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1.  A whole genome association study of neuroticism using DNA pooling 
Molecular psychiatry  2007;13(3):302-312.
We describe a multistage approach to identify single nucleotide polymorphisms (SNPs) associated with neuroticism, a personality trait that shares genetic determinants with major depression and anxiety disorders. Whole genome association with 452 574 SNPs was performed on DNA pools from ~2000 individuals selected on extremes of neuroticism scores from a cohort of 88 142 people from southwest England. The most significant SNPs were then genotyped on independent samples to replicate findings. We were able to replicate association of one SNP within the PDE4D gene in a second sample collected by our laboratory and in a family-based test in an independent sample; however, the SNP was not significantly associated with neuroticism in two other independent samples. We also observed an enrichment of low P-values in known regions of copy number variations. Simulation indicates that our study had ~80% power to identify neuroticism loci in the genome with odds ratio (OR) > 2, and ~50% power to identify small effects (OR = 1.5). Since we failed to find any loci accounting for more than 1% of the variance, the heritability of neuroticism probably arises from many loci each explaining much less than 1%. Our findings argue the need for much larger samples than anticipated in genetic association studies and that the biological basis of emotional disorders is extremely complex.
doi:10.1038/sj.mp.4002048
PMCID: PMC4004964  PMID: 17667963
neuroticism; whole genome association; DNA pooling; personality; quantitative trait; PDE4D
2.  Oscillating and Pulsed Gradient Diffusion Magnetic Resonance Microscopy Over an Extended b-value Range: Implications for the Characterization of Tissue Microstructure 
Oscillating gradient spin-echo (OGSE) pulse sequences have been proposed for acquiring diffusion data with very short diffusion times, which probe tissue structure at the subcellular scale. OGSE sequences are an alternative to pulsed gradient (PGSE) measurements, which typically probe longer diffusion times due to gradient limitations. In this investigation, a high-strength (6600 G/cm) gradient designed for small-sample microscopy was used to acquire OGSE and PGSE data in a rat hippocampal specimen at microscopic resolution. Measurements covered a broad range of diffusion times (TDeff=1.2–15.0 ms), frequencies (ω=67–1000 Hz) and b-values (b=0–3.2 ms/μm2). Variations in ADC with frequency and diffusion time provided microstructural information at a scale much smaller than the imaging resolution.
For a more direct comparison of the techniques, OGSE and PGSE data were acquired with similar effective diffusion times. Measurements with similar TDeff were consistent at low b-value (b<1 ms/um2), but diverged at higher b-values. Experimental observations suggest that the effective diffusion time can be helpful in the interpretation of low b-value OGSE data. However, caution is required at higher b, where enhanced sensitivity to restriction and exchange render the effective diffusion time an unsuitable representation. Oscillating and pulsed gradient diffusion techniques offer unique, complementary information. In combination, the two methods provide a powerful tool for characterizing complex diffusion within biological tissues.
doi:10.1002/mrm.24325
PMCID: PMC3421060  PMID: 22576352
diffusion; microscopy; oscillating gradient; pulsed gradient; kurtosis; hippocampus
3.  The p53 Protein Does Not Facilitate Adenovirus Type 5 Replication in Normal Human Cells 
Journal of Virology  2013;87(10):6044-6046.
Although several adenovirus type 5 (Ad5) proteins prevent deleterious consequences of activation of p53, it has been reported that viral replication proceeds more efficiently when human tumor cells produce wild-type compared to mutant p53. We have now exploited RNA interference and lentiviral vectors to achieve essentially complete knockdown of p53 in normal human cells: no effects on the kinetics or efficiency of viral gene expression or production of infectious particles were observed.
doi:10.1128/JVI.00129-13
PMCID: PMC3648133  PMID: 23487462
4.  The Repression Domain of the E1B 55-Kilodalton Protein Participates in Countering Interferon-Induced Inhibition of Adenovirus Replication 
Journal of Virology  2013;87(8):4432-4444.
