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author:("luxu, Truong")
1.  Genome Sequencing of 15 Clinical Vibrio Isolates, Including 13 Non-O1/Non-O139 Serogroup Strains 
Genome Announcements  2014;2(5):e00893-14.
We present draft genome sequences of 15 clinical Vibrio isolates of various serogroups. These are valuable data for use in studying Vibrio cholerae genetic diversity, epidemic potential, and strain attribution.
PMCID: PMC4161747  PMID: 25212618
Addiction biology  2012;18(3):480-495.
Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations we utilized a behavior genetics strategy designed to associate contingent intravenous drug self-administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a yoked-control paradigm, C57BL/6J mice showed clear morphine-reinforced behavior whereas DBA/2J mice did not. Moreover, the yoked-control paradigm revealed the powerful consequences of self-administration versus passive administration at the level of gene expression. Morphine self-administration in the C57BL/6J mice uniquely up- or down-regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self-administration genes (<3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent- and genotype-dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and microRNAs (miRNAs) were among the key themes associated with drug self-administration. Noteworthy were the primary miRNA genes H19 and microRNA containing gene (Mirg), processed respectively to mature miRNAs miR-675 and miR-154, since they are prime candidates to mediate network-like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu-opioid receptor regulation. The strategic approach designed to focus on reinforcement-associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction.
PMCID: PMC3477247  PMID: 22804800
Opioid; addiction; neuroadaptation; vulnerability; reward; microRNA; H19
3.  Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood 
BMC Genomics  2014;15:96.
The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown.
In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis.
The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.
PMCID: PMC3922542  PMID: 24495417
4.  Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody 
PLoS Pathogens  2013;9(10):e1003684.
The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.
Author Summary
Since their initial emergence, henipaviruses have continued to cause spillover events in both human and livestock populations, posing significant biothreats. Currently there are no licensed or approved therapies for treatment of henipavirus infection and the human case mortality rates average >70%. We used X-ray crystallography to determine the high-resolution structures of the Hendra virus G glycoprotein in complex with a cross-reactive neutralizing human monoclonal antibody. The structures provide detailed insight into the mechanism of HeV and NiV neutralization by this potent and clinically-relevant human monoclonal antibody that is currently in development for use in humans. This monoclonal antibody was recently shown to be an effective post-exposure therapy in non-human models of lethal Hendra virus infection. Indeed, it has already been used in four people on a compassionate use request, three in Australia and one in the United States, as a therapeutic agent. Furthermore, we identified and characterized two escape mutants generated in vitro and evaluated their mechanism of escape. Our results serve as a blueprint for further optimization of this antibody and for the development of novel entry inhibitors and vaccines. This report also supports the additional pre-clinical data required for eventual licensure by detailing the antibody's mechanism of henipavirus neutralization.
PMCID: PMC3795035  PMID: 24130486
6.  Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2 
PLoS ONE  2012;7(11):e48228.
In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.
PMCID: PMC3486847  PMID: 23133618
7.  Whole genome sequencing of phage resistant Bacillus anthracis mutants reveals an essential role for cell surface anchoring protein CsaB in phage AP50c adsorption 
Virology Journal  2012;9:246.
Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix.
Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection.
Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains.
Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).
PMCID: PMC3545897  PMID: 23098174
Phage resistance; WGS; Mutation mapping; SNP; B. anthracis
8.  Persistent gene expression changes in ventral tegmental area of adolescent but not adult rats in response to chronic nicotine 
Neuroscience  2010;170(2):503-513.
