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1.  Nonmelanoma skin cancer is associated with reduced Alzheimer disease risk 
Neurology  2013;80(21):1966-1972.
Objective:
To explore the association of nonmelanoma skin cancer (NMSC) and Alzheimer disease (AD) in the Einstein Aging Study, an epidemiologic study of aging in New York City.
Methods:
Community-residing volunteers aged 70 years or older were assessed annually, followed by multidisciplinary diagnostic consensus. Cancer status and type was obtained by self-report. Cox proportional hazards models were used to test associations between NMSC and subsequent risk of developing a neurocognitive disorder. To deduce a biologically specific association between AD and NMSC, we considered 3 nested outcomes groups: only AD (probable or possible AD as the sole diagnosis), any AD (probable AD or possible AD, as well as mixed AD/vascular dementia), and all-cause dementia.
Results:
We followed 1,102 adults with a mean age of 79 years at enrollment. Prevalent NMSC was associated with reduced risk of only AD (hazard ratio = 0.21; 95% confidence interval = 0.051–0.87; p = 0.031) among subjects after adjustment for demographics, hypertension, diabetes, and coronary heart disease. APOE ε4 genotypes were available in 769 individuals. The association was similar in magnitude, but nonsignificant, when the number of APOE ε4 alleles was included in the model. No significant association was found between NMSC and subsequent development of any AD or all-cause dementia.
Conclusions:
This population-based longitudinal study shows that individuals older than 70 years with NMSC have a significantly reduced risk of developing AD compared with individuals without NMSC. We deduce Alzheimer-specific neuroprotection, because the effect is attenuated or eliminated when considering less-specific diagnoses such as AD with another diagnosis (any AD) or all-cause dementia.
doi:10.1212/WNL.0b013e3182941990
PMCID: PMC3716346  PMID: 23677746
2.  Brain Arteriovenous Malformations associated with Hereditary Hemorrhagic Telangiectasia: Gene-Phenotype Correlations 
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disease with a wide spectrum of vascular malformations involving multiple organs. Nine-16% of patients with HHT harbor brain arteriovenous malformations (AVMs), which can cause intracranial hemorrhage. Our objective was to study clinical manifestations of brain AVM in patients with HHT and correlate these with the specific gene mutated. We reviewed records of 171 patients with HHT and brain AVMs. A history of intracranial hemorrhage was found in 27% (41/152) patients, with a mean (range) age of 26 +/− 18 (0–68) years. All patients with intracranial hemorrhage were neurologically asymptomatic prior to intracranial hemorrhage. Multiple brain AVMs were found in 23% (170/39) of patients on initial examination. Genetic test results were available in 109 (64%) patients. Mutations in ENG, ACVRL1, and SMAD4 were present in 75 (69%), 18 (17%) and 2 (2%), respectively. A history of intracranial hemorrhage was reported in 24% of patients with an ENG mutation and 27% of patients with an ACVRL1 mutation, with a mean (range) age of 26 +/− 16 (2–50) and 18 +/− 21 (0–48) years, respectively. No statistically significant differences in age at first brain AVM diagnosis, prevalence of intracranial hemorrhage history, age at intracranial hemorrhage, or other manifestations of brain AVMs were observed among gene groups. In conclusion, no evidence for differences in brain AVM characteristics was observed among HHT gene groups, although we cannot exclude clinically important differences. Larger studies are needed to further guide brain AVM screening decisions in patients with HHT.
doi:10.1002/ajmg.a.35622
PMCID: PMC3610331  PMID: 22991266
Hereditary Hemorrhagic Telangiectasia; Brain Arteriovenous Malformation; Genotype; Intracranial Hemorrhage
3.  Genome-Wide and Cell-Specific Epigenetic Analysis Challenges the Role of Polycomb in Drosophila Spermatogenesis 
PLoS Genetics  2013;9(10):e1003842.
The Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. We investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model we expected to see a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes in tTAF mutant testes. However, we find little evidence for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, we further tested the model by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although we find Polycomb associated with its canonical targets, we find little or no evidence of Polycomb at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation.
