Publications

2019

  • [DOI] J. Allen, S. Hao, C. L. Sears, and W. Timp, “Epigenetic changes induced by Bacteroides fragilis toxin (BFT),” Infection and Immunity, 2019.
    [Bibtex]
    @article{allen_epigenetic_2019,
    author = {Allen, Jawara and Hao, Stephanie and Sears, Cynthia L. and Timp, Winston},
    copyright = {Copyright © 2019 American Society for Microbiology.. All Rights Reserved.},
    doi = {10.1128/IAI.00447-18},
    issn = {0019-9567, 1098-5522},
    journal = {Infection and Immunity},
    month = {March},
    pmid = {30885929},
    title = {Epigenetic changes induced by {Bacteroides} fragilis toxin ({BFT})},
    url = {https://iai.asm.org/content/early/2019/03/14/IAI.00447-18},
    year = {2019}
    }
  • [DOI] B. A. Avin, Y. Wang, T. Gilpatrick, R. E. Workman, I. Lee, W. Timp, C. B. Umbricht, and M. A. Zeiger, “Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines,” Genes, Chromosomes & Cancer, 2019.
    [Bibtex]
    @article{avin_characterization_2019,
    abstract = {Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.},
    author = {Avin, Brittany A. and Wang, Yongchun and Gilpatrick, Timothy and Workman, Rachael E. and Lee, Isac and Timp, Winston and Umbricht, Christopher B. and Zeiger, Martha A.},
    doi = {10.1002/gcc.22735},
    issn = {1098-2264},
    journal = {Genes, Chromosomes \& Cancer},
    keywords = {DNA methylation, promoter, telomerase, thyroid cancer},
    month = {January},
    pmid = {30664813},
    title = {Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines},
    year = {2019}
    }
  • [DOI] P. D. Tamma, Y. Fan, Y. Bergman, G. Pertea, A. Kazmi, S. Lewis, K. C. Carroll, M. C. Schatz, W. Timp, and P. J. Simner, “Applying Rapid Whole Genome Sequencing to Predict Phenotypic Antimicrobial Susceptibility Testing Results Among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates,” Antimicrobial Agents and Chemotherapy, vol. 63, iss. 1, p. e01923–18, 2019.
    [Bibtex]
    @article{TammaAAC.01923-18,
    abstract = {Objective: Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. We sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole genome sequencing in predicting AST results.Methods: Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae, 10 non-carbapenemase-producing carbapenem resistant K. pneumoniae, and 9 carbapenem-susceptible K. pneumoniae), three separate sequencing and analysis pipelines were performed: (1) a real-time Nanopore analysis approach identifying acquired AMR genes, (2) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (3) an approach using short read correction of Nanopore assemblies. The short read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results.Results: With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 hours of subcultured isolates. Assemblies sufficient for full resistance gene and single nucleotide polymorphism annotation were available within 14 hours from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 77\% (range 30-100\%) and 92\% (range 80-100\%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 hours and 26 hours, respectively, compared to standard AST.Conclusions: Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms as well as predict AST results for K. pneumoniae isolates. Bioinformatics improvements enabling real-time alignment coupled with rapid extraction and library preparation will further enhance the accuracy and workflow of the Nanopore real-time approach.},
    author = {Tamma, Pranita D. and Fan, Yunfan and Bergman, Yehudit and Pertea, Geo and Kazmi, Abida and Lewis, Shawna and Carroll, Karen C. and Schatz, Michael C. and Timp, Winston and Simner, Patricia J.},
    doi = {10.1128/AAC.01923-18},
    issn = {0066-4804},
    journal = {Antimicrobial Agents and Chemotherapy},
    month = {January},
    number = {1},
    pages = {e01923--18},
    pmcid = {PMC6325187},
    pmid = {30373801},
    publisher = {American Society for Microbiology Journals},
    title = {Applying Rapid Whole Genome Sequencing to Predict Phenotypic Antimicrobial Susceptibility Testing Results Among Carbapenem-Resistant {Klebsiella pneumoniae} Clinical Isolates},
    url = {https://aac.asm.org/content/early/2018/10/23/AAC.01923-18},
    volume = {63},
    year = {2019}
    }
  • [DOI] E. Briem, Z. Budkova, A. K. Sigurdardottir, B. Hilmarsdottir, J. Kricker, W. Timp, M. K. Magnusson, G. A. Traustadottir, and T. Gudjonsson, “MiR-203a is differentially expressed during branching morphogenesis and EMT in breast progenitor cells and is a repressor of peroxidasin,” Mechanisms of Development, vol. 155, p. 34–47, 2019.
    [Bibtex]
    @article{briem_mir-203a_2019,
    author = {Briem, Eirikur and Budkova, Zuzana and Sigurdardottir, Anna Karen and Hilmarsdottir, Bylgja and Kricker, Jennifer and Timp, Winston and Magnusson, Magnus Karl and Traustadottir, Gunnhildur Asta and Gudjonsson, Thorarinn},
    doi = {10.1016/j.mod.2018.11.002},
    issn = {0925-4773},
    journal = {Mechanisms of Development},
    month = {February},
    pages = {34--47},
    title = {{MiR}-203a is differentially expressed during branching morphogenesis and {EMT} in breast progenitor cells and is a repressor of peroxidasin},
    url = {http://www.sciencedirect.com/science/article/pii/S0925477318300972},
    volume = {155},
    year = {2019}
    }
  • [DOI] N. P. Roach, N. Sadowski, A. F. Alessi, W. Timp, J. Taylor, and J. K. Kim, “The full-length transcriptome of C. elegans using direct RNA sequencing,” bioRxiv, 2019.
    [Bibtex]
    @article{Roach598763,
    abstract = {Current transcriptome annotations have largely relied on short read lengths intrinsic to most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans. Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 20,902 splice isoforms across 14,115 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 2,188 are novel splice isoforms not present in the Wormbase WS265 annotation. Furthermore, we identified 16,325 3{\textquoteright} untranslated region (3{\textquoteright}UTR) isoforms, 2,304 of which are novel and do not fall within 10 bp of existing 3{\textquoteright}UTR datasets and annotations. Combining 3{\textquoteright}UTRs and splice isoforms we identified 25,944 full-length isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3{\textquoteright}UTR length. Finally, we have formatted this data as a publically accessible track hub, enabling researchers to explore this dataset easily in a genome browser.},
    author = {Roach, Nathan P. and Sadowski, Norah and Alessi, Amelia F. and Timp, Winston and Taylor, James and Kim, John K.},
    doi = {10.1101/598763},
    elocation-id = {598763},
    entrysubtype = {preprint},
    eprint = {https://www.biorxiv.org/content/early/2019/04/04/598763.full.pdf},
    journal = {{b}ioRxiv},
    month = {April},
    publisher = {Cold Spring Harbor Laboratory},
    title = {The full-length transcriptome of C. elegans using direct RNA sequencing},
    url = {https://www.biorxiv.org/content/early/2019/04/04/598763},
    year = {2019}
    }
  • [DOI] T. Gilpatrick, I. Lee, J. E. Graham, E. Raimondeau, R. Bowen, A. Heron, F. Sedlazeck, and W. Timp, “Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants and mutations,” bioRxiv, 2019.
    [Bibtex]
    @article{Gilpatrick604173,
    abstract = {Nanopore sequencing technology offers a significant advancement through its ability to rapidly and directly interrogate native DNA molecules. Often we are interested only in interrogating specific areas at high depth, but this has proved challenging for long read sequencing with conventional enrichment methods. Existing strategies are currently limited by high input DNA requirements, low yield, short (\<5kb) reads, time-intensive protocols, and/or amplification or cloning (losing base modification information). In this paper, we describe a technique utilizing the ability of Cas9 to introduce cuts at specific locations and ligating nanopore sequencing adaptors directly to those sites, a method we term {\textquoteright}nanopore Cas9 Targeted-Sequencing{\textquoteright} (nCATS). We have demonstrated the ability of this method to generate median 165X coverage at 10 genomic loci with a median length of 18kb from a single flow cell, which represents a several hundred fold improvement over the 2-3X coverage achieved without enrichment. Using a panel of guide RNAs, we show that the high coverage data from this method enables us to (1) profile DNA methylation patterns at cancer driver genes, (2) detect structural variations at known hot spots, and (3) survey for the presence of single nucleotide mutations. Together, this provides a low-cost method that can be applied even in low resource settings to directly examine cellular DNA. This technique has extensive clinical applications for assessing medically relevant genes and has the versatility to be a rapid and comprehensive diagnostic tool. We demonstrate applications of this technique by examining the well characterized GM12878 cell line as well as three breast cell lines (MCF-10A, MCF-7, MDA-MB-231) with varying tumorigenic potential as a model for cancer.},
    author = {Gilpatrick, Timothy and Lee, Isac and Graham, James E. and Raimondeau, Etienne and Bowen, Rebecca and Heron, Andrew and Sedlazeck, Fritz and Timp, Winston},
    doi = {10.1101/604173},
    elocation-id = {604173},
    entrysubtype = {preprint},
    eprint = {https://www.biorxiv.org/content/early/2019/04/11/604173.full.pdf},
    journal = {bioRxiv},
    publisher = {Cold Spring Harbor Laboratory},
    title = {Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants and mutations},
    url = {https://www.biorxiv.org/content/early/2019/04/11/604173},
    year = {2019}
    }

