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1.  A conditional knockout resource for the genome–wide study of mouse gene function 
Nature  2011;474(7351):337-342.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
doi:10.1038/nature10163
PMCID: PMC3572410  PMID: 21677750
2.  Non-Hodgkin Lymphoma after Treatment with Extended Dosing Temozolomide and Radiotherapy for a Glioblastoma: A Case Report 
Case Reports in Oncology  2013;6(1):45-49.
Temozolomide (TMZ) is an alkylating agent, used for the treatment of high-grade gliomas. This case report describes the development of a non-Hodgkin lymphoma in a patient treated with extended-dose temozolomide and radiotherapy. In addition to the possible mutagenic effect of temozolomide – as described for all alkylating agents – there might have been an immunosuppressive effect of TMZ. The pathological appearance of the lymphoma as well as the presence of a grade 3 lymphopenia early in treatment supports this hypothesis. As the use of TMZ increases, the awareness that TMZ may induce secondary malignancies should increase as well.
doi:10.1159/000346614
PMCID: PMC3573799  PMID: 23466981
Temozolomide; B-cell lymphoma; Diffuse large B-cell lymphoma; Lymphopenia; Epstein-Barr virus
3.  Temperature, Not Fine Particulate Matter (PM2.5), is Causally Associated with Short-Term Acute Daily Mortality Rates: Results from One Hundred United States Cities 
Dose-Response  2012;11(3):319-343.
Exposures to fine particulate matter (PM2.5) in air (C) have been suspected of contributing causally to increased acute (e.g., same-day or next-day) human mortality rates (R). We tested this causal hypothesis in 100 United States cities using the publicly available NMMAPS database. Although a significant, approximately linear, statistical C-R association exists in simple statistical models, closer analysis suggests that it is not causal. Surprisingly, conditioning on other variables that have been extensively considered in previous analyses (usually using splines or other smoothers to approximate their effects), such as month of the year and mean daily temperature, suggests that they create strong, nonlinear confounding that explains the statistical association between PM2.5 and mortality rates in this data set. As this finding disagrees with conventional wisdom, we apply several different techniques to examine it. Conditional independence tests for potential causation, non-parametric classification tree analysis, Bayesian Model Averaging (BMA), and Granger-Sims causality testing, show no evidence that PM2.5 concentrations have any causal impact on increasing mortality rates. This apparent absence of a causal C-R relation, despite their statistical association, has potentially important implications for managing and communicating the uncertain health risks associated with, but not necessarily caused by, PM2.5 exposures.
doi:10.2203/dose-response.12-034.Cox
PMCID: PMC3748846  PMID: 23983662
Air pollution health effects; PM2.5; time series; mortality rates; cardiovascular disease; causality; Granger-Sims; conditional independence tests; J-shaped dose-response curve
4.  DNA methylation profiling of human chromosomes 6, 20 and 22 
Nature genetics  2006;38(12):1378-1385.
DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methylation and a core region surrounding the transcriptional start site as informative surrogate for promoter methylation. We find 17% of the 873 analyzed genes differentially methylated in their 5′-untranslated regions (5′-UTR) and about one third of the differentially methylated 5′-UTRs to be inversely correlated with transcription. While our study was controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.
doi:10.1038/ng1909
PMCID: PMC3082778  PMID: 17072317
5.  Genome-wide end-sequenced BAC resources for the NOD/MrkTac☆ and NOD/ShiLtJ☆☆ mouse genomes 
Genomics  2010;95(2):105-110.
Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.
doi:10.1016/j.ygeno.2009.10.004
PMCID: PMC2824108  PMID: 19909804
Bacterial artificial chromosome; NOD/MrkTac; NOD/ShiLtJ; Mouse genome; Non-obese diabetic (NOD); Type 1 diabetes; T1D; Insulin-dependent diabetes; IDD
8.  Large-Scale Mutagenesis in p19ARF- and p53-Deficient Mice Identifies Cancer Genes and Their Collaborative Networks 
Cell  2008;133(4):727-741.
Summary
p53 and p19ARF are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19ARF deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19ARF-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.
doi:10.1016/j.cell.2008.03.021
PMCID: PMC2405818  PMID: 18485879
SYSBIO; SIGNALING; HUMDISEASE
9.  Chromhome: A rich internet application for accessing comparative chromosome homology maps 
BMC Bioinformatics  2008;9:168.
Background
Comparative genomics has become a significant research area in recent years, following the availability of a number of sequenced genomes. The comparison of genomes is of great importance in the analysis of functionally important genome regions. It can also be used to understand the phylogenetic relationships of species and the mechanisms leading to rearrangement of karyotypes during evolution. Many species have been studied at the cytogenetic level by cross species chromosome painting. With the large amount of such information, it has become vital to computerize the data and make them accessible worldwide. Chromhome is a comprehensive web application that is designed to provide cytogenetic comparisons among species and to fulfil this need.
Results
The Chromhome application architecture is multi-tiered with an interactive client layer, business logic and database layers. Enterprise java platform with open source framework OpenLaszlo is used to implement the Rich Internet Chromhome Application. Cross species comparative mapping raw data are collected and the processed information is stored into MySQL Chromhome database. Chromhome Release 1.0 contains 109 homology maps from 51 species. The data cover species from 14 orders and 30 families. The homology map displays all the chromosomes of the compared species as one image, making comparisons among species easier. Inferred data also provides maps of homologous regions that could serve as a guideline for researchers involved in phylogenetic or evolution based studies.