To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55-kDa protein protects against the antiviral effects of type 1 interferon (IFN) (J. S. Chahal, J. Qi, and S. J. Flint, PLoS Pathog. 8:e1002853, 2012 [doi:10.1371/journal.ppat.1002853]), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution of residues 443 to 448 of E1B for alanine (E1B Sub19) specifically impaired production of progeny virus and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild-type-infected cells and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5′-ethynyluridine labeling and click chemistry. These results indicated that the mutations created by substitution of residues 443 to 448 for alanine (Sub19) impair repression of transcription of IFN-inducible genes, by the E1B, 55-kDa protein, consistent with their location in a segment required for repression of p53-dependent transcription. However, when synthesized alone, the E1B 55-kDa protein inhibited expression of the p53-regulated genes BAX and MDM2 but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55-kDa protein with protection against inhibition of viral genome replication and indicate that the E1B 55-kDa protein is not sufficient to establish such transcriptional repression.
doi:10.1128/JVI.03387-12
PMCID: PMC3624377  PMID: 23388716
5.  Reduced Infectivity of Adenovirus Type 5 Particles and Degradation of Entering Viral Genomes Associated with Incomplete Processing of the Preterminal Protein 
Journal of Virology  2012;86(24):13554-13565.
To investigate further the contribution of the adenovirus type 5 (Ad5) E1B 55-kDa protein to genome replication, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected with Ad5 or the E1B 55-kDa-null mutant Hr6. Unexpectedly, all cell types were observed to contain a significantly higher concentration of entering Hr6 than of Ad5 DNA, as did an infectious unit of Hr6. However, the great majority of the Hr6 genomes were degraded soon after entry. As this unusual phenotype cannot be ascribed to the Hr6 E1B frameshift mutation (J. S. Chahal and S. J. Flint, J. Virol. 86:3064–3072, 2012), the sequences of the Ad5 and Hr6 genomes were compared by using high-throughput sequencing. Seven previously unrecognized mutations were identified in the Hr6 genome, two of which result in substitutions in virion proteins, G315V in the preterminal protein (preTP) and A406V in fiber protein IV. Previous observations and the visualization by immunofluorescence of greater numbers of viral genomes entering the cytosol of Hr6-infected cells than of Ad5-infected cells indicated that the fiber mutation could not be responsible for the low-infectivity phenotype of Hr6. However, comparison of the forms of terminal protein present in purified virus particles indicated that the production of mature terminal protein from a processing intermediate is impaired in Hr6 particles. We therefore propose that complete processing of preTP within virus particles is necessary for the ability of viral genomes to become localized at appropriate sites and persist in infected cells.
doi:10.1128/JVI.02337-12
PMCID: PMC3503036  PMID: 23035217
6.  Direct Blood PCR in Combination with Nucleic Acid Lateral Flow Immunoassay for Detection of Plasmodium Species in Settings Where Malaria Is Endemic 
Journal of Clinical Microbiology  2012;50(11):3520-3525.
Declining malaria transmission and known difficulties with current diagnostic tools for malaria, such as microscopy and rapid diagnostic tests (RDTs) in particular at low parasite densities, still warrant the search for sensitive diagnostic tests. Molecular tests need substantial simplification before implementation in clinical settings in countries where malaria is endemic. Direct blood PCR (db-PCR), circumventing DNA extraction, to detect Plasmodium was developed and adapted to be visualized by nucleic acid lateral flow immunoassay (NALFIA). The assay was evaluated in the laboratory against samples from confirmed Sudanese patients (n = 51), returning travelers (n = 214), samples from the Dutch Blood Bank (n = 100), and in the field in Burkina Faso (n = 283) and Thailand (n = 381) on suspected malaria cases and compared to RDT and microscopy. The sensitivity and specificity of db-PCR-NALFIA compared to the initial diagnosis in the laboratory were 94.4% (95% confidence interval [CI] = 0.909 to 0.969) and 97.4% (95% CI = 0.909 to 0.969), respectively. In Burkina Faso, the sensitivity was 94.8% (95% CI = 0.88.7 to 97.9%), and the specificity was 82.4% (95% CI = 75.4 to 87.7%) compared to microscopy and 93.3% (95% CI = 87.4 to 96.7%) and 91.4% (95% CI = 85.2 to 95.3%) compared to RDT. In Thailand, the sensitivity and specificity were 93.4% (CI = 86.4 to 97.1%) and 90.9 (95% CI = 86.7 to 93.9%), respectively, compared to microscopy and 95.6% (95% CI = 88.5 to 98.6%) and 87.1% (95% CI = 82.5 to 90.6) compared to RDT. db-PCR-NALFIA is highly sensitive and specific for easy and rapid detection of Plasmodium parasites and can be easily used in countries where malaria is endemic. The inability of the device to discriminate Plasmodium species requires further investigation.
doi:10.1128/JCM.01426-12
PMCID: PMC3486265  PMID: 22915610
7.  Monitoring dynamics of human adenovirus disassembly induced by mechanical fatigue 
Scientific Reports  2013;3:1434.
The standard pathway for virus infection of eukaryotic cells requires disassembly of the viral shell to facilitate release of the viral genome into the host cell. Here we use mechanical fatigue, well below rupture strength, to induce stepwise disruption of individual human adenovirus particles under physiological conditions, and simultaneously monitor disassembly in real time. Our data show the sequence of dismantling events in individual mature (infectious) and immature (noninfectious) virions, starting with consecutive release of vertex structures followed by capsid cracking and core exposure. Further, our experiments demonstrate that vertex resilience depends inextricably on maturation, and establish the relevance of penton vacancies as seeding loci for virus shell disruption. The mechanical fatigue disruption route recapitulates the adenovirus disassembly pathway in vivo, as well as the stability differences between mature and immature virions.
doi:10.1038/srep01434
PMCID: PMC3595926  PMID: 23486377
8.  Timely Synthesis of the Adenovirus Type 5 E1B 55-Kilodalton Protein Is Required for Efficient Genome Replication in Normal Human Cells 
Journal of Virology  2012;86(6):3064-3072.
Previous studies have indicated that the adenovirus type 5 E1B 55-kDa protein facilitates viral DNA synthesis in normal human foreskin fibroblasts (HFFs) but not in primary epithelial cells. To investigate this apparent difference further, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected by the mutant AdEasyE1Δ2347, which carries the Hr6 frameshift mutation that prevents production of the E1B 55-kDa protein, in an E1-containing derivative of AdEasy. Impaired viral DNA synthesis was observed in normal HFFs but not in normal human bronchial epithelial cells infected by this mutant. However, acceleration of progression through the early phase, which is significantly slower in HFFs than in epithelial cells, eliminated the dependence of efficient viral DNA synthesis in HFFs on the E1B 55-kDa protein. These observations suggest that timely synthesis of the E1B 55-kDa protein protects normal cells against a host defense that inhibits adenoviral genome replication. One such defense is mediated by the Mre11-Rad50-Nbs1 complex. Nevertheless, examination of the localization of Mre11 and viral proteins by immunofluorescence suggested that this complex is inactivated similarly in AdEasyE1Δ2347 mutant-infected and AdEasyE1-infected HFFs.
doi:10.1128/JVI.06764-11
PMCID: PMC3302336  PMID: 22278242
9.  Role of the RNA recognition motif of the E1B 55 kDa protein in the adenovirus type 5 infectious cycle 
Virology  2011;417(1):9-17.
Although the adenovirus type 5 (Ad5) E1B 55 kDa protein can bind to RNA in vitro, no UV-light-induced crosslinking of this E1B protein to RNA could be detected in infected cells, under conditions in which RNA binding by a known viral RNA-binding protein (the L4 100kDa protein) was observed readily. Substitution mutations, including substitutions reported to inhibit RNA binding in vitro, did not impair synthesis of viral early or late proteins, or alter significantly the efficiency of viral replication in transformed or normal human cells. However, substitutions of conserved residues in the C-terminal segment of an RNA recognition motif specifically inhibited degradation of Mre11. We conclude that, if the E1B 55 kDa protein binds to RNA in infected cells in the same manner as in in vitro assays, this activity is not required for such well established functions as induction of selective export of viral late mRNAs.
doi:10.1016/j.virol.2011.04.014
PMCID: PMC3377160  PMID: 21605885
10.  The Human Adenovirus Type 5 E1B 55 kDa Protein Obstructs Inhibition of Viral Replication by Type I Interferon in Normal Human Cells 
PLoS Pathogens  2012;8(8):e1002853.
Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN) was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT) virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.
Author Summary
The most frequently used therapeutic vectors for gene transfer or cancer treatment are derived from human adenovirus type 5 (Ad5). We have observed previously that the E1B 55 kDa protein encoded by a gene routinely deleted from these vectors represses expression of numerous cellular genes regulated by interferon (IFN) α and β, which are important components of the innate immune response to viral infection. We therefore compared synthesis of pre-mRNA from IFN-inducible genes, viral yields and early reactions in the infectious cycle in normal human cells exposed to exogenous IFN and infected by wild-type or E1B 55 kDa null-mutant viruses. We report that the E1B 55 kDa protein is a potent repressor of expression of IFN-regulated genes, and protects viral replication against anti-viral actions of IFN by blocking inhibition of formation of viral replication centers and genome replication. These observations provide the first information about the function of the transcription repression activity of E1B during the infectious cycle. Importantly, they also suggest new design considerations for adenoviral vectors that can circumvent induction of innate immune responses, currently a major therapeutic limitation.
doi:10.1371/journal.ppat.1002853
PMCID: PMC3415460  PMID: 22912576
11.  Cytoarchitectural disruption of the superior colliculus and an enlarged acoustic startle response in the Tuba1a mutant mouse 
Neuroscience  2011;195(4):191-200.
The Jenna mutant mouse harbours an S140G mutation in Tuba1a that impairs tubulin heterodimer formation resulting in defective neuronal migration during development. The consequence of decreased neuronal motility is a fractured pyramidal cell layer in the hippocampus and wave-like perturbations in the cerebral cortex. Here, we extend our characterisation of this mouse investigating the laminar architecture of the superior colliculus (SC). Our results reveal that the structure of the SC in mutant animals is intact; however, it is significantly thinner with an apparent fusion of the intermediate grey and white layers. Birthdate labelling at E12.5 and E13.5 showed that the S140G mutation impairs the radial migration of neurons in the SC. A quantitative assessment of neuronal number in adulthood reveals a massive reduction in postmitotic neurons in mutant animals, which we attribute to increased apoptotic cell death. Consistent with the role of the SC in modulating sensorimotor gating, and the circuitry that modulates this behaviour, we find that Jenna mutants exhibit an exaggerated acoustic startle response. Our results highlight the importance of Tuba1a for correct neuronal migration and implicate postnatal apoptotic cell death in the pathophysiological mechanisms underlying the tubulinopathies.
Highlights
▶We investigate the superior colliculus and behaviour of a mouse model of lissencephaly. ▶We find that there is abnormal lamination and impairment in neuronal migration. ▶This is accompanied by a massive increase in postnatal neuronal cell death. ▶This is associated with an exaggerated acoustic startle response. ▶This study aids our understanding of disease mechanisms.
doi:10.1016/j.neuroscience.2011.08.035
PMCID: PMC3188702  PMID: 21875651
tubulin lissencephaly; superior colliculus; acoustic startle response; BrdU, 5-bromo-2-deoxyuridine; DAPI, 4',6-diamidino-2-phenylindole; DpG, deep grey layer; DpW, deep white layer; InG, intermediate grey layer; InW, intermediate white layer; Jenna, Jna/+; Op, optic layer; PAG, periaqueductal grey; SC, superior colliculus; SuG, superficial grey layer; Tpd52L1, tumour protein D52-like-1; Zn, zonal layer
12.  Export of Adenoviral Late mRNA from the Nucleus Requires the Nxf1/Tap Export Receptor ▿  
Journal of Virology  2010;85(4):1429-1438.