2018

  • [DOI] K. G. Vanaja, W. Timp, A. P. Feinberg, and A. Levchenko, “A Loss of Epigenetic Control Can Promote Cell Death through Reversing the Balance of Pathways in a Signaling Network,” Molecular Cell, vol. 72, iss. 1, p. 60–70.e3, 2018.
    [Bibtex]
    @article{vanaja2018loss,
    abstract = {Epigenetic control of regulatory networks is only partially understood. Expression of Insulin-like growth factor-II (IGF2) is controlled by genomic imprinting, mediated by silencing of the maternal allele. Loss of imprinting of IGF2 (LOI) is linked to intestinal and colorectal cancers, causally in murine models and epidemiologically in humans. However, the molecular underpinnings of the LOI phenotype are not clear. Surprisingly, in LOI cells, we find a reversal of the relative activities of two canonical signaling pathways triggered by IGF2, causing further rebalancing between pro- and anti-apoptotic signaling. A predictive mathematical model shows that this network rebalancing quantitatively accounts for the effect of receptor tyrosine kinase inhibition in both WT and LOI cells. This mechanism also quantitatively explains both the stable LOI phenotype and the therapeutic window for selective killing of LOI cells, and thus prevention of epigenetically controlled cancers. These findings suggest a framework for understanding epigenetically modified cell signaling.},
    author = {Vanaja, Kiran G. and Timp, Winston and Feinberg, Andrew P. and Levchenko, Andre},
    doi = {10.1016/j.molcel.2018.08.025},
    issn = {1097-4164},
    journal = {Molecular Cell},
    keywords = {AKT, apoptosis, computational model, death plane, epigenetics, ERK, imprinting, kinase signaling, network rewiring, oncogene addiction},
    month = {October},
    number = {1},
    pages = {60--70.e3},
    pmcid = {PMC6219618},
    pmid = {30244832},
    title = {A Loss of Epigenetic Control Can Promote Cell Death through Reversing the Balance of Pathways in a Signaling Network},
    volume = {72},
    year = {2018}
    }
  • [DOI] R. E. Workman, A. M. Myrka, W. G. Wong, E. Tseng, K. C. Welch, and W. Timp, “Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris,” GigaScience, vol. 7, iss. 3, 2018.
    [Bibtex]
    @article{workman_single-molecule_2018,
    abstract = {BackgroundHummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation.FindingsWe generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes.ConclusionsWe confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.},
    author = {Workman, Rachael E. and Myrka, Alexander M. and Wong, G. William and Tseng, Elizabeth and Welch, Kenneth C. and Timp, Winston},
    doi = {10.1093/gigascience/giy009},
    journal = {GigaScience},
    month = {March},
    number = {3},
    title = {Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird {{Archilochus} colubris}},
    url = {https://academic.oup.com/gigascience/article/7/3/giy009/4860431},
    volume = {7},
    year = {2018}
    }
  • [DOI] N. D. Olson, S. M. Kumar, S. Li, S. Hao, W. Timp, M. L. Salit, C. O. Stine, and H. Corrada Bravo, “Assessing 16S marker gene survey data analysis methods using mixtures of human stool sample DNA extracts.,” bioRxiv, 2018.
    [Bibtex]
    @article{Olson400226,
    abstract = {Background: Analysis of 16S rRNA marker-gene surveys, used to characterize prokaryotic microbial communities, may be performed by numerous bioinformatic pipelines and downstream analysis methods. However, there is limited guidance on how to decide between methods, appropriate data sets and statistics for assessing these methods are needed. We developed a mixture dataset with real data complexity and an expected value for assessing 16S rRNA bioinformatic pipelines and downstream analysis methods. We generate an assessment dataset using a two-sample titration mixture design. The sequencing data were processed using multiple bioinformatic pipelines, i) DADA2 a sequence inference method, ii)Mothur a de novo clustering method, and iii) QIIME with open-reference clustering. The mixture dataset was used to qualitatively and quantitatively assess count tables generated using the pipelines. Results: The qualitative assessment was used to evaluate features only present in unmixed samples and titrations. The abundance of Mothur and QIIME features specific to unmixed samples and titrations were explained by sampling alone. However, for DADA2 over a third of the unmixed sample and titration specific feature abundance could not be explained by sampling alone. The quantitative assessment evaluated pipeline performance by comparing observed to expected relative and differential abundance values. Overall the observed relative abundance and differential abundance values were consistent with the expected values. Though outlier features were observed across all pipelines. Conclusions: Using a novel mixture dataset and assessment methods we quantitatively and qualitatively evaluated count tables generated using three bioinformatic pipelines. The dataset and methods developed for this study will serve as a valuable community resource for assessing 16S rRNA marker-gene survey bioinformatic methods.},
    author = {Olson, Nathan D and Kumar, M. Senthil and Li, Shan and Hao, Stephanie and Timp, Winston and Salit, Marc L and Stine, O. Colin and Corrada Bravo, Hector},
    doi = {10.1101/400226},
    elocation-id = {400226},
    entrysubtype = {preprint},
    eprint = {https://www.biorxiv.org/content/early/2018/08/25/400226.full.pdf},
    journal = {{b}ioRxiv},
    month = {August},
    publisher = {Cold Spring Harbor Laboratory},
    title = {Assessing 16S marker gene survey data analysis methods using mixtures of human stool sample DNA extracts.},
    url = {https://www.biorxiv.org/content/early/2018/08/25/400226},
    year = {2018}
    }
  • [DOI] P. J. Simner, A. A. R. Antar, S. Hao, J. Gurtowski, P. D. Tamma, C. Rock, B. N. A. Opene, T. Tekle, K. C. Carroll, M. C. Schatz, and W. Timp, “Antibiotic pressure on the acquisition and loss of antibiotic resistance genes in Klebsiella pneumoniae,” The Journal of Antimicrobial Chemotherapy, 2018.
    [Bibtex]
    @article{simner_antibiotic_2018,
    abstract = {Objectives: In this study, we characterize a concurrent disseminated infection with a virulent hypermucoviscous (HMV) Klebsiella pneumoniae and an OXA-181-producing XDR K. pneumoniae from a patient with recent hospitalization in India. During exposure to meropenem therapy, the highly susceptible HMV K. pneumoniae became resistant to carbapenems, consistent with the acquisition of blaOXA-181.
    Methods: Twelve K. pneumoniae isolates were recovered from the patient and the hospital room environment over a 3 month hospitalization. Phenotypic and molecular studies were completed to characterize the isolates. Oxford Nanopore and Illumina MiSeq WGS were performed to study phylogeny (MLST and SNPs), plasmids and virulence genes and demonstrate changes in the organism's resistome that occurred over time.
    Results: WGS revealed that the HMV K. pneumoniae belonged to ST23 and harboured an IncH1B virulence plasmid, while the XDR K. pneumoniae belonged to ST147 and possessed two MDR plasmids (IncR and IncFII), the blaOXA-181-bearing ColKP3 plasmid and chromosomal mutations conferring the XDR phenotype. Sequential isolates demonstrated plasmid diversification (fusion of the IncR and IncFII plasmids), mobilization of resistance elements (ompK35 inactivation by ISEcp1-blaCTX-M-15 mobilization, varying numbers of resistance genes on plasmid scaffolds) and chromosomal mutations (mutations in mgrB) leading to further antibiotic resistance that coincided with antibiotic pressure. Importantly, the HMV strain in this study was unable to preserve the carbapenem-resistant phenotype without the selective pressure of meropenem.
    Conclusions: To the best of our knowledge, we are the first to report a carbapenem-resistant HMV K. pneumoniae strain in the USA. Ultimately, this case demonstrates the role of antibiotic pressure in the acquisition and loss of important genetic elements.},
    author = {Simner, Patricia J. and Antar, Annukka A. R. and Hao, Stephanie and Gurtowski, James and Tamma, Pranita D. and Rock, Clare and Opene, Belita N. A. and Tekle, Tsigereda and Carroll, Karen C. and Schatz, Michael C. and Timp, Winston},
    doi = {10.1093/jac/dky121},
    entrysubtype = {casereport},
    issn = {1460-2091},
    journal = {The Journal of Antimicrobial Chemotherapy},
    month = {April},
    pmid = {29648629},
    title = {Antibiotic pressure on the acquisition and loss of antibiotic resistance genes in {Klebsiella pneumoniae}},
    year = {2018}
    }