Author Summary
How are developmental gene expression programs activated? An interesting activation mechanism has been suggested by studies of the switch-on of the genes required for sperm differentiation in Drosophila; the spermatogenesis genes. These studies indicated that removal of the Polycomb protein from spermatogenesis genes was a key event in their activation. This was interesting because Polycomb is a central component of a gene regulatory complex that works at the level of chromatin and is best known for its role in stable heritable gene silencing. We have further analysed this system by examining Polycomb binding at the genome-wide scale and specifically in the precursor cells where Polycomb is proposed to repress the spermatogenesis genes. Surprisingly, this cell-specific analysis found no clear evidence for Polycomb binding at the spermatogenesis genes before their activation. This argues against the current model where Polycomb removal is the key mechanism involved in the activation of these genes. Further work is needed to identify the actual mechanism of gene activation in this system; however, our results fit with previous work suggesting that lamin-association rather than Polycomb binding may be responsible for repression of spermatogenesis genes prior to their activation.
doi:10.1371/journal.pgen.1003842
PMCID: PMC3798269  PMID: 24146626
4.  Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells 
Developmental Biology  2013;382(2):504-516.
Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.
highlights
•Expression of S1PR3 is associated with non-cycling myoblasts.•Constitutive expression of S1PR3 leads to reduced cell proliferation.•Satellite cells lacking S1PR3 have enhanced proliferation.•Muscle regeneration is improved in the absence of S1PR3.•The dystrophic phenotype in mdx mice is improved by the absence of S1PR3.
doi:10.1016/j.ydbio.2013.07.006
PMCID: PMC3898928  PMID: 23911934
Stem cell; Satellite cell; Skeletal muscle; Sphingosine-1-phosphate; S1PR3; Muscular dystrophy; mdx; Regeneration; Cell cycle; Proliferation; Quiescence
5.  Nanosensor dosimetry of mouse blood proteins after exposure to ionizing radiation 
Scientific Reports  2013;3:2234.
Giant magnetoresistive (GMR) nanosensors provide a novel approach for measuring protein concentrations in blood for medical diagnosis. Using an in vivo mouse radiation model, we developed protocols for measuring Flt3 ligand (Flt3lg) and serum amyloid A1 (Saa1) in small amounts of blood collected during the first week after X-ray exposures of sham, 0.1, 1, 2, 3, or 6 Gy. Flt3lg concentrations showed excellent dose discrimination at ≥ 1 Gy in the time window of 1 to 7 days after exposure except 1 Gy at day 7. Saa1 dose response was limited to the first two days after exposure. A multiplex assay with both proteins showed improved dose classification accuracy. Our magneto-nanosensor assay demonstrates the dose and time responses, low-dose sensitivity, small volume requirements, and rapid speed that have important advantages in radiation triage biodosimetry.
doi:10.1038/srep02234
PMCID: PMC3715761  PMID: 23868657
7.  Bacteria-reactive immune response may induce RANKL-expressing T-cells in the mouse periapical bone loss lesion 
Journal of Endodontics  2012;38(3):346-350.
Introduction
The present study investigated if T-cells infiltrating the periapical lesion produce RANKL and whether bacteria infecting the root canal can activate T-cells to produce RANKL.
Methods
Using a mouse model of periapical lesion induced by artificial dental pulp exposure, the presence of RANKL-positive T-cells and osteoclasts in the periapical lesion was examined by an immuno-histochemical approach. The bacteria colonizing the exposed root canal were identified by 16S ribosomal RNA (rRNA) sequence analysis. The isolated endodontic bacteria were further immunized to normal mice, and sRANKL production by the T-cells isolated from the immunized mice was evaluated by ex vivo culture system.
Results
RANKL-positive T-cells, along with TARP+ osteoclasts, were identified in periapical bone resorption lesions. The Gram-negative bacterium Pasterurella pnumotropica (P. pnumotropica), which was most frequently detected from root canal of exposed pulp, showed remarkably elevated serum IgG antibody response in pulp-exposed mice compared to control non-treated mice. Immunization of mice with P. pneumotropica induced not only serum IgG antibody but also primed bacteria reactive T-cells that produced sRANKL in response to ex vivo exposure to P. pneumotropica.