One important function of the human adenovirus E1B 55-kDa protein is induction of selective nuclear export of viral late mRNAs. This protein interacts with the viral E4 Orf6 and four cellular proteins to form an infected-cell-specific E3 ubiquitin ligase. The assembly of this enzyme is required for efficient viral late mRNA export, but neither the relevant substrates nor the cellular pathway that exports viral late mRNAs has been identified. We therefore examined the effects on viral late gene expression of inhibition of the synthesis or activity of the mRNA export receptor Nxf1, which was observed to colocalize with the E1B 55-kDa protein in infected cells. When production of Nxf1 was impaired by using RNA interference, the efficiency of viral late mRNA export was reduced to a corresponding degree. Furthermore, synthesis of a dominant-negative derivative of Nxf1 during the late phase of infection interfered with production of a late structural protein. These observations indicate that the Nxf1 pathway is responsible for export of viral late mRNAs. As the infected-cell-specific E3 ubiquitin ligase targets its known substrates for proteasomal degradation, we compared the concentrations of several components of this pathway (Nxf1, Thox1, and Thoc4) in infected cells that did or did not contain this enzyme. Although the concentration of a well-established substrate, Mre11, decreased significantly in cells infected by adenovirus type 5 (Ad5), but not in those infected by the E1B 55-kDa protein-null mutant Hr6, no E1B 55-kDa protein-dependent degradation of the Nxf1 pathway proteins was observed.
doi:10.1128/JVI.02108-10
PMCID: PMC3028892  PMID: 21123381
13.  Optically based-indentation technique for acute rat brain tissue slices and thin biomaterials 
Currently, micro-indentation testing of soft biological materials is limited in its capability to test over long time scales due to accumulated instrumental drift errors. As a result, there is a paucity of measures for mechanical properties such as the equilibrium modulus. In this study, indentation combined with optical coherence tomography (OCT) was used for mechanical testing of thin tissue slices. OCT was used to measure the surface deformation profiles by placing spherical beads onto submerged test samples. Agarose-based hydrogels at low-concentrations (w/v, 0.3–0.6 %) and acute rat brain tissue slices were tested using this technique over a 30 min time window. To establish that tissue slices maintained cell viability, allowable testing times were determined by measuring neuronal death or degeneration as a function of incubation time with Fluor-Jade C (FJC) staining. Since large deformations at equilibrium were measured, displacements of surface beads were compared with finite element elastic contact simulations to predict the equilibrium modulus, μ∞. Values of μ∞ for the low- concentration hydrogels ranged from 0.07–1.8 kPa, and μ∞ for acute rat brain tissue slices was 0.13 ± 0.04 kPa for the cortex and 0.09 ± 0.015 kPa for the hippocampus (for Poisson ratio=0.35). This indentation technique offers a localized, real-time, and high resolution method for long-time scale mechanical testing of very soft materials. This test method may also be adapted for viscoelasticity, for testing of different tissues and biomaterials, and for analyzing changes in internal structures with loading.
doi:10.1002/jbm.b.31789
PMCID: PMC3140429  PMID: 21290586
14.  Pulmonary actinomycosis complicating infliximab therapy for Crohn's disease 
Thorax  2007;62(11):1013-1014.
The use of anti‐tumour necrosis factor (TNF) agents has expanded significantly over the past few years, particularly for rheumatological diseases and Crohn's disease. A number of associated opportunistic infections have been observed as a result of suppression of T cell‐mediated immunity, the most frequent being tuberculosis. We report the first case of pulmonary actinomycosis in a patient receiving regular infusions of infliximab, an anti‐TNF agent, for Crohn's disease.
doi:10.1136/thx.2006.075150
PMCID: PMC2117133  PMID: 17965080
15.  The Adenoviral E1B 55-Kilodalton Protein Controls Expression of Immune Response Genes but Not p53-Dependent Transcription▿  
Journal of Virology  2009;83(8):3591-3603.
The human adenovirus type 5 (Ad5) E1B 55-kDa protein modulates several cellular processes, including activation of the tumor suppressor p53. Binding of the E1B protein to the activation domain of p53 inhibits p53-dependent transcription. This activity has been correlated with the transforming activity of the E1B protein, but its contribution to viral replication is not well understood. To address this issue, we used microarray hybridization methods to examine cellular gene expression in normal human fibroblasts (HFFs) infected by Ad5, the E1B 55-kDa-protein-null mutant Hr6, or a mutant carrying substitutions that impair repression of p53-dependent transcription. Comparison of the changes in cellular gene expression observed in these and our previous experiments (D. L. Miller et al., Genome Biol. 8:R58, 2007) by significance analysis of microarrays indicated excellent reproducibility. Furthermore, we again observed that Ad5 infection led to efficient reversal of the p53-dependent transcriptional program. As this same response was also induced in cells infected by the two mutants, we conclude that the E1B 55-kDa protein is not necessary to block activation of p53 in Ad5-infected cells. However, groups of cellular genes that were altered in expression specifically in the absence of the E1B protein were identified by consensus k-means clustering of the hybridization data. Statistical analysis of the enrichment of genes associated with specific functions in these clusters established that the E1B 55-kDa protein is necessary for repression of genes encoding proteins that mediate antiviral and immune defenses.
doi:10.1128/JVI.02269-08
PMCID: PMC2663238  PMID: 19211769
16.  The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription 
Molecular cell  2008;30(1):51-60.
Summary
Post-translational histone modifications are crucial for the modulation of chromatin structure and regulation of transcription. Bromodomains present in many chromatin-associated proteins recognize acetylated lysines in the unstructured N-terminal regions of histones. Here, we report that the double bromodomain proteins Brd2 and Brd3 associate preferentially in vivo with hyper-acetylated chromatin along the entire lengths of transcribed genes. Brd2 and Brd3 associated chromatin is significantly enriched in H4K5, H4K12 and H3K14 acetylation and contains relatively little dimethylated H3K9. Both Brd2 and Brd3 allowed RNA polymerase II to transcribe through nucleosomes in a defined transcription system. Such activity depended on specific histone H4 modifications known to be recognized by the Brd proteins. We also demonstrate that Brd2 has intrinsic histone chaperone activity, and is required for transcription of the cyclin D1 gene in vivo. These data identify proteins that render nucleosomes marked by acetylation permissive to the passage of elongating RNA polymerase II.
doi:10.1016/j.molcel.2008.01.018
PMCID: PMC2387119  PMID: 18406326
17.  Women in UK cardiology: report of a Working Group of the British Cardiac Society 
Heart  2005;91(3):283-289.
doi:10.1136/hrt.2004.047340
PMCID: PMC1768743  PMID: 15710701
18.  Idiopathic diffuse pulmonary ossification 
Thorax  2004;59(11):1004.
doi:10.1136/thx.2004.031682
PMCID: PMC1746871  PMID: 15516484
19.  Nucleolin Is Required for RNA Polymerase I Transcription In Vivo▿  
Molecular and Cellular Biology  2006;27(3):937-948.
Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin.
doi:10.1128/MCB.01584-06
PMCID: PMC1800701  PMID: 17130237
20.  The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription▿  
Journal of Virology  2006;81(3):1327-1338.
The adenovirus late IVa2 protein is required for maximally efficient transcription from the viral major late (ML) promoter, and hence, the synthesis of the majority of viral late proteins. This protein is a sequence-specific DNA-binding protein that also promotes the assembly of progeny virus particles. Previous studies have established that a IVa2 protein dimer (DEF-B) binds specifically to an intragenic ML promoter sequence necessary for late phase-specific stimulation of ML transcription. However, activation of transcription from the ML promoter correlates with binding of at least one additional infected-cell-specific protein, termed DEF-A, to the promoter. Using an assay for the DNA-binding activity of DEF-A, we identified the unknown protein by using conventional purification methods, purification of FLAG-tagged IVa2-protein-containing complexes, and transient synthesis of viral late proteins. The results of these experiments established that the viral L4 33-kDa protein is the only component of DEF-A: the IVa2 and L4 33-kDa proteins are necessary and sufficient for formation of all previously described complexes in the intragenic control region of the ML promoter. Furthermore, the L4 33-kDa protein binds to the promoter with the specificity characteristic of DEF-A and stimulates transcription from the ML promoter in transient-expression assays.
doi:10.1128/JVI.01584-06
PMCID: PMC1797539  PMID: 17093188
21.  Calcifying fibrous pseudotumour of the lung 
Thorax  2003;58(12):1018-1019.
doi:10.1136/thorax.58.12.1018
PMCID: PMC1746547  PMID: 14645962
22.  Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts 
Journal of Virology  2006;80(2):964-974.
The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).
doi:10.1128/JVI.80.2.964-974.2006
PMCID: PMC1346875  PMID: 16378998
23.  Genetic effects on human cognition: lessons from the study of mental retardation syndromes 
The molecular basis of human cognition is still poorly understood, but recent advances in finding genetic mutations that result in cognitive impairment may provide insights into the neurobiology of cognitive function. Here we review the progress that has been made so far and assess what has been learnt from this work on the relation between genes and cognitive processes. We review evidence that the pathway from genetic lesion to cognitive impairment can be dissected, that some genetic effects on cognition are relatively direct and we argue that the study of mental retardation syndromes is giving us new clues about the biological bases of cognition.
doi:10.1136/jnnp.72.3.287
PMCID: PMC1737778  PMID: 11861682
24.  Clinical studies on submicroscopic subtelomeric rearrangements: a checklist 
Journal of Medical Genetics  2001;38(3):145-150.
BACKGROUND—Submicroscopic subtelomeric chromosome defects have been found in 7.4% of children with moderate to severe mental retardation and in 0.5% of children with mild retardation. Effective clinical preselection is essential because of the technical complexities and cost of screening for subtelomere deletions.
METHODS—We studied 29 patients with a known subtelomeric defect and assessed clinical variables concerning birth history, facial dysmorphism, congenital malformations, and family history. Controls were 110 children with mental retardation of unknown aetiology with normal G banded karyotype and no detectable submicroscopic subtelomeric abnormalities.
RESULTS—Prenatal onset of growth retardation was found in 37% compared to 9% of the controls (p<0.0005). A higher percentage of positive family history for mental retardation was reported in the study group than the controls (50% v 21%, p=0.002). Miscarriage(s) were observed in only 8% of the mothers of subtelomeric cases compared to 30% of controls (p=0.028) which was, however, not significant after a Bonferroni correction. Common features (>30%) among subtelomeric deletion cases were microcephaly, short stature, hypertelorism, nasal and ear anomalies, hand anomalies, and cryptorchidism. Two or more facial dysmorphic features were observed in 83% of the subtelomere patients. None of these features was significantly different from the controls. Using the results, a five item checklist was developed which allowed exclusion from further testing in 20% of the mentally retarded children (95% CI 13-28%) in our study without missing any subtelomere cases. As our control group was selected for the "chromosomal phenotype", the specificity of the checklist is likely to be higher in an unselected group of mentally retarded subjects.
CONCLUSIONS—Our results suggest that good indicators for subtelomeric defects are prenatal onset of growth retardation and a positive family history for mental retardation. These clinical criteria, in addition to features suggestive of a chromosomal phenotype, resulted in the development of a five item checklist which will improve the diagnostic pick up rate of subtelomeric defects among mentally retarded subjects.


Keywords: submicroscopic subtelomeric rearrangements; clinical preselection; checklist; chromosome deletion.
doi:10.1136/jmg.38.3.145
PMCID: PMC1734836  PMID: 11238680
25.  Submicroscopic subtelomeric 1qter deletions: a recognisable phenotype? 
Journal of Medical Genetics  2001;38(3):175-178.
doi:10.1136/jmg.38.3.175
PMCID: PMC1734828  PMID: 11303509

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