  • [DOI] P. D. Tamma, Y. Fan, Y. Bergman, A. C. Sick-Samuels, A. J. Hsu, W. Timp, and P. J. Simner, “Successful Treatment of Persistent Burkholderia cepacia Complex Bacteremia with Ceftazidime-Avibactam,” Antimicrobial Agents and Chemotherapy, vol. 62, iss. 4, 2018.
    [Bibtex]
    @article{tamma_successful_2018,
    abstract = {We report our clinical experience treating a 2-month-old infant with congenital diaphragmatic hernia who experienced prolonged bacteremia with Burkholderia cepacia complex (Bcc) despite conventional antibiotic therapy and appropriate source control measures. The infection resolved after initiation of ceftazidime-avibactam. Whole-genome sequencing revealed that the isolate most closely resembled B. contaminans and identified the mechanism of resistance that likely contributed to clinical cure with this agent. Ceftazidime-avibactam should be considered salvage therapy for Bcc infections if other treatment options have been exhausted.},
    author = {Tamma, Pranita D. and Fan, Yunfan and Bergman, Yehudit and Sick-Samuels, Anna C. and Hsu, Alice J. and Timp, Winston and Simner, Patricia J.},
    doi = {10.1128/AAC.02213-17},
    entrysubtype = {casereport},
    issn = {1098-6596},
    journal = {Antimicrobial Agents and Chemotherapy},
    keywords = {bacteremia, Burkholderia cepacia complex, ceftazidime-avibactam, whole-genome sequencing},
    month = {April},
    number = {4},
    pmcid = {PMC5913954},
    pmid = {29588357},
    title = {Successful {Treatment} of {Persistent} {Burkholderia cepacia} {Complex} {Bacteremia} with {Ceftazidime}-{Avibactam}},
    volume = {62},
    year = {2018}
    }
  • R. Workman, R. Fedak, D. Kilburn, S. Hao, K. Liu, and W. Timp, “High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing,” Nature Protocols Exchange, 2018.
    [Bibtex]
    @article{workman_high_2018,
    abstract = {Single molecule sequencing requires optimized sample and library preparation protocols to obtain long-read lengths and high sequencing yields. Numerous protocols exist for the extraction of DNA from plant species, but the genomic DNA from these extractions is either too low yield, of insufficient purity for sensitive sequencing platforms, e.g. nanopore sequencing, too fragmented to achieve long reads, or otherwise unattainable from recalcitrant adult tissue. This renders many plant sequencing projects cost prohibitive or methodologically intractable. Existing protocols are also labor intensive, taking days to complete. Our protocol described here yields micrograms of high molecular weight gDNA from a single gram of adult or seedling leaf tissue in only a few hours, and produces high quality sequencing libraries for the Oxford Nanopore system, with typical yields ranging from 3-10 Gb per R9.4.1 flowcell and producing reads averaging 5-8 kb, with read length N50s ranging from 6-30 kb depending on the style},
    author = {Workman, Rachael and Fedak, Renee and Kilburn, Duncan and Hao, Stephanie and Liu, Kelvin and Timp, Winston},
    entrysubtype = {preprint},
    eprint = {https://www.nature.com/protocolexchange/protocols/6785},
    journal = {Nature Protocols Exchange},
    month = {April},
    title = {High Molecular Weight {DNA} Extraction from Recalcitrant Plant Species for Third Generation Sequencing},
    url = {https://www.nature.com/protocolexchange/protocols/6785},
    year = {2018}
    }
  • [DOI] T. Xiong, D. Rohm, R. E. Workman, L. Roundtree, C. D. Novina, W. Timp, and M. Ostermeier, “Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria,” PLoS One, vol. 13, iss. 12, pp. 1-18, 2018.
    [Bibtex]
    @article{10.1371/journal.pone.0209408,
    abstract = {Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo gene regulation could have therapeutic and research applications. We have previously developed a dCas9-MC/MN protein for targeting CpG methylation. dCas9-MC/MN is composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused to a nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed dCas9-MC/MN to methylate target sites in E. coli and human cells but also caused some low-level off-target methylation. Here, in E. coli, we show that shortening the dCas9-MC linker increases methylation of CpG sites located at select distances from the dCas9 binding site. Although a shortened linker decreased methylation of other CpGs proximal to the target site, it did not reduce off-target methylation of more distant CpG sites. Instead, targeted mutagenesis of the methyltransferase’s DNA binding domain, designed to reduce DNA affinity, significantly and preferentially reduced methylation of such sites.},
    author = {Xiong, Tina AND Rohm, Dahlia AND Workman, Rachael E. AND Roundtree, Lauren AND Novina, Carl D. AND Timp, Winston AND Ostermeier, Marc},
    doi = {10.1371/journal.pone.0209408},
    journal = {PLoS One},
    month = {12},
    number = {12},
    pages = {1-18},
    publisher = {Public Library of Science},
    title = {Protein engineering strategies for improving the selective methylation of target {CpG} sites by a {dCas9-directed} cytosine methyltransferase in bacteria},
    url = {https://doi.org/10.1371/journal.pone.0209408},
    volume = {13},
    year = {2018}
    }
  • [DOI] I. Lee, R. Razaghi, T. Gilpatrick, N. Sadowski, F. Sedlazeck, and W. Timp, “Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing,” bioRxiv, 2018.
    [Bibtex]
    @article{Lee504993,
    abstract = {Understanding how the genome and the epigenome work together to control gene transcription has applications in our understanding of diseases such as human cancer. In this study, we combine the ability of NOMe-seq to simultaneously evaluate CpG and chromatin accessibility, with long-read nanopore sequencing technology, a method we call nanoNOMe. We generated \>60Gb whole-genome nanopore sequencing data for each of four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231) including normally poorly mapped repetitive regions. Using the long reads, we find that we can observe phased methylation and chromatin accessibility, large scale pattern changes, and genetic changes such as structural variations from a single assay.},
    author = {Lee, Isac and Razaghi, Roham and Gilpatrick, Timothy and Sadowski, Norah and Sedlazeck, Fritz and Timp, Winston},
    doi = {10.1101/504993},
    elocation-id = {504993},
    entrysubtype = {preprint},
    eprint = {https://www.biorxiv.org/content/early/2018/12/22/504993.full.pdf},
    journal = {{b}ioRxiv},
    month = {12},
    publisher = {Cold Spring Harbor Laboratory},
    title = {Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing},
    url = {https://www.biorxiv.org/content/early/2018/12/22/504993},
    year = {2018}
    }
  • [DOI] R. E. Workman, A. Tang, P. S. Tang, M. Jain, J. R. Tyson, P. C. Zuzarte, T. Gilpatrick, R. Razaghi, J. Quick, N. Sadowski, N. Holmes, J. Goes de Jesus, K. Jones, T. P. Snutch, N. J. Loman, B. Paten, M. W. Loose, J. T. Simpson, H. E. Olsen, A. N. Brooks, M. Akeson, and W. Timp, “Nanopore native RNA sequencing of a human poly(A) transcriptome,” bioRxiv, 2018.
    [Bibtex]
    @article{Workman459529,
    abstract = {High throughput RNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these cDNA-based methods lose information contained in biological RNA because the copied reads are short or because modifications are not carried forward in cDNA. Here we address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA isolated from the human cell line GM12878, from which we sequenced 4p74ximately 9.9 million individual aligned strands. These native RN78]\-=[79A sequence reads had an N50 length of 1334 bases, and a maximum length of 22,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. Among these isoforms, over 50\% are not present in GENCODE v24. We describe strategies for assessing 3{\textquoteright}poly(A) tail length, base modifications and transcript haplotypes using this single molecule technology. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.},
    author = {Workman, Rachael E and Tang, Alison and Tang, Paul S. and Jain, Miten and Tyson, John R and Zuzarte, Philip C and Gilpatrick, Timothy and Razaghi, Roham and Quick, Joshua and Sadowski, Norah and Holmes, Nadine and Goes de Jesus, Jaqueline and Jones, Karen and Snutch, Terrance P and Loman, Nicholas James and Paten, Benedict and Loose, Matthew W and Simpson, Jared T and Olsen, Hugh E. and Brooks, Angela N and Akeson, Mark and Timp, Winston},
    doi = {10.1101/459529},
    elocation-id = {459529},
    entrysubtype = {preprint},
    eprint = {https://www.biorxiv.org/content/early/2018/11/09/459529.full.pdf},
    journal = {{b}ioRxiv},
    month = {11},
    publisher = {Cold Spring Harbor Laboratory},
    title = {Nanopore native {RNA} sequencing of a human {poly(A)} transcriptome},
    url = {https://www.biorxiv.org/content/early/2018/11/09/459529},
    year = {2018}
    }

2017

  • [DOI] J. T. Simpson, R. E. Workman, P. C. Zuzarte, M. David, L. J. Dursi, and W. Timp, “Detecting DNA cytosine methylation using nanopore sequencing,” Nature Methods, vol. 14, iss. 4, p. 407–410, 2017.
    [Bibtex]
    @article{simpson_detecting_2017,
    abstract = {In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.},
    author = {Simpson, Jared T. and Workman, Rachael E. and Zuzarte, P. C. and David, Matei and Dursi, L. J. and Timp, Winston},
    copyright = {© 2017 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.},
    doi = {10.1038/nmeth.4184},
    issn = {1548-7091},
    journal = {Nature Methods},
    keywords = {DNA sequencing, Epigenomics, nanopores, Software},
    month = {April},
    number = {4},
    pages = {407--410},
    title = {Detecting {DNA} cytosine methylation using nanopore sequencing},
    url = {http://www.nature.com/nmeth/journal/v14/n4/full/nmeth.4184.html},
    volume = {14},
    year = {2017}
    }
  • [DOI] J. J. Credle, C. Y. Itoh, T. Yuan, R. Sharma, E. R. Scott, R. E. Workman, Y. Fan, F. Housseau, N. J. Llosa, R. W. Bell, H. Miller, S. X. Zhang, W. Timp, and B. H. Larman, “Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization,” Nucleic Acids Research, vol. 45, iss. 14, p. e128–e128, 2017.
    [Bibtex]
    @article{credle_multiplexed_2017,
    author = {Credle, Joel J. and Itoh, Christopher Y. and Yuan, Tiezheng and Sharma, Rajni and Scott, Erick R. and Workman, Rachael E. and Fan, Yunfan and Housseau, Franck and Llosa, Nicolas J. and Bell, W. Robert and Miller, Heather and Zhang, Sean X. and Timp, Winston and Larman, H. Benjamin},
    doi = {10.1093/nar/gkx471},
    journal = {Nucleic Acids Research},
    number = {14},
    pages = {e128--e128},
    shortjournal = {Nucleic Acids Res},
    title = {Multiplexed analysis of fixed tissue {RNA} using {Ligation} {in situ} {Hybridization}},
    volume = {45},
    year = {2017}
    }
  • [DOI] A. J. Kandathil, F. P. Breitwieser, J. Sachithanandham, M. Robinson, S. H. Mehta, W. Timp, S. L. Salzberg, D. L. Thomas, and A. Balagopal, “Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs,” Annals of Internal Medicine, vol. 167, iss. 1, p. 1–7, 2017.
    [Bibtex]
    @article{kandathil_presence_2017,
    abstract = {Background: Next-generation metagenomic sequencing ({NGMS}) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings.
    Objective: To explore the plasma virome of persons who inject drugs and to characterize the sensitivity and accuracy of {NGMS} compared with quantitative clinical standards.
    Design: Longitudinal and cross-sectional studies.
    Setting: A clinical trial ({ClinicalTrials}.gov: {NCT}01285050) and a well-characterized cohort study of persons who have injected drugs.
    Participants: Persons co-infected with hepatitis C virus ({HCV}) and {HIV}.
    Measurements: Viral nucleic acid in plasma by {NGMS} and quantitative polymerase chain reaction ({PCR}).
    Results: Next-generation metagenomic sequencing generated a total of 600 million reads, which included the expected {HIV} and {HCV} {RNA} sequences. {HIV} and {HCV} reads were consistently identified only when samples contained more than 10000 copies/{mL} or {IU}/{mL}, respectively, as determined by quantitative {PCR}. A novel {RNA} virus, human hepegivirus-1 ({HHpgV}-1), was also detected by {NGMS} in 4 samples from 2 persons in the clinical trial. Through use of a quantitative {PCR} assay for {HHpgV}-1, infection was also detected in 17 (10.9\%) of 156 members of a cohort of persons who injected drugs. In these persons, {HHpgV}-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses, such as {HCV} {RNA} and {SEN} virus D.
    Limitation: The medical importance of {HHpgV}-1 infection is unknown.
    Conclusion: Although {NGMS} is insensitive for detection of viruses with relatively low plasma nucleic acid concentrations, it may have broad potential for discovery of new viral infections of possible medical importance, such as {HHpgV}-1.
    Primary Funding Source: National Institutes of Health.},
    author = {Kandathil, Abraham J. and Breitwieser, Florian P. and Sachithanandham, Jaiprasath and Robinson, Matthew and Mehta, Shruti H. and Timp, Winston and Salzberg, Steven L. and Thomas, David L. and Balagopal, Ashwin},
    date = {2017-06-06},
    doi = {10.7326/M17-0085},
    issn = {1539-3704},
    journal = {Annals of Internal Medicine},
    number = {1},
    pages = {1--7},
    pmid = {28586923},
    shortjournal = {Ann. Intern. Med.},
    title = {Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs},
    volume = {167},
    year = {2017}
    }
  • [DOI] I. Lee, B. A. Rasoul, A. S. Holub, A. Lejeune, R. A. Enke, and W. Timp, “Whole genome DNA methylation sequencing of the chicken retina, cornea and brain,” Scientific Data, vol. 4, p. sdata2017148, 2017.
    [Bibtex]
    @article{lee_whole_2017,
    abstract = {Data Descriptor},
    author = {Lee, Isac and Rasoul, Bejan A. and Holub, Ashton S. and Lejeune, Alannah and Enke, Raymond A. and Timp, Winston},
    date = {2017-10-10},
    doi = {10.1038/sdata.2017.148},
    issn = {2052-4463},
    journal = {Scientific Data},
    langid = {english},
    pages = {sdata2017148},
    rights = {2017 Nature Publishing Group},
    title = {Whole genome {DNA} methylation sequencing of the chicken retina, cornea and brain},
    url = {https://www.nature.com/articles/sdata2017148},
    volume = {4},
    year = {2017}
    }
  • [DOI] R. Luo, A. Zimin, R. Workman, Y. Fan, G. Pertea, N. Grossman, M. P. Wear, B. Jia, H. Miller, A. Casadevall, W. Timp, S. X. Zhang, and S. L. Salzberg, “First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans),” G3: Genes, Genomes, Genetics, vol. 7, iss. 11, p. 3831–3836, 2017.
    [Bibtex]
    @article{Luog3.300107.2017,
    abstract = {Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. The resulting assembly is highly contiguous, containing a total of 37,627,092 bp with over 98\% of the sequence in just 26 scaffolds. Annotation identified 8,896 protein-coding genes. Pulsed-field gel analysis suggests that this organism contains at least 7 and possibly 11 chromosomes, the two longest of which have sizes corresponding closely to the sizes of the longest scaffolds, at 6.6 and 5.7 Mb.},
    author = {Luo, Ruibang and Zimin, Aleksey and Workman, Rachael and Fan, Yunfan and Pertea, Geo and Grossman, Nina and Wear, Maggie P. and Jia, Bei and Miller, Heather and Casadevall, Arturo and Timp, Winston and Zhang, Sean X. and Salzberg, Steven L.},
    doi = {10.1534/g3.117.300107},
    journal = {G3: Genes, Genomes, Genetics},
    number = {11},
    pages = {3831--3836},
    pmcid = {PMC5677167},
    pmid = {28963165},
    publisher = {G3: Genes, Genomes, Genetics},
    title = {First Draft Genome Sequence of the Pathogenic Fungus {Lomentospora prolificans} (formerly {Scedosporium prolificans})},
    volume = {7},
    year = {2017}
    }
  • [DOI] T. Xiong, G. E. Meister, R. E. Workman, N. C. Kato, M. J. Spellberg, F. Turker, W. Timp, M. Ostermeier, and C. D. Novina, “Targeted DNA methylation in human cells using engineered dCas9-methyltransferases,” Scientific Reports, vol. 7, 2017.
    [Bibtex]
    @article{xiong_targeted_2017,
    abstract = {Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by {DNA} methylation. The advent of engineered {DNA} methyltransferases ({MTases}) to target {DNA} methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted {MTases} require clear design rules to direct site-specific {DNA} methylation and minimize the unintended effects of off-target {DNA} methylation. Here we report a targeted {MTase} composed of an artificially split {CpG} {MTase} ({sMTase}) with one fragment fused to a catalytically-inactive Cas9 ({dCas}9) that directs the functional assembly of {sMTase} fragments at the targeted {CpG} site. We precisely map {RNA}-programmed {DNA} methylation to targeted {CpG} sites as a function of distance and orientation from the protospacer adjacent motif ({PAM}). Expression of the {dCas}9-{sMTase} in mammalian cells led to predictable and efficient (up to {\textasciitilde}70\%) {DNA} methylation at targeted sites. Multiplexing {sgRNAs} enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo {MTase} tool might be used for studying mechanisms of initiation, spreading and inheritance of {DNA} methylation, and for therapeutic gene silencing.},
    author = {Xiong, Tina and Meister, Glenna E. and Workman, Rachael E. and Kato, Nathaniel C. and Spellberg, Michael J. and Turker, Fulya and Timp, Winston and Ostermeier, Marc and Novina, Carl D.},
    date = {2017-07-27},
    doi = {10.1038/s41598-017-06757-0},
    issn = {2045-2322},
    journal = {Scientific Reports},
    pmcid = {PMC5532369},
    pmid = {28751638},
    shortjournal = {Sci Rep},
    title = {Targeted {DNA} methylation in human cells using engineered {dCas}9-methyltransferases},
    url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532369/},
    volume = {7},
    year = {2017}
    }

2016

  • [DOI] A. L. Norris, R. E. Workman, Y. Fan, J. R. Eshleman, and W. Timp, “Nanopore sequencing detects structural variants in cancer,” Cancer Biology & Therapy, vol. 17, iss. 3, p. 246–253, 2016.
    [Bibtex]
    @article{norris_nanopore_2016,
    abstract = {Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length ({\textless}300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency. We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.},
    author = {Norris, Alexis L. and Workman, Rachael E. and Fan, Yunfan and Eshleman, James R. and Timp, Winston},
    doi = {10.1080/15384047.2016.1139236},
    issn = {1555-8576},
    journal = {Cancer Biology \& Therapy},
    keywords = {3rd Generation Sequencing, Cancer Diagnostics, Deletions, DNA sequencing, Inversions, nanopore sequencing, next generation sequencing, Structural variation, Translocations, tumor suppressor gene},
    month = {March},
    number = {3},
    pages = {246--253},
    pmid = {26787508},
    title = {Nanopore sequencing detects structural variants in cancer},
    volume = {17},
    year = {2016}
    }

2014

  • [DOI] W. Timp, A. M. Nice, E. M. Nelson, V. Kurz, K. McKelvey, and G. Timp, “Think Small: Nanopores for Sensing and Synthesis,” IEEE Access, vol. 2, p. 1396–1408, 2014.
    [Bibtex]
    @article{timp_think_2014,
    author = {Timp, W. and Nice, A.M. and Nelson, E.M. and Kurz, V. and McKelvey, K. and Timp, G.},
    doi = {10.1109/ACCESS.2014.2369506},
    entrysubtype = {review},
    issn = {2169-3536},
    journal = {IEEE Access},
    keywords = {AFM, Cells (biology), DNA, DNA sequencing, genomics, Nanobioscience, Nanoparticles, Nanopore, Proteins, scanning ion conductance microscopy, single cell transfection, single molecule force spectroscopy},
    month = {November},
    pages = {1396--1408},
    shorttitle = {Think {Small}},
    title = {Think {Small}: {Nanopores} for {Sensing} and {Synthesis}},
    volume = {2},
    year = {2014}
    }
  • [DOI] W. Timp, H. C. Bravo, O. G. McDonald, M. Goggins, C. Umbricht, M. Zeiger, A. P. Feinberg, and R. A. Irizarry, “Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors,” Genome Medicine, vol. 6, iss. 8, p. 61, 2014.
    [Bibtex]
    @article{timp_large_2014,
    author = {Timp, Winston and Bravo, Hector Corrada and McDonald, Oliver G. and Goggins, Michael and Umbricht, Chris and Zeiger, Martha and Feinberg, Andrew P. and Irizarry, Rafael A.},
    doi = {10.1186/s13073-014-0061-y},
    issn = {1756-994X},
    journal = {Genome Medicine},
    month = {August},
    number = {8},
    pages = {61},
    pmcid = {PMC4154522},
    pmid = {25191524},
    title = {Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors},
    volume = {6},
    year = {2014}
    }