Conclusion
T-cells infiltrating the periapical region express RANKL, and the endodontic bacteria colonizing the root canal appear to induce RANKL expression from bacteria-reactive T-cells, suggesting the possible pathogenic engagement of immune response to endodontic bacteria in the context of developing boneresorptive periapical lesions.
doi:10.1016/j.joen.2011.12.029
PMCID: PMC3291477  PMID: 22341072
8.  Induction of p16INK4a Is the Major Barrier to Proliferation when Epstein-Barr Virus (EBV) Transforms Primary B Cells into Lymphoblastoid Cell Lines 
PLoS Pathogens  2013;9(2):e1003187.
To explore the role of p16INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16INK4a and p14ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16INK4a protein but expressing a functional 14ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16INK4a and growth arrest, EBNA3C inactivation in the p16INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs.
Author Summary
Epstein-Barr virus (EBV) is a causative agent of several types of B cell lymphoma. In human B cells, EBV reduces protein levels of at least two tumour suppressors that would otherwise be activated in response to over-expressed oncogenes. These proteins are BIM, which induces cell death and p16INK4a, which prevents cell proliferation. Repression of both is via epigenetic methylation of histones and is dependent on expression of both EBNA3A and EBNA3C – two EBV proteins required for the transformation of normal B cells into lymphoblastoid cell lines (LCLs). In this report we have used EBV with a conditionally active EBNA3C – active only in the presence of 4-hydroxytamoxifen – together with B cells from an individual carrying a homozygous deletion of p16INK4a to confirm that regulation of p16INK4a expression is a major function of EBNA3C and demonstrate that if B cells lack p16INK4a, then EBNA3C is no longer required for EBV-driven proliferation of LCLs. Furthermore we show that early after the infection of normal B cells, EBV induces p16INK4a accumulation that – if unchecked by EBNA3C (and EBNA3A) – prevents LCL outgrowth. Formal proof that p16INK4a is the main target of EBNA3C comes with the production of p16-null LCLs that have never expressed functional EBNA3C.
doi:10.1371/journal.ppat.1003187
PMCID: PMC3578823  PMID: 23436997
9.  Functional Promiscuity of the COG0720 Family 
ACS Chemical Biology  2011;7(1):197-209.
The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function.
doi:10.1021/cb200329f
PMCID: PMC3262898  PMID: 21999246
Queuosine; archaeosine; tetrahydrofolate; biopterin; tRNA modification; riboflavin; 6-pyruvoyl-tetrahydropterin synthase
10.  tRNA genes protect a reporter gene from epigenetic silencing in mouse cells 
Cell Cycle  2011;10(16):2779-2791.
It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.
doi:10.4161/cc.10.16.17092
PMCID: PMC3219543  PMID: 21822054
barrier elements; tRNA genes; Pol III-transcribed genes
11.  Silicon Nano-well Arrays for Reliable Pattern Transfer and Locally Confined High Temperature Reactions 
Nanotechnology  2011;22(30):305304.
Si nano-well arrays, with precisely controlled undercut Si sidewall profiles and flat bottomed pockets, enable uniform nanoscale pattern transfer from resists to metal deposits without degradation of the initial lithographic resolution, as verified by formation of arrays of Au nano-dots with 10 nm diameter. An additional functionality of the Si nano-wells as local nano-reactors, where the patterned material is enclosed in a Si pocket during high temperature reaction, is demonstrated by thermally inducing a phase transformation of the as-deposited A1 phase of FePt nano-dots to the high coercivity, chemically ordered L10 phase.
doi:10.1088/0957-4484/22/30/305304
PMCID: PMC3167870  PMID: 21709347
12.  BIM promoter directly targeted by EBNA3C in polycomb-mediated repression by EBV 
Nucleic Acids Research  2012;40(15):7233-7246.
Detailed analyses of the chromatin around the BIM promoter has revealed that latent Epstein–Barr virus (EBV) triggers the recruitment of polycomb repressive complex 2 (PRC2) core subunits and the trimethylation of histone H3 lysine 27 (H3K27me3) at this locus. The recruitment is absolutely dependent on nuclear proteins EBNA3A and EBNA3C; what is more, epitope-tagged EBNA3C could be shown bound near the transcription start site (TSS). EBV induces no consistent changes in the steady-state expression of PRC2 components, but lentivirus delivery of shRNAs against PRC2 and PRC1 subunits disrupted EBV repression of BIM. The activation mark H3K4me3 is largely unaltered at this locus irrespective of H3K27me3 status, suggesting the establishment of a ‘bivalent’ chromatin domain. Consistent with the ‘poised’ nature of these domains, RNA polymerase II (Pol II) occupancy was not altered by EBV at the BIM TSS, but analysis of phospho-serine 5 on Pol II indicated that EBNA3A and EBNA3C together inhibit initiation of BIM transcripts. B cell lines carrying EBV encoding a conditional EBNA3C-oestrogen receptor-fusion revealed that this epigenetic repression of BIM was reversible, but took more than 3 weeks from when EBNA3C was inactivated.
doi:10.1093/nar/gks391
PMCID: PMC3424555  PMID: 22584624
13.  Genomic Binding Profiles of Functionally Distinct RNA Polymerase III Transcription Complexes in Human Cells 
Genome-wide occupancy profiles of five components of the RNA Polymerase III (Pol III) machinery in human cells identified the expected tRNA and non-coding RNA targets and revealed many additional Pol III-associated loci, mostly near SINEs. Several genes are targets of an alternative TFIIIB containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike non-expressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.
doi:10.1038/nsmb.1794
PMCID: PMC3350333  PMID: 20418883
14.  Pol III binding in six mammalian genomes shows high conservation among amino acid isotypes, despite divergence in tRNA gene usage 
Nature genetics  2011;43(10):948-955.
RNA polymerase III (pol III) transcription of transfer RNA (tRNA) genes is essential for generating the tRNA adapter molecules that link genetic sequence and protein translation. By mapping pol III occupancy genome-wide in the livers of mouse, rat, human, macaque, dog and opossum, we found that pol III binding to individual tRNA genes varies substantially in strength and location. However, taking into account tRNA redundancies by grouping pol III occupancy into 46 anticodon isoacceptor families or 21 amino acid-based isotype classes shows strong conservation. Similarly, pol III occupancy of amino-acid isotypes is almost invariant among transcriptionally and evolutionarily diverse tissues in mouse. Thus, synthesis of functional tRNA isotypes has been highly constrained, though the usage of individual tRNA genes has evolved rapidly.
doi:10.1038/ng.906
PMCID: PMC3184141  PMID: 21873999
15.  EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors 
The Journal of Clinical Investigation  2012;122(4):1487-1502.
Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc–/– mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell–chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell–mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.
doi:10.1172/JCI58092
PMCID: PMC3314448  PMID: 22406538
16.  High Cell-Free Virus Load and Robust Autologous Humoral Immune Responses in Breast Milk of Simian Immunodeficiency Virus-Infected African Green Monkeys ▿ 
Journal of Virology  2011;85(18):9517-9526.
The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4+ T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.
doi:10.1128/JVI.00796-11
PMCID: PMC3165740  PMID: 21734053
17.  An ATM/Chk2-mediated DNA damage responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells 
Cell host & microbe  2010;8(6):510-522.
SUMMARY
Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication nor did the DDR marks co-localize with latent episomes. Rather, a transient period of EBV-induced hyper-proliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoproteins EBNA3C was required to attenuate the EBV-induced DNA damage response We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR which is attenuated by viral latency products to induce cell immortalization.
doi:10.1016/j.chom.2010.11.004
PMCID: PMC3049316  PMID: 21147465
18.  Purine biosynthesis in archaea: variations on a theme 
Biology Direct  2011;6:63.
Background
The ability to perform de novo biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains.
Results
We searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11 steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function.
Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified.
Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%) assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified.
Conclusions
The patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a need to identify further pathway components even in well-studied pathways.
Reviewers
This article was reviewed by Dr. Céline Brochier-Armanet, Dr Kira S Makarova (nominated by Dr. Eugene Koonin), and Dr. Michael Galperin.
doi:10.1186/1745-6150-6-63
PMCID: PMC3261824  PMID: 22168471
19.  Latent Epstein-Barr Virus Can Inhibit Apoptosis in B Cells by Blocking the Induction of NOXA Expression 
PLoS ONE  2011;6(12):e28506.