Because adolescent brains are undergoing extensive developmental changes, they may be uniquely sensitive to effects of addictive drugs like nicotine. We exposed adolescent and adult rats to nicotine infusion for two weeks, and then used whole genome microarray analysis to determine effects on gene expression in the ventral tegmental area. We examined brains immediately after two weeks of nicotine or saline, and also four weeks after termination of nicotine exposure. After identifying genes with a significant age X treatment interaction, we employed template matching to find specific patterns of expression across age and treatment. Of those genes that were transiently regulated (up- or down-regulated immediately following the end of nicotine treatment, but back to saline baseline 30 days later), two-thirds were specific to adult animals, while only 30% were specific to adolescents and 4% were shared across the two ages. In contrast, significant genes that were persistently regulated (altered following nicotine treatment and still altered 30 days later) were more likely (59%) to be adolescent, with only 32% in adults and 8% shared. The greatest number of significant genes was late-regulated (no change immediately after nicotine, but regulated 30 days later). Again, most were in adolescents (54%), compared to adults (10%) or shared (36%). Pathway analysis revealed that adolescent-specific genes were over-represented in several biological functions and canonical pathways, including nervous system development and function and long-term potentiation. Furthermore, adolescent-specific genes formed extensive interaction networks, unlike those specific for adults or shared. This age-specific expression pattern may relate to the heightened vulnerability of adolescents to the effects of addictive drugs. In particular, the propensity of adolescents to show persistent alterations in gene expression corresponds to the persistence of drug dependence among smokers who began their habit as adolescents. These findings support a model whereby adolescent brains are uniquely vulnerable to long-term changes in gene expression in the brain’s reward pathway caused by early exposure to nicotine.
PMCID: PMC2962594  PMID: 20633606
Nicotine addiction; Adolescent drug abuse; Long-term potentiation; Nervous system development; Nicotinic receptors
9.  Voltage-gated Na+ channel SCN5A is a key regulator of a gene transcriptional network that controls colon cancer invasion 
Cancer research  2010;70(17):6957-6967.
Voltage-gated Na+ channels (VGSCs) have been implicated in the metastatic potential of human breast, prostate and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Nav1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiological recordings. Na+ channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by siRNAs specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, where there was strong Nav1.5 protein staining in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion.
PMCID: PMC2936697  PMID: 20651255
voltage-gated Na+ channels; invasion; colon cancer; gene network
10.  TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells 
Molecular Cancer  2010;9:181.
The homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11) is inappropriately expressed in a major subgroup of T cell acute lymphoblastic leukemia (T-ALL) where it is strongly associated with activating NOTCH1 mutations. Despite the recognition that these genetic lesions cooperate in leukemogenesis, there have been no mechanistic studies addressing how TLX1 and NOTCH1 functionally interact to promote the leukemic phenotype.
Global gene expression profiling after downregulation of TLX1 and inhibition of the NOTCH pathway in ALL-SIL cells revealed that TLX1 synergistically regulated more than 60% of the NOTCH-responsive genes. Structure-function analysis demonstrated that TLX1 binding to Groucho-related TLE corepressors was necessary for maximal transcriptional regulation of the NOTCH-responsive genes tested, implicating TLX1 modulation of the NOTCH-TLE regulatory network. Comparison of the dataset to publicly available biological databases indicated that the TLX1/NOTCH-coregulated genes are frequently targeted by MYC. Gain- and loss-of-function experiments confirmed that MYC was an essential mediator of TLX1/NOTCH transcriptional output and growth promotion in ALL-SIL cells, with TLX1 contributing to the NOTCH-MYC regulatory axis by posttranscriptional enhancement of MYC protein levels. Functional classification of the TLX1/NOTCH-coregulated targets also showed enrichment for genes associated with other human cancers as well as those involved in developmental processes. In particular, we found that TLX1, NOTCH and MYC coregulate CD1B and RAG1, characteristic markers of early cortical thymocytes, and that concerted downregulation of the TLX1 and NOTCH pathways resulted in their irreversible repression.
We found that TLX1 and NOTCH synergistically regulate transcription in T-ALL, at least in part via the sharing of a TLE corepressor and by augmenting expression of MYC. We conclude that the TLX1/NOTCH/MYC network is a central determinant promoting the growth and survival of TLX1+ T-ALL cells. In addition, the TLX1/NOTCH/MYC transcriptional network coregulates genes involved in T cell development, such as CD1 and RAG family members, and therefore may prescribe the early cortical stage of differentiation arrest characteristic of the TLX1 subgroup of T-ALL.
PMCID: PMC2913983  PMID: 20618946
11.  Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NFκB and microRNA network 
Molecular Cancer  2010;9:98.
Diminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted.
Colon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NFκB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified ~700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NFκB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NFκB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity.
Our findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NFκB in the cytoplasm with consequential changes in the expression of microRNA pathway components.
PMCID: PMC2883962  PMID: 20433755
12.  Gene Expression Profiling Differentiates Autism Case–Controls and Phenotypic Variants of Autism Spectrum Disorders: Evidence for Circadian Rhythm Dysfunction in Severe Autism 
Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by delayed/abnormal language development, deficits in social interaction, repetitive behaviors and restricted interests. The heterogeneity in clinical presentation of ASD, likely due to different etiologies, complicates genetic/biological analyses of these disorders. DNA microarray analyses were conducted on 116 lymphoblastoid cell lines (LCL) from individuals with idiopathic autism who are divided into three phenotypic subgroups according to severity scores from the commonly used Autism Diagnostic Interview-Revised questionnaire and age-matched, nonautistic controls. Statistical analyses of gene expression data from control LCL against that of LCL from ASD probands identify genes for which expression levels are either quantitatively or qualitatively associated with phenotypic severity. Comparison of the significant differentially expressed genes from each subgroup relative to the control group reveals differentially expressed genes unique to each subgroup as well as genes in common across subgroups. Among the findings unique to the most severely affected ASD group are 15 genes that regulate circadian rhythm, which has been shown to have multiple effects on neurological as well as metabolic functions commonly dysregulated in autism. Among the genes common to all three subgroups of ASD are 20 novel genes mostly in putative noncoding regions, which appear to associate with androgen sensitivity and which may underlie the strong 4:1 bias toward affected males.
PMCID: PMC2737477  PMID: 19418574
autism phenotypes; gene expression profiling; circadian rhythm; novel genes
13.  Gene Expression Profiling of Lymphoblasts from Autistic and Nonaffected Sib Pairs: Altered Pathways in Neuronal Development and Steroid Biosynthesis 
PLoS ONE  2009;4(6):e5775.
Despite the identification of numerous autism susceptibility genes, the pathobiology of autism remains unknown. The present “case-control” study takes a global approach to understanding the molecular basis of autism spectrum disorders based upon large-scale gene expression profiling. DNA microarray analyses were conducted on lymphoblastoid cell lines from over 20 sib pairs in which one sibling had a diagnosis of autism and the other was not affected in order to identify biochemical and signaling pathways which are differentially regulated in cells from autistic and nonautistic siblings. Bioinformatics and gene ontological analyses of the data implicate genes which are involved in nervous system development, inflammation, and cytoskeletal organization, in addition to genes which may be relevant to gastrointestinal or other physiological symptoms often associated with autism. Moreover, the data further suggests that these processes may be modulated by cholesterol/steroid metabolism, especially at the level of androgenic hormones. Elevation of male hormones, in turn, has been suggested as a possible factor influencing susceptibility to autism, which affects ∼4 times as many males as females. Preliminary metabolic profiling of steroid hormones in lymphoblastoid cell lines from several pairs of siblings reveals higher levels of testosterone in the autistic sibling, which is consistent with the increased expression of two genes involved in the steroidogenesis pathway. Global gene expression profiling of cultured cells from ASD probands thus serves as a window to underlying metabolic and signaling deficits that may be relevant to the pathobiology of autism.
PMCID: PMC2685981  PMID: 19492049
14.  Positional identification of variants of Adamts16 linked to inherited hypertension 
Human Molecular Genetics  2009;18(15):2825-2838.
A previously reported blood pressure (BP) quantitative trait locus on rat Chromosome 1 was isolated in a short congenic segment spanning 804.6 kb. The 804.6 kb region contained only two genes, LOC306664 and LOC306665. LOC306664 is predicted to translate into A Disintegrin-like and Metalloproteinase with Thrombospondin Motifs-16 (Adamts16). LOC306665 is a novel gene. All predicted exons of both LOC306664 and LOC306665 were sequenced. Non-synonymous variants were identified in only one of these genes, LOC306664. These variants were naturally existing polymorphisms among inbred, outbred and wild rats. The full-length rat transcript of Adamts16 was detected in multiple tissues. Similar to ADAMTS16 in humans, expression of Adamts16 was prominent in the kidney. Renal transcriptome analysis suggested that a network of genes related to BP was differential between congenic and S rats. These genes were also differentially expressed between kidney cell lines with or without knock-down of Adamts16. Adamts16 is conserved between rats and humans. It is a candidate gene within the homologous region on human Chromosome 5, which is linked to systolic and diastolic BP in the Quebec Family Study. Multiple variants, including an Ala to Pro variant in codon 90 (rs2086310) of human ADAMTS16, were associated with human resting systolic BP (SBP). Replication study in GenNet confirmed the association of two variants of ADAMTS16 with SBP, including rs2086310. Overall, our report represents a high resolution positional cloning and translational study for Adamts16 as a candidate gene controlling BP.
PMCID: PMC2706685  PMID: 19423552
15.  A Factor Graph Nested Effects Model To Identify Networks from Genetic Perturbations 
PLoS Computational Biology  2009;5(1):e1000274.
Complex phenotypes such as the transformation of a normal population of cells into cancerous tissue result from a series of molecular triggers gone awry. We describe a method that searches for a genetic network consistent with expression changes observed under the knock-down of a set of genes that share a common role in the cell, such as a disease phenotype. The method extends the Nested Effects Model of Markowetz et al. (2005) by using a probabilistic factor graph to search for a network representing interactions among these silenced genes. The method also expands the network by attaching new genes at specific downstream points, providing candidates for subsequent perturbations to further characterize the pathway. We investigated an extension provided by the factor graph approach in which the model distinguishes between inhibitory and stimulatory interactions. We found that the extension yielded significant improvements in recovering the structure of simulated and Saccharomyces cerevisae networks. We applied the approach to discover a signaling network among genes involved in a human colon cancer cell invasiveness pathway. The method predicts several genes with new roles in the invasiveness process. We knocked down two genes identified by our approach and found that both knock-downs produce loss of invasive potential in a colon cancer cell line. Nested effects models may be a powerful tool for inferring regulatory connections and genes that operate in normal and disease-related processes.
Author Summary
Biological processes are the result of the actions and interactions of many genes and the proteins that they encode. Our knowledge of interactions for many biological processes is limited, especially for cancer where genomic alterations may create entirely novel pathways not present in normal tissue. Perturbing gene expression (for example, by deleting a gene) has long been used as a tool in molecular biology to elucidate interactions but is very expensive and labor intensive. The search for new genes that may participate can be a daunting “fishing expedition.” We have devised a tool that automatically infers interactions using high-throughput gene expression data. When a gene is silenced, it causes other genes to be switched on or off, which provide clues about the pathway(s) in which the gene acts. Our method uses the genomewide on/off states as a fingerprint to detect interactions among a set of silenced genes. We were able to elucidate a network of interactions for several genes implicated in metastatic colon cancer. Genes newly connected to the network were found to operate in cancer cell invasion in human cells, validating the approach. Thus, the method enables an efficient discovery of the networks that underlie biological processes such as carcinogenesis.
PMCID: PMC2613752  PMID: 19180177
16.  Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays 
Genome Biology  2003;4(1):R5.
Long oligonucleotide microarrays are potentially more cost- and management efficient than cDNA microarrays. Unmodified sense and antisense 70-mer oligonucleotides were synthesized and compared with PCR-amplified cDNA clones corresponding to the same genes. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80.
Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear.
Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 μm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR.
Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.
PMCID: PMC151289  PMID: 12540297

Results 1-16 (16)