Conclusion
Chromhome provides a useful resource for comparative genomics, holding graphical homology maps of a wide range of species. It brings together cytogenetic data of many genomes under one roof. Inferred painting can often determine the chromosomal homologous regions between two species, if each has been compared with a common third species. Inferred painting greatly reduces the need to map entire genomes and helps focus only on relevant regions of the chromosomes of the species under study. Future releases of Chromhome will accommodate more species and their respective gene and BAC maps, in addition to chromosome painting data. Chromhome application provides a single-page interface (SPI) with desktop style layout, delivering a better and richer user experience.
doi:10.1186/1471-2105-9-168
PMCID: PMC2323974  PMID: 18366796
10.  A Novel CpG Island Set Identifies Tissue-Specific Methylation at Developmental Gene Loci 
PLoS Biology  2008;6(1):e22.
CpG islands (CGIs) are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%–8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.
Author Summary
The human genome contains about 22,000 genes, each encoding one of the proteins required for human life. A particular cell type (e.g., blood, skin, etc.) expresses a specific subset of protein genes and silences the remainder. To shed light on the mechanisms that cause genes to be activated or shut down, we studied DNA sequences called “CpG islands” (CGIs). These sequences are found at over half of all human genes and can exist in either the active or silent state depending on the presence or absence of methyl groups on the DNA. We devised a method for purifying all CGIs and showed that, unexpectedly, only half occur at the beginning of genes near the promoter, the rest occurring within or between genes. Notably, methylation of CGIs causes stable gene silencing. We tested 17,000 CGIs in four human tissues and found that 6%–8% were methylated in each. Genes whose protein products play an essential role during embryonic development were preferentially methylated, suggesting that gene expression during development could be regulated by CGI methylation.
CpG island methylation, an epigenetic phenomenon usually associated with abnormality in disease, is little characterised in the context of "normal" human cells. Here we highlight tissue-specific CpG Island methylation, which frequently associates with developmental genes.
doi:10.1371/journal.pbio.0060022
PMCID: PMC2214817  PMID: 18232738
11.  Priorities for nucleotide trace, sequence and annotation data capture at the Ensembl Trace Archive and the EMBL Nucleotide Sequence Database 
Nucleic Acids Research  2007;36(Database issue):D5-D12.
The Ensembl Trace Archive (http://trace.ensembl.org/) and the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/), known together as the European Nucleotide Archive, continue to see growth in data volume and diversity. Selected major developments of 2007 are presented briefly, along with data submission and retrieval information. In the face of increasing requirements for nucleotide trace, sequence and annotation data archiving, data capture priority decisions have been taken at the European Nucleotide Archive. Priorities are discussed in terms of how reliably information can be captured, the long-term benefits of its capture and the ease with which it can be captured.
doi:10.1093/nar/gkm1018
PMCID: PMC2238915  PMID: 18039715
12.  Efficacy and Safety of AM-101 in the Treatment of Acute Inner Ear Tinnitus—A Double-Blind, Randomized, Placebo-Controlled Phase II Study 
Otology & Neurotology  2014;35(4):589-597.
Objective
To evaluate the efficacy and safety of intratympanic AM-101 in patients with persistent acute inner ear tinnitus after acute acoustic trauma, idiopathic sudden sensorineural hearing loss (ISSNHL), or acute otitis media.
Study Design
Prospective, double-blind, randomized, placebo-controlled study with follow-up visits on Days 7, 30, and 90.
Setting
Twenty-eight European sites (academic tertiary referral centers and private ENT practices).
Patients
248 patients aged 16 to 65 years.
Interventions
Three intratympanic injections of AM-101 (0.27 or 0.81 mg/ml) or placebo over 3 consecutive days.
Main Outcome Measures
Efficacy was assessed by changes in minimum masking level (MML; primary end point), loudness match, tinnitus loudness, tinnitus annoyance, and sleep difficulties on a 0 to 100 numerical rating scale, THI-12 questionnaire, and patient global impression of change. Safety was evaluated using the frequency of clinically relevant hearing deterioration and adverse events.
Results
The study overall failed to demonstrate a treatment benefit based on the change in MML. However, AM-101 0.81 mg/ml showed statistically significantly better improvement for tinnitus loudness, annoyance, sleep difficulties, and tinnitus impact in patients with tinnitus after noise trauma or otitis media. The subgroup of ISSNHL-related tinnitus patients did not show conclusive results. The study drug and I.T. injections were well tolerated.
Conclusion
The study established proof of concept for AM-101 in the treatment of tinnitus arising from cochlear glutamate excitotoxicity. Patient-reported outcomes seem to be more relevant and reliable efficacy measures for assessing treatment-related changes in tinnitus than psychoacoustic tests.
doi:10.1097/MAO.0000000000000268
PMCID: PMC3966923  PMID: 24603353
Acute acoustic trauma; Cochlea; Idiopathic sudden sensorineural hearing loss; Intratympanic; Otitis Media; Therapy; Tinnitus

Results 1-12 (12)