Latent Epstein-Barr virus (EBV) has been shown to protect Burkitt's lymphoma-derived B cells from apoptosis induced by agents that cause damage to DNA, in the context of mutant p53. This protection requires expression of the latency-associated nuclear proteins EBNA3A and EBNA3C and correlates with their ability to cooperate in the repression of the gene encoding the pro-apoptotic, BH3-only protein BIM. Here we confirm that latent EBV in B cells also inhibits apoptosis induced by two other agents – ionomycin and staurosporine – and show that these act by a distinct pathway that involves a p53-independent increase in expression of another pro-apoptotic, BH3-only protein, NOXA. Analyses employing a variety of B cells infected with naturally occurring EBV or B95.8 EBV-BAC recombinant mutants indicated that the block to NOXA induction does not depend on the well-characterized viral latency-associated genes (EBNAs 1, 2, 3A, 3B, 3C, the LMPs or the EBERs) or expression of BIM. Regulation of NOXA was shown to be at least partly at the level of mRNA and the requirement for NOXA to induce cell death in this context was demonstrated by NOXA-specific shRNA-mediated depletion experiments. Although recombinant EBV with a deletion removing the BHRF1 locus – that encodes the BCL2-homologue BHRF1 and three microRNAs – partially abrogates protection against ionomycin and staurosporine, the deletion has no effect on the EBV-mediated block to NOXA accumulation.
doi:10.1371/journal.pone.0028506
PMCID: PMC3235132  PMID: 22174825
20.  Cellular Gene Expression That Correlates with EBER Expression in Epstein-Barr Virus-Infected Lymphoblastoid Cell Lines ▿ †  
Journal of Virology  2011;85(7):3535-3545.
Novel Epstein-Barr Virus (EBV) strains with deletion of either EBER1 or EBER2 and corresponding revertant viruses were constructed and used to infect B lymphocytes to make lymphoblastoid cell lines (LCLs). The LCLs were used in microarray expression profiling to identify genes whose expression correlates with the presence of EBER1 or EBER2. Functions of regulated genes identified in the microarray analysis include membrane signaling, regulation of apoptosis, and the interferon/antiviral response. Although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cell gene expression were greater with EBER2 deletion. In this system, deletion of EBER1 or EBER2 had little effect on the EBV transformation frequency of primary B cells or the growth of the resulting LCLs. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed, but in LCLs almost all of the cells had a predominantly cytoplasmic expression of this ribosomal protein, which was not detectably changed by EBER1. The changes in LCL gene expression identified here will provide a basis for identifying the mechanisms of action of EBER RNAs.
doi:10.1128/JVI.02086-10
PMCID: PMC3067860  PMID: 21248031
21.  A Cis-Regulatory Map of the Drosophila Genome 
Nature  2011;471(7339):527-531.
Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide 1,2 has successfully identified specific subtypes of regulatory elements 3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb-Response Elements 4, chromatin states 5, transcription factor binding sites (TFBS) 6–9, PolII regulation 8, and insulator elements 10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome based on more than 300 chromatin immuno-precipitation (ChIP) datasets for eight chromatin features, five histone deacetylases (HDACs) and thirty-eight site-specific transcription factors (TFs) at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and we validated a subset of predictions for promoters, enhancers, and insulators in vivo. We also identified nearly 2,000 genomic regions of dense TF binding associated with chromatin activity and accessibility. We discovered hundreds of new TF co-binding relationships and defined a TF network with over 800 potential regulatory relationships.
doi:10.1038/nature09990
PMCID: PMC3179250  PMID: 21430782
22.  A one-year economic evaluation of six alternative strategies for the management of uninvestigated upper gastrointestinal symptoms in Canadian primary care 
BACKGROUND:
The cost-effectiveness of initial strategies in managing Canadian patients with uninvestigated upper gastrointestinal symptoms remains controversial.
OBJECTIVE:
To assess the cost-effectiveness of six management approaches to uninvestigated upper gastrointestinal symptoms in the Canadian setting.
METHODS:
The present study analyzed data from four randomized trials assessing homogeneous and complementary populations of Canadian patients with uninvestigated upper gastrointestinal symptoms with comparable outcomes. Symptom-free months, quality-adjusted life-years (QALYs) and direct costs in Canadian dollars of two management approaches based on the Canadian Dyspepsia Working Group (CanDys) Clinical Management Tool, and four additional strategies (two empirical antisecretory agents, and two prompt endoscopy) were examined and compared. Prevalence data, probabilities, utilities and costs were included in a Markov model, while sensitivity analysis used Monte Carlo simulations. Incremental cost-effectiveness ratios and cost-effectiveness acceptability curves were determined.
RESULTS:
Empirical omeprazole cost $226 per QALY ($49 per symptom-free month) per patient. CanDys omeprazole and endoscopy approaches were more effective than empirical omeprazole, but more costly. Alternatives using H2-receptor antagonists were less effective than those using a proton pump inhibitor. No significant differences were found for most incremental cost-effectiveness ratios. As willingness to pay (WTP) thresholds rose from $226 to $24,000 per QALY, empirical antisecretory approaches were less likely to be the most cost-effective choice, with CanDys omeprazole progressively becoming a more likely option. For WTP values ranging from $24,000 to $70,000 per QALY, the most clinically relevant range, CanDys omeprazole was the most cost-effective strategy (32% to 46% of the time), with prompt endoscopy-proton pump inhibitor favoured at higher WTP values.
CONCLUSIONS:
Although no strategy was the indisputable cost-effective option, CanDys omeprazole may be the strategy of choice over a clinically relevant range of WTP assumptions in the initial management of Canadian patients with uninvestigated dyspepsia.
PMCID: PMC2947002  PMID: 20711528
Antisecretory therapy; Cost-effectiveness; Dyspepsia; Economic modelling; Endoscopy; Helicobacter pylori
23.  Genome-Wide Analysis of the Binding of the Hox Protein Ultrabithorax and the Hox Cofactor Homothorax in Drosophila 
PLoS ONE  2011;6(4):e14778.
Hox genes encode a family of transcription factors that are key developmental regulators with a highly conserved role in specifying segmental diversity along the metazoan body axis. Although they have been shown to regulate a wide variety of downstream processes, direct transcriptional targets have been difficult to identify and this has been a major obstacle to our understanding of Hox gene function. We report the identification of genome-wide binding sites for the Hox protein Ultrabithorax (Ubx) using a YFP-tagged Drosophila protein-trap line together with chromatin immunoprecipitation and microarray analysis. We identify 1,147 genes bound by Ubx at high confidence in chromatin from the haltere imaginal disc, a prominent site of Ubx function where it specifies haltere versus wing development. The functional relevance of these genes is supported by their overlap with genes differentially expressed between wing and haltere imaginal discs. The Ubx-bound gene set is highly enriched in genes involved in developmental processes and contains both high-level regulators as well as genes involved in more basic cellular functions. Several signalling pathways are highly enriched in the Ubx target gene set and our analysis supports the view that Hox genes regulate many levels of developmental pathways and have targets distributed throughout the gene network. We also performed genome-wide analysis of the binding sites for the Hox cofactor Homothorax (Hth), revealing a striking similarity with the Ubx binding profile. We suggest that these binding profiles may be strongly influenced by chromatin accessibility and provide evidence of a link between Ubx/Hth binding and chromatin state at genes regulated by Polycomb silencing. Overall, we define a set of direct Ubx targets in the haltere imaginal disc and suggest that chromatin accessibility has important implications for Hox target selection and for transcription factor binding in general.
doi:10.1371/journal.pone.0014778
PMCID: PMC3071696  PMID: 21483667
24.  Identification, Source, and Metabolism of N-Ethylglutamate in Escherichia coli▿  
Journal of Bacteriology  2010;192(20):5437-5440.
N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-2H4]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.
doi:10.1128/JB.00721-10
PMCID: PMC2950513  PMID: 20709899
25.  Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy 
PLoS ONE  2011;6(2):e16651.
LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting.
doi:10.1371/journal.pone.0016651
PMCID: PMC3043058  PMID: 21364987

Results 1-25 (105)