2013

  • [DOI] V. Kurz, E. M. Nelson, N. Perry, W. Timp, and G. Timp, “Epigenetic Memory Emerging from Integrated Transcription Bursts,” Biophysical Journal, vol. 105, iss. 6, p. 1526–1532, 2013.
    [Bibtex]
    @article{kurz_epigenetic_2013,
    abstract = {Some autonomous bacteria coordinate their actions using quorum-sensing (QS) signals to affect gene expression. However, noise in the gene environment can compromise the cellular response. By exercising precise control over a cell’s genes and its microenvironment, we have studied the key positive autoregulation element by which the lux QS system integrates noisy signals into an epigenetic memory. We observed transcriptional bursting of the lux receptor in cells stimulated by near-threshold levels of QS ligand. The bursts are integrated over time into an epigenetic memory that confers enhanced sensitivity to the ligand. An emergent property of the system is manifested in pattern formation among phenotypes within a chemical gradient.},
    author = {Kurz, Volker and Nelson, Edward M. and Perry, Nicolas and Timp, Winston and Timp, Gregory},
    doi = {10.1016/j.bpj.2013.08.010},
    issn = {0006-3495},
    journal = {Biophysical Journal},
    keywords = {Acyl-Butyrolactones, Epigenesis, Genetic, Escherichia coli, Models, Genetic, Phenotype, Stochastic Processes, Transcription, Genetic},
    month = {September},
    number = {6},
    pages = {1526--1532},
    pmcid = {PMC3785891},
    pmid = {24048004},
    title = {Epigenetic {Memory} {Emerging} from {Integrated} {Transcription} {Bursts}},
    url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785891/},
    volume = {105},
    year = {2013}
    }
  • [DOI] W. Timp and A. P. Feinberg, “Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host,” Nature Reviews Cancer, vol. 13, iss. 7, p. 497–510, 2013.
    [Bibtex]
    @article{timp_cancer_2013,
    abstract = {Although at the genetic level cancer is caused by diverse mutations, epigenetic modifications are characteristic of all cancers, from apparently normal precursor tissue to advanced metastatic disease, and these epigenetic modifications drive tumour cell heterogeneity. We propose a unifying model of cancer in which epigenetic dysregulation allows rapid selection for tumour cell survival at the expense of the host. Mechanisms involve both genetic mutations and epigenetic modifications that disrupt the function of genes that regulate the epigenome itself. Several exciting recent discoveries also point to a genome-scale disruption of the epigenome that involves large blocks of DNA hypomethylation, mutations of epigenetic modifier genes and alterations of heterochromatin in cancer (including large organized chromatin lysine modifications (LOCKs) and lamin-associated domains (LADs)), all of which increase epigenetic and gene expression plasticity. Our model suggests a new approach to cancer diagnosis and therapy that focuses on epigenetic dysregulation and has great potential for risk detection and chemoprevention.},
    author = {Timp, Winston and Feinberg, Andrew P.},
    doi = {10.1038/nrc3486},
    entrysubtype = {review},
    issn = {1474-1768},
    journal = {Nature Reviews Cancer},
    keywords = {Cancer models, Chromatin Assembly and Disassembly, Epigenesis, Genetic, Epigenetics, genetics, Heterochromatin, Humans, Nature Reviews Cancer, Neoplasms},
    month = {July},
    number = {7},
    pages = {497--510},
    pmid = {23760024},
    title = {Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host},
    volume = {13},
    year = {2013}
    }

2012

  • [DOI] W. Timp, J. Comer, and A. Aksimentiev, “DNA base-calling from a nanopore using a Viterbi algorithm,” Biophysical Journal, vol. 102, iss. 10, p. L37–39, 2012.
    [Bibtex]
    @article{timp_dna_2012,
    author = {Timp, Winston and Comer, Jeffrey and Aksimentiev, Aleksei},
    doi = {10.1016/j.bpj.2012.04.009},
    issn = {1542-0086},
    journal = {Biophysical Journal},
    keywords = {Algorithms, Base Sequence, DNA, Electricity, Genome, Human, Humans, Markov Chains, Nanopores, Sequence Analysis, DNA, Signal-To-Noise Ratio},
    month = {May},
    number = {10},
    pages = {L37--39},
    pmcid = {PMC3353060},
    pmid = {22677395},
    title = {{DNA} base-calling from a nanopore using a {Viterbi} algorithm},
    volume = {102},
    year = {2012}
    }
  • [DOI] E. M. Nelson, V. Kurz, J. Shim, W. Timp, and G. Timp, “Using a Nanopore for Single Molecule Detection and Single Cell Transfection,” The Analyst, vol. 137, iss. 13, p. 3020–3027, 2012.
    [Bibtex]
    @article{nelson_using_2012,
    abstract = {We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.},
    author = {Nelson, Edward M. and Kurz, Volker and Shim, Jiwook and Timp, Winston and Timp, Gregory},
    doi = {10.1039/c2an35571j},
    issn = {0003-2654},
    journal = {The Analyst},
    month = {July},
    number = {13},
    pages = {3020--3027},
    pmcid = {PMC3384492},
    pmid = {22645737},
    title = {Using a {Nanopore} for {Single} {Molecule} {Detection} and {Single} {Cell} {Transfection}},
    url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384492/},
    urldate = {2019-04-27},
    volume = {137},
    year = {2012}
    }

2011

  • [DOI] K. D. Hansen, W. Timp, H. C. Bravo, S. Sabunciyan, B. Langmead, O. G. McDonald, B. Wen, H. Wu, Y. Liu, D. Diep, E. Briem, K. Zhang, R. A. Irizarry, and A. P. Feinberg, “Increased methylation variation in epigenetic domains across cancer types,” Nature Genetics, vol. 43, iss. 8, p. 768–775, 2011.
    [Bibtex]
    @article{hansen_increased_2011,
    abstract = {Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal, in colon, lung, breast, thyroid, and Wilms tumors, with intermediate variation in adenomas. Whole genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. These data suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer and could contribute to tumor heterogeneity.},
    author = {Hansen, Kasper Daniel and Timp, Winston and Bravo, Héctor Corrada and Sabunciyan, Sarven and Langmead, Benjamin and McDonald, Oliver G. and Wen, Bo and Wu, Hao and Liu, Yun and Diep, Dinh and Briem, Eirikur and Zhang, Kun and Irizarry, Rafael A. and Feinberg, Andrew P.},
    doi = {10.1038/ng.865},
    issn = {1061-4036},
    journal = {Nature Genetics},
    month = {June},
    number = {8},
    pages = {768--775},
    pmcid = {PMC3145050},
    pmid = {21706001},
    title = {Increased methylation variation in epigenetic domains across cancer types},
    url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145050/},
    urldate = {2019-04-27},
    volume = {43},
    year = {2011}
    }
  • [DOI] O. G. McDonald, H. Wu, W. Timp, A. Doi, and A. P. Feinberg, “Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition,” Nature Structural & Molecular Biology, vol. 18, iss. 8, p. 867–874, 2011.
    [Bibtex]
    @article{mcdonald_genome-scale_2011,
    abstract = {Epithelial-to-mesenchymal transition (EMT) is an extreme example of cell plasticity that is important for normal development, injury repair and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during EMT mediated by transforming growth factor beta. Although DNA methylation was unchanged during EMT, we found a global reduction in the heterochromatin mark H3 Lys9 dimethylation (H3K9Me2), an increase in the euchromatin mark H3 Lys4 trimethylation (H3K4Me3) and an increase in the transcriptional mark H3 Lys36 trimethylation (H3K36Me3). These changes depended largely on lysine-specific demethylase-1 (Lsd1), and loss of Lsd1 function had marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping showed that chromatin changes were mainly specific to large organized heterochromatin K9 modifications (LOCKs), which suggests that EMT is characterized by reprogramming of specific chromatin domains across the genome.},
    author = {McDonald, Oliver G. and Wu, Hao and Timp, Winston and Doi, Akiko and Feinberg, Andrew P.},
    doi = {10.1038/nsmb.2084},
    issn = {1545-9985},
    journal = {Nature Structural \& Molecular Biology},
    keywords = {Animals, Cell Line, Cell Movement, Chromatin Assembly and Disassembly, Chromatin immunoprecipitation, DNA methylation, Epigenesis, Genetic, Epithelial Cells, Epithelial-mesenchymal transition, Histones, Mesenchymal Stromal Cells, Mice, Neoplastic Stem Cells, Oxidoreductases, N-Demethylating, Transforming Growth Factor beta},
    month = {August},
    number = {8},
    pages = {867--874},
    pmcid = {PMC3150339},
    pmid = {21725293},
    title = {Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition},
    volume = {18},
    year = {2011}
    }

2010

  • [DOI] W. Timp, U. M. Mirsaidov, D. Wang, J. Comer, A. Aksimentiev, and G. Timp, “Nanopore Sequencing: Electrical Measurements of the Code of Life,” IEEE Transactions on Nanotechnology, vol. 9, iss. 3, p. 281–294, 2010.
    [Bibtex]
    @article{timp_nanopore_2010,
    abstract = {Sequencing a single molecule of deoxyribonucleic acid (DNA) using a nanopore is a revolutionary concept because it combines the potential for long read lengths ({\textgreater}5 kbp) with high speed (1 bp/10 ns), while obviating the need for costly amplification procedures due to the exquisite single molecule sensitivity. The prospects for implementing this concept seem bright. The cost savings from the removal of required reagents, coupled with the speed of nanopore sequencing places the \$1000 genome within grasp. However, challenges remain: high fidelity reads demand stringent control over both the molecular configuration in the pore and the translocation kinetics. The molecular configuration determines how the ions passing through the pore come into contact with the nucleotides, while the translocation kinetics affect the time interval in which the same nucleotides are held in the constriction as the data is acquired. Proteins like α-hemolysin and its mutants offer exquisitely precise self-assembled nanopores and have demonstrated the facility for discriminating individual nucleotides, but it is currently difficult to design protein structure ab initio, which frustrates tailoring a pore for sequencing genomic DNA. Nanopores in solid-state membranes have been proposed as an alternative because of the flexibility in fabrication and ease of integration into a sequencing platform. Preliminary results have shown that with careful control of the dimensions of the pore and the shape of the electric field, control of DNA translocation through the pore is possible. Furthermore, discrimination between different base pairs of DNA may be feasible. Thus, a nanopore promises inexpensive, reliable, high-throughput sequencing, which could thrust genomic science into personal medicine.},
    author = {Timp, Winston and Mirsaidov, Utkur M. and Wang, Deqiang and Comer, Jeff and Aksimentiev, Aleksei and Timp, Gregory},
    doi = {10.1109/TNANO.2010.2044418},
    issn = {1536-125X},
    journal = {IEEE Transactions on Nanotechnology},
    language = {eng},
    month = {May},
    number = {3},
    pages = {281--294},
    pmcid = {PMC3092306},
    pmid = {21572978},
    shorttitle = {Nanopore {Sequencing}},
    title = {Nanopore {Sequencing}: {Electrical} {Measurements} of the {Code} of {Life}},
    volume = {9},
    year = {2010}
    }

2009

  • [DOI] U. Mirsaidov, W. Timp, X. Zou, V. Dimitrov, K. Schulten, A. P. Feinberg, and G. Timp, “Nanoelectromechanics of methylated DNA in a synthetic nanopore,” Biophysical Journal, vol. 96, iss. 4, p. L32–34, 2009.
    [Bibtex]
    @article{mirsaidov_nanoelectromechanics_2009,
    abstract = {Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a {\textless}2 nm diameter pore, which we attribute to the stretching transition; this can differ by {\textgreater}1 V/20 nm depending on the methylation level, but not the DNA sequence.},
    author = {Mirsaidov, U. and Timp, W. and Zou, X. and Dimitrov, V. and Schulten, K. and Feinberg, A. P. and Timp, G.},
    doi = {10.1016/j.bpj.2008.12.3760},
    issn = {1542-0086},
    journal = {Biophysical Journal},
    keywords = {Algorithms, Base Sequence, DNA, DNA Methylation, Electrophoresis, Agar Gel, Genes, BRCA1, Insulin-Like Growth Factor II, Membranes, Artificial, Permeability, Polymerase Chain Reaction, Protein Conformation},
    language = {eng},
    month = {February},
    number = {4},
    pages = {L32--34},
    pmcid = {PMC2717226},
    pmid = {19217843},
    title = {Nanoelectromechanics of methylated {DNA} in a synthetic nanopore},
    volume = {96},
    year = {2009}
    }
  • [DOI] W. Timp, A. Levchenko, and A. P. Feinberg, “A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction,” Cell Cycle (Georgetown, Tex.), vol. 8, iss. 3, p. 383–390, 2009.
    [Bibtex]
    @article{timp_new_2009,
    abstract = {Our recent study of the mechanism by which an epigenetic alteration, loss of imprinting (LOI) of Igf2, increases tumor risk, revealed a strong relationship between IGF2 dosage, the dynamics of signaling along the IGF2 axis, cell proliferation and tumor risk.(1) Colon epithelia in a mouse model with LOI of Igf2 showed increased sensitivity to IGF1R blockade and abrogation of premalignant lesion development in LOI(+) mice. These results are consistent with the epigenetic progenitor model of cancer,(2) in which epigenetic changes precede and heighten risk of cancer in response to oncogenic mutations. Thus, one can envision a highly targeted and focused chemoprevention strategy targeted to signaling pathways in nonmalignant cells that have undergone an epigenetic lesion, rather than a broad approach toward reversing epigenetic lesions that may have unintended consequences affecting the whole epigenome.},
    author = {Timp, Winston and Levchenko, Andre and Feinberg, Andrew P.},
    doi = {10.4161/cc.8.3.7542},
    issn = {1551-4005},
    journal = {Cell Cycle (Georgetown, Tex.)},
    keywords = {Animals, beta Catenin, Colonic Neoplasms, Epigenesis, Genetic, Humans, Insulin-Like Growth Factor II, Neoplasms, Signal Transduction, Wnt Proteins},
    language = {eng},
    month = {February},
    number = {3},
    pages = {383--390},
    pmcid = {PMC6275123},
    pmid = {19177016},
    title = {A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction},
    volume = {8},
    year = {2009}
    }
  • [DOI] W. Timp, U. Mirsaidov, P. Matsudaira, and G. Timp, “Jamming prokaryotic cell-to-cell communications in a model biofilm,” Lab on a Chip, vol. 9, iss. 7, p. 925–934, 2009.
    [Bibtex]
    @article{timp_jamming_2009,
    abstract = {We report on the physical parameters governing prokaryotic cell-to-cell signaling in a model biofilm. The model biofilm is comprised of bacteria that are genetically engineered to transmit and receive quorum-sensing (QS) signals. The model is formed using arrays of time-shared, holographic optical traps in conjunction with microfluidics to precisely position bacteria, and then encapsulated within a hydrogel that mimics the extracellular matrix. Using fluorescent protein reporters functionally linked to QS genes, we assay the intercellular signaling. We find that there isn't a single cell density for which QS-regulated genes are induced or repressed. On the contrary, cell-to-cell signaling is largely governed by diffusion, and is acutely sensitive to mass-transfer to the surroundings and the cell location. These observations are consistent with the view that QS-signals act simply as a probe measuring mixing, flow, or diffusion in the microenvironment of the cell.},
    author = {Timp, Winston and Mirsaidov, Utkur and Matsudaira, Paul and Timp, Gregory},
    doi = {10.1039/b810157d},
    issn = {1473-0197},
    journal = {Lab on a Chip},
    keywords = {Bacteria, Bacterial Proteins, Biofilms, Cell Communication, Diffusion, Equipment Design, Fluorescein, Fluorescent Dyes, Gene Expression Regulation, Bacterial, Genes, Reporter, Genetic Engineering, Green Fluorescent Proteins, Holography, Hydrogel, Polyethylene Glycol Dimethacrylate, Microfluidics, Models, Biological, Oligonucleotide Array Sequence Analysis, Optical Tweezers, Prokaryotic Cells, Quorum Sensing, Signal Transduction},
    language = {eng},
    month = {April},
    number = {7},
    pages = {925--934},
    pmid = {19294303},
    title = {Jamming prokaryotic cell-to-cell communications in a model biofilm},
    volume = {9},
    year = {2009}
    }

2008

  • [DOI] U. Mirsaidov, J. Scrimgeour, W. Timp, K. Beck, M. Mir, P. Matsudaira, and G. Timp, “Live cell lithography: using optical tweezers to create synthetic tissue,” Lab on a Chip, vol. 8, iss. 12, p. 2174–2181, 2008.
    [Bibtex]
    @article{mirsaidov_live_2008,
    abstract = {We demonstrate a new method for creating synthetic tissue that has the potential to capture the three-dimensional (3D) complexity of a multi-cellular organism with submicron precision. Using multiple laminar fluid flows in a microfluidic network, we convey cells to an assembly area where multiple, time-shared optical tweezers are used to organize them into a complex array. The cells are then encapsulated in a 30 microm x 30 microm x 45 microm volume of photopolymerizable hydrogel that mimicks an extra-cellular matrix. To extend the size, shape and constituency of the array without loss of viability, we then step to an adjacent location while maintaining registration with the reference array, and repeat the process. Using this step-and-repeat method, we formed a heterogeneous array of E. coli genetically engineered with a lac switch that is functionally linked to fluorescence reporters. We then induced the array using ligands through a microfluidic network and followed the space-time development of the fluorescence to evaluate viability and metabolic activity.},
    author = {Mirsaidov, Utkur and Scrimgeour, Jan and Timp, Winston and Beck, Kaethe and Mir, Mustafa and Matsudaira, Paul and Timp, Gregory},
    doi = {10.1039/b807987k},
    issn = {1473-0197},
    journal = {Lab on a Chip},
    keywords = {Biomimetic Materials, Escherichia, Hydrogel, Polyethylene Glycol Dimethacrylate, Microfluidics, Optical Tweezers, Surface Properties},
    language = {eng},
    month = {December},
    number = {12},
    pages = {2174--2181},
    pmid = {19023484},
    shorttitle = {Live cell lithography},
    title = {Live cell lithography: using optical tweezers to create synthetic tissue},
    volume = {8},
    year = {2008}
    }
  • [DOI] U. Mirsaidov, W. Timp, K. Timp, M. Mir, P. Matsudaira, and G. Timp, “Optimal optical trap for bacterial viability,” Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics, vol. 78, iss. 2 Pt 1, p. 21910, 2008.
    [Bibtex]
    @article{mirsaidov_optimal_2008,
    abstract = {Optical trapping is a powerful tool for the micromanipulation of living cells--especially bacteria--but photodamage induced by the laser beam can adversely affect viability. We have explored optical trapping conditions in the near infrared (840-930 nm) that preserve the viability of E. coli, as measured by gene expression of green fluorescent protein. We have found that time-sharing the optical traps, i.e., dwelling only 10 micros-1 ms on the cell, improves viability relative to continuous wave (CW) exposure for the same exposure time. We have also observed that similar to CW traps the photodamage in a time-shared trap depends weakly on wavelength, but linearly on peak power, implying an effect induced by single photon absorption. Taken altogether, integrating the exposure time and peak power, the data indicate that there is a lethal energy dose of about 5 J for E. coli. Thus a single parameter--the energy--can be used to describe the limitation on viability.},
    author = {Mirsaidov, Utkur and Timp, Winston and Timp, Kaethe and Mir, Mustafa and Matsudaira, Paul and Timp, Gregory},
    doi = {10.1103/PhysRevE.78.021910},
    issn = {1539-3755},
    journal = {Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics},
    keywords = {Escherichia coli, Green Fluorescent Proteins, Microbial Viability, Optical Tweezers, Plasmids},
    language = {eng},
    month = {August},
    number = {2 Pt 1},
    pages = {021910},
    pmid = {18850868},
    title = {Optimal optical trap for bacterial viability},
    volume = {78},
    year = {2008}
    }
  • [DOI] W. Timp and P. Matsudaira, “Chapter 14: Electron microscopy of hydrated samples,” Methods in Cell Biology, vol. 89, p. 391–407, 2008.
    [Bibtex]
    @article{timp_chapter_2008,
    abstract = {Conventional electron microscopy offers a substantial resolution advantage over light microscopy, but requires difficult and often destructive preparation techniques. Recent advances in electron microscopy allow for imaging of hydrated samples, retaining the resolution advantage while removing the difficulty in preparation. Two new techniques, environmental scanning electron microscopy and wet electron microscopy offer this advantage, allowing for new possibilities in biological imaging.},
    author = {Timp, Winston and Matsudaira, Paul},
    doi = {10.1016/S0091-679X(08)00614-6},
    issn = {0091-679X},
    journal = {Methods in Cell Biology},
    keywords = {Animals, Cell Line, Chondrocytes, Epithelial Cells, Microscopy, Electron, Scanning, Water},
    language = {eng},
    pages = {391--407},
    pmid = {19118683},
    shorttitle = {Chapter 14},
    title = {Chapter 14: {Electron} microscopy of hydrated samples},
    volume = {89},
    year = {2008}
    }

2007

  • [DOI] B. J. Zeskind, C. D. Jordan, W. Timp, L. Trapani, G. Waller, V. Horodincu, D. J. Ehrlich, and P. Matsudaira, “Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy,” Nature Methods, vol. 4, iss. 7, p. 567–569, 2007.
    [Bibtex]
    @article{zeskind_nucleic_2007,
    abstract = {We developed a deep-ultraviolet (UV) microscope capable of imaging cell mitosis and motility at 280 nm for 45 min with minimal UV-induced toxicity, and for 6 h before the onset of visible cell death in cultured human and mouse cells. Combined with computational methods that convert the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid mass, protein mass and fluorescence yield in unlabeled cells.},
    author = {Zeskind, Benjamin J. and Jordan, Caroline D. and Timp, Winston and Trapani, Linda and Waller, Guichy and Horodincu, Victor and Ehrlich, Daniel J. and Matsudaira, Paul},
    doi = {10.1038/nmeth1053},
    issn = {1548-7091},
    journal = {Nature Methods},
    keywords = {Animals, Cell Movement, Humans, Image Processing, Computer-Assisted, Mice, Microscopy, Ultraviolet, Mitosis, Nucleic Acids, Proteins},
    language = {eng},
    month = {July},
    number = {7},
    pages = {567--569},
    pmid = {17546037},
    title = {Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy},
    volume = {4},
    year = {2007}
    }
  • [DOI] A. Kaneda, C. J. Wang, R. Cheong, W. Timp, P. Onyango, B. Wen, C. A. Iacobuzio-Donahue, R. Ohlsson, R. Andraos, M. A. Pearson, A. A. Sharov, D. L. Longo, M. S. H. Ko, A. Levchenko, and A. P. Feinberg, “Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, iss. 52, p. 20926–20931, 2007.
    [Bibtex]
    @article{kaneda_enhanced_2007,
    abstract = {Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(-) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(-) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60\% increase in premalignant aberrant crypt foci (ACF) formation over LOI(-) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30\% lower even than found in exposed LOI(-) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are "IGF-II addicted" and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.},
    author = {Kaneda, Atsushi and Wang, Chiaochun J. and Cheong, Raymond and Timp, Winston and Onyango, Patrick and Wen, Bo and Iacobuzio-Donahue, Christine A. and Ohlsson, Rolf and Andraos, Rita and Pearson, Mark A. and Sharov, Alexei A. and Longo, Dan L. and Ko, Minoru S. H. and Levchenko, Andre and Feinberg, Andrew P.},
    doi = {10.1073/pnas.0710359105},
    issn = {1091-6490},
    journal = {Proceedings of the National Academy of Sciences of the United States of America},
    keywords = {Animals, Anticarcinogenic Agents, Azoxymethane, Cell Proliferation, DNA Methylation, Epigenesis, Genetic, Gene Expression Regulation, Neoplastic, Genomic Imprinting, Insulin-Like Growth Factor II, Mice, Mice, Inbred C57BL, Neoplasms, Oligonucleotide Array Sequence Analysis, Pyrimidines, Pyrroles, Signal Transduction},
    language = {eng},
    month = {December},
    number = {52},
    pages = {20926--20931},
    pmcid = {PMC2409243},
    pmid = {18087038},
    title = {Enhanced sensitivity to {IGF}-{II} signaling links loss of imprinting of {IGF}2 to increased cell proliferation and tumