 02 14;15(2):437-445. Epub 2020 Jan 14.

The Ferrier Research Institute , Victoria University of Wellington , Wellington , New Zealand.

Mucosal-associated invariant T (MAIT) cells are antibacterial effector T cells that react to pyrimidines derived from bacterial riboflavin synthesis presented by the monomorphic molecule MR1. A major challenge in MAIT cell research is that the commonly used MAIT agonist precursor, 5-amino-6-d-ribitylaminouracil (5-A-RU), is labile to autoxidation, resulting in a loss of biological activity. Here, we characterize two independent autoxidation processes by LCMS. To overcome the marked instability, we report the synthesis of a 5-A-RU prodrug generated by modification of the 5-amino group with a cleavable valine-citrulline--aminobenzyl carbamate. The compound is stable in prodrug form, with the parent amine (i.e., 5-A-RU) released only after enzymatic cleavage. Analysis of the prodrug and showed an enhanced MAIT cell activation profile compared to 5-A-RU, which was associated with preferential loading within recycling endosomes, a route used by some natural agonists. This prodrug design therefore overcomes the difficulties associated with 5-A-RU in biological studies and provides an opportunity to explore different presentation pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.9b00902DOI Listing
February 2020

Enhancing T cell responses and tumour immunity by vaccination with peptides conjugated to a weak NKT cell agonist.

Org Biomol Chem 2019 01;17(5):1225-1237

The Ferrier Research Institute, Victoria University of Wellington, Lower Hutt, New Zealand.

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c8ob02982bDOI Listing
January 2019

Transcriptomic analysis of CIC and ATXN1L reveal a functional relationship exploited by cancer.

Oncogene 2019 01 9;38(2):273-290. Epub 2018 Aug 9.

Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.

Aberrations in Capicua (CIC) have recently been implicated as a negative prognostic factor in a multitude of cancer types through activation of the MAPK signalling cascade and derepression of oncogenic ETS transcription factors. The Ataxin-family protein ATXN1L has previously been reported to interact with CIC in developmental and disease contexts to facilitate the repression of CIC target genes. To further investigate this relationship, we performed functional in vitro studies utilizing ATXN1L and CIC human cell lines and characterized a reciprocal functional relationship between CIC and ATXN1L. Transcriptomic interrogation of the CIC-ATXN1-ATXN1L axis in low-grade glioma, prostate adenocarcinoma and stomach adenocarcinoma TCGA cohorts revealed context-dependent convergence of gene sets and pathways related to mitotic cell cycle and division. This study highlights the CIC-ATXN1-ATXN1L axis as a more potent regulator of the cell cycle than previously appreciated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-018-0427-5DOI Listing
January 2019

Comparative transcriptome analysis of isogenic cell line models and primary cancers links capicua (CIC) loss to activation of the MAPK signalling cascade.

J Pathol 2017 06 26;242(2):206-220. Epub 2017 Apr 26.

Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada.

CIC encodes a transcriptional repressor, capicua (CIC), whose disrupted activity appears to be involved in several cancer types, including type I low-grade gliomas (LGGs) and stomach adenocarcinomas (STADs). To explore human CIC's transcriptional network in an isogenic background, we developed novel isogenic CIC knockout cell lines as model systems, and used these in transcriptome analyses to study the consequences of CIC loss. We also compared our results with analyses of transcriptome data from TCGA for type I LGGs and STADs. We identified 39 candidate targets of CIC transcriptional regulation, and confirmed seven of these as direct targets. We showed that, although many CIC targets appear to be context-specific, the effects of CIC loss converge on the dysregulation of similar biological processes in different cancer types. For example, we found that CIC deficiency was associated with disruptions in the expression of genes involved in cell-cell adhesion, and in the development of several cell and tissue types. We also showed that loss of CIC leads to overexpression of downstream members of the mitogen-activated protein kinase (MAPK) signalling cascade, indicating that CIC deficiency may present a novel mechanism for activation of this oncogenic pathway. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.4894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485162PMC
June 2017

Characterization and Synthesis of Eudistidine C, a Bioactive Marine Alkaloid with an Intriguing Molecular Scaffold.

J Org Chem 2016 11 10;81(22):10631-10640. Epub 2016 Nov 10.

National Center for Natural Products Research, School of Pharmacy, University of Mississippi , Mississippi 38677, United States.

An extract of Eudistoma sp. provided eudistidine C (1), a heterocyclic alkaloid with a novel molecular framework. Eudistidine C (1) is a racemic natural product composed of a tetracyclic core structure further elaborated with a p-methoxyphenyl group and a phenol-substituted aminoimidazole moiety. This compound presented significant structure elucidation challenges due to the large number of heteroatoms and fully substituted carbons. These issues were mitigated by application of a new NMR pulse sequence (LR-HSQMBC) optimized to detect four- and five-bond heteronuclear correlations and the use of computer-assisted structure elucidation software. Synthesis of eudistidine C (1) was accomplished in high yield by treating eudistidine A (2) with 4(2-amino-1H-imidazol-5-yl)phenol (4) in DMSO. Synthesis of eudistidine C (1) confirmed the proposed structure and provided material for further biological characterization. Treatment of 2 with various nitrogen heterocycles and electron-rich arenes provided a series of analogues (5-10) of eudistidine C. Chiral-phase HPLC resolution of epimeric eudistidine C provided (+)-(R)-eudistidine C (1a) and (-)-(S)-eudistidine C (1b). The absolute configuration of these enantiomers was assigned by ECD analysis. (-)-(S)-Eudistidine C (1b) modestly inhibited interaction between the protein binding domains of HIF-1α and p300. Compounds 1, 2, and 6-10 exhibited significant antimalarial activity against Plasmodium falciparum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.joc.6b02380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350249PMC
November 2016

Biologically Active Acetylenic Amino Alcohol and N-Hydroxylated 1,2,3,4-Tetrahydro-β-carboline Constituents of the New Zealand Ascidian Pseudodistoma opacum.

J Nat Prod 2016 Mar 15;79(3):607-10. Epub 2015 Dec 15.

Laboratoire Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Muséum National d'Histoire Naturelle , 57 Rue Cuvier (C.P. 54), 75005 Paris, France.

The first occurrence of an acetylenic 1-amino-2-alcohol, distaminolyne A (1), isolated from the New Zealand ascidian Pseudodistoma opacum, is reported. The isolation and structure elucidation of 1 and assignment of absolute configuration using the exciton coupled circular dichroism technique are described. In addition, a new N-9 hydroxy analogue (2) of the known P. opacum metabolite 7-bromohomotrypargine is also reported. Antimicrobial screening identified modest activity of 1 toward Escherichia coli, Staphylococcus aureus, and Mycobacterim tuberculosis, while 2 exhibited a moderate antimalarial activity (IC50 3.82 μM) toward a chloroquine-resistant strain (FcB1) of Plasmodium falciparum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.5b00770DOI Listing
March 2016

Structural Elucidation and Synthesis of Eudistidine A: An Unusual Polycyclic Marine Alkaloid that Blocks Interaction of the Protein Binding Domains of p300 and HIF-1α.

J Am Chem Soc 2015 Apr 20;137(16):5569-75. Epub 2015 Apr 20.

†Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702-1201, United States.

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A (1) and B (2) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (1) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)pyrimidin-2-amine (3) and 4-methoxy-phenylglyoxal (4), while eudistidine B (2) was synthesized in a similar fashion with glyoxylic acid (5) in place of 4. Naturally occurring eudistidine A (1) effectively inhibited CH1/C-TAD binding with an IC50 of 75 μM, and synthetic 1 had similar activity. The eudistidine A (1) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein-protein binding events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.5b02156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318789PMC
April 2015

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity.

Oncotarget 2014 Sep;5(17):7960-79

Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada. Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism bof reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202173PMC
http://dx.doi.org/10.18632/oncotarget.2401DOI Listing
September 2014

Anti-inflammatory and antimalarial meroterpenoids from the New Zealand ascidian Aplidium scabellum.

J Org Chem 2011 Nov 3;76(21):9151-6. Epub 2011 Oct 3.

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.

Bioassay-directed fractionation of an extract of the New Zealand ascidian Aplidium scabellum has afforded the anti-inflammatory secondary metabolite 2-geranyl-6-methoxy-1,4-hydroquinone-4-sulfate (1) and a family of pseudodimeric meroterpenoids scabellones A (2)-D (5). The benzo[c]chromene-7,10-dione scaffold contained within scabellones A-D is particularly rare among natural products. The structures were elucidated by interpretation of NMR data. Scabellone B was also identified as a moderately potent, nontoxic inhibitor of Plasmodium falciparum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jo201654hDOI Listing
November 2011

Antimalarial β-carbolines from the New Zealand ascidian Pseudodistoma opacum.

J Nat Prod 2011 Sep 16;74(9):1972-9. Epub 2011 Aug 16.

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.

One tetrahydro-β-carboline, (-)-7-bromohomotrypargine (1), and three alkylguanidine-substituted β-carbolines, opacalines A, B, and C (2-4), have been isolated from the New Zealand ascidian Pseudodistoma opacum. The structures of the metabolites were determined by analysis of mass spectrometric and 2D NMR spectroscopic data. Natural products 2 and 3, synthetic debromo analogues 8 and 9, and intermediate 16 exhibited moderate antimalarial activity toward a chloroquine-resistant strain of Plasmodium falciparum, with an IC50 range of 2.5-14 μM. The biosynthesis of 1-4 is proposed to proceed via a Pictet-Spengler condensation of 6-bromotryptamine and the α-keto acid transamination product of either arginine or homoarginine. Cell separation and 1H NMR analysis of P. opacum identified tetrahydro-β-carboline 1 to be principally located in the zooids, while fully aromatized analogues 2-4 were localized to the test.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/np200509gDOI Listing
September 2011

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Nature 2011 Jul 27;476(7360):298-303. Epub 2011 Jul 27.

Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature10351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210554PMC
July 2011

Comparison of genome-wide array genomic hybridization platforms for the detection of copy number variants in idiopathic mental retardation.

BMC Med Genomics 2011 Mar 25;4:25. Epub 2011 Mar 25.

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

Background: Clinical laboratories are adopting array genomic hybridization as a standard clinical test. A number of whole genome array genomic hybridization platforms are available, but little is known about their comparative performance in a clinical context.

Methods: We studied 30 children with idiopathic MR and both unaffected parents of each child using Affymetrix 500 K GeneChip SNP arrays, Agilent Human Genome 244 K oligonucleotide arrays and NimbleGen 385 K Whole-Genome oligonucleotide arrays. We also determined whether CNVs called on these platforms were detected by Illumina Hap550 beadchips or SMRT 32 K BAC whole genome tiling arrays and tested 15 of the 30 trios on Affymetrix 6.0 SNP arrays.

Results: The Affymetrix 500 K, Agilent and NimbleGen platforms identified 3061 autosomal and 117 X chromosomal CNVs in the 30 trios. 147 of these CNVs appeared to be de novo, but only 34 (22%) were found on more than one platform. Performing genotype-phenotype correlations, we identified 7 most likely pathogenic and 2 possibly pathogenic CNVs for MR. All 9 of these putatively pathogenic CNVs were detected by the Affymetrix 500 K, Agilent, NimbleGen and the Illumina arrays, and 5 were found by the SMRT BAC array. Both putatively pathogenic CNVs identified in the 15 trios tested with the Affymetrix 6.0 were identified by this platform.

Conclusions: Our findings demonstrate that different results are obtained with different platforms and illustrate the trade-off that exists between sensitivity and specificity. The large number of apparently false positive CNV calls on each of the platforms supports the need for validating clinically important findings with a different technology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1755-8794-4-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076225PMC
March 2011

Alternative expression analysis by RNA sequencing.

Nat Methods 2010 Oct 12;7(10):843-7. Epub 2010 Sep 12.

Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada.

In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.1503DOI Listing
October 2010

Detection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridization.

BMC Genomics 2009 Nov 16;10:526. Epub 2009 Nov 16.

Department of Medical Genetics, University of British Columbia, Vancouver, Canada.

Background: Array genomic hybridization is being used clinically to detect pathogenic copy number variants in children with intellectual disability and other birth defects. However, there is no agreement regarding the kind of array, the distribution of probes across the genome, or the resolution that is most appropriate for clinical use.

Results: We performed 500 K Affymetrix GeneChip array genomic hybridization in 100 idiopathic intellectual disability trios, each comprised of a child with intellectual disability of unknown cause and both unaffected parents. We found pathogenic genomic imbalance in 16 of these 100 individuals with idiopathic intellectual disability. In comparison, we had found pathogenic genomic imbalance in 11 of 100 children with idiopathic intellectual disability in a previous cohort who had been studied by 100 K GeneChip array genomic hybridization. Among 54 intellectual disability trios selected from the previous cohort who were re-tested with 500 K GeneChip array genomic hybridization, we identified all 10 previously-detected pathogenic genomic alterations and at least one additional pathogenic copy number variant that had not been detected with 100 K GeneChip array genomic hybridization. Many benign copy number variants, including one that was de novo, were also detected with 500 K array genomic hybridization, but it was possible to distinguish the benign and pathogenic copy number variants with confidence in all but 3 (1.9%) of the 154 intellectual disability trios studied.

Conclusion: Affymetrix GeneChip 500 K array genomic hybridization detected pathogenic genomic imbalance in 10 of 10 patients with idiopathic developmental disability in whom 100 K GeneChip array genomic hybridization had found genomic imbalance, 1 of 44 patients in whom 100 K GeneChip array genomic hybridization had found no abnormality, and 16 of 100 patients who had not previously been tested. Effective clinical interpretation of these studies requires considerable skill and experience.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-10-526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781027PMC
November 2009

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data.

BMC Bioinformatics 2007 Oct 2;8:368. Epub 2007 Oct 2.

Genome Sciences Centre, BC Cancer Agency, British Columbia Cancer Agency, Suite 100, 570 West 7th Avenue, Vancouver, BC, V5Z 4S6, Canada.

Background: Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results: We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion: We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2105-8-368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148068PMC
October 2007

Does intensive rehabilitation improve the functional outcome of patients with traumatic brain injury (TBI)? A randomized controlled trial.

Brain Inj 2007 Jun;21(7):681-90

Division of Neurosurgery, Department of Surgery, Prince of Wales Hospital, Chinese University of Hong Kong, Hong Kong, PR China.

Objective: To evaluate the effects of an increase in the intensity of rehabilitation on the functional outcome of patients with traumatic brain injury (TBI).

Design And Methods: Sixty-eight patients (age 12-65 years) with moderate-to-severe TBI were included. They were randomized into high (4-hour/day) or control (2-hour/day) intensity rehabilitation programmes at an average of 20 days after the injury. The programmes ended when the patients achieved independence in daily activities or when 6 months had passed.

Outcome And Results: No significant differences were found in the Functional Independence Measure (FIM) (primary outcome) and Neurobehavioural Cognitive Status Examination (NCSE) total scores between the two groups. There were significantly more patients in the high intensity group than in the control group who achieved a maximum FIM total score at the third month (47% vs. 19%, p = 0.015) and a maximum Glasgow Outcome Scale (GOS) score at the second (28% vs. 8%, p = 0.034) and third months (34% vs. 14%, p = 0.044).

Conclusions: Early intensive rehabilitation may improve the functional outcome of patients with TBI in the early months post-injury and hence increase the chance of their returning to work early. Intensive rehabilitation in this study speeded up recovery rather than changed the final outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/02699050701468941DOI Listing
June 2007

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation.

Plant J 2007 Jun 3;50(6):1063-78. Epub 2007 May 3.

Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 +/- 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-313X.2007.03112.xDOI Listing
June 2007

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics.

Genome Biol 2006 ;7(12):R126

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Background: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes.

Results: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5.

Conclusion: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gb-2006-7-12-r126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1794439PMC
May 2007

Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation.

Am J Hum Genet 2006 Sep 25;79(3):500-13. Epub 2006 Jul 25.

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

The cause of mental retardation in one-third to one-half of all affected individuals is unknown. Microscopically detectable chromosomal abnormalities are the most frequently recognized cause, but gain or loss of chromosomal segments that are too small to be seen by conventional cytogenetic analysis has been found to be another important cause. Array-based methods offer a practical means of performing a high-resolution survey of the entire genome for submicroscopic copy-number variants. We studied 100 children with idiopathic mental retardation and normal results of standard chromosomal analysis, by use of whole-genome sampling analysis with Affymetrix GeneChip Human Mapping 100K arrays. We found de novo deletions as small as 178 kb in eight cases, de novo duplications as small as 1.1 Mb in two cases, and unsuspected mosaic trisomy 9 in another case. This technology can detect at least twice as many potentially pathogenic de novo copy-number variants as conventional cytogenetic analysis can in people with mental retardation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1086/507471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1559542PMC
September 2006

Do geriatricians stay in geriatrics?.

Gerontol Geriatr Educ 2006 ;27(1):57-65

Parker Institute for Health Care and Rehabilitation, New Hyde Park, NY 10040, USA.

To evaluate whether formally trained geriatricians remain in the field of Geriatrics, and to determine their job satisfaction and perceived quality of life, we surveyed the 107 fellows trained over the last 25 years in one accredited geriatric program. Of the 88 physicians who consented to participate, 75% devoted at least half of their practice to the care of the elderly. On an academic level, 89.5% had, or planned to pursue, recertification in geriatric medicine. Ninety-five percent of these geriatricians felt that the impact of a formal geriatric fellowship was positive on their medical career and satisfaction index. Sixty-four percent had yearly incomes between $100 and $200k, and 25.6% had income greater than $200k. Eighty-seven percent would recommend pursuing geriatric fellowship training. We need to further explore how recruitment process and job opportunities are presented to potential geriatric fellows.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1300/J021v27n01_04DOI Listing
October 2006

A set of BAC clones spanning the human genome.

Nucleic Acids Res 2004 9;32(12):3651-60. Epub 2004 Jul 9.

BC Cancer Agency Genome Sciences Center and BC Cancer Agency, Vancouver, BC V5Z 4E6, Canada.

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkh700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC484185PMC
July 2004

Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

Genome Res 2004 Apr;14(4):766-79

Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada V5Z 4E6.

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.2336604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC383324PMC
April 2004

Predictors of site of death of end-of-life patients: the importance of specificity in advance directives.

J Palliat Med 2004 Feb;7(1):9-17

Parker Jewish Institute for Health Care and Rehabilitation, New Hyde Park, New York 11040, USA.

Despite the compelling reasons for advance directives and their endorsement by the public and medical professions, little is known about their actual use and impact on site of death. This study was conducted to examine the role of advance directives and other "drivers" of hospitalization of the long-term care end-of-life patient. The medical records of 100 deceased consecutive nursing home residents, stratified by site of death (skilled nursing facility or acute care hospital), were reviewed by a team of geriatric researchers to obtain patient information in the following domains: sociodemographic, advance directives, transfer and death information, patient diagnoses at admission, discharge, and other time intervals; medication usage and signs and symptoms precipitating death. Severity of illness was assessed using the Cumulative Illness Rating Scale-G (CIRS-G). In testing for differences between patients by site of death, sociodemographic variables (gender, age, race, payer at discharge, cognitive capacity) did not significantly differ between the two groups of patients. Strong similarities between the groups were also found in terms of severity of illness and medication usage. Significantly higher proportions of patients dying in the nursing home had specific advance directives (do not resuscitate, do not intubate, do not artificially feed, do not hydrate, and do not hospitalize), as opposed to those dying in the hospital. The findings of this study demonstrate the impact of the explicit advance directive on the decision to transfer the patient to the acute care setting at the end of life.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/109662104322737205DOI Listing
February 2004

The Genome sequence of the SARS-associated coronavirus.

Science 2003 May 1;300(5624):1399-404. Epub 2003 May 1.

British Columbia Cancer Agency (BCCA) Genome Sciences Centre, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada.

We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1085953DOI Listing
May 2003

A physical map of the mouse genome.

Nature 2002 Aug 4;418(6899):743-50. Epub 2002 Aug 4.

The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature00957DOI Listing
August 2002

An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones.

Nucleic Acids Res 2002 Jun;30(11):2460-8

Genome Sciences Centre, BC Cancer Agency, 600 West 10th Avenue, Vancouver, BC V5Z 4E6, Canada.

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC117194PMC
http://dx.doi.org/10.1093/nar/30.11.2460DOI Listing
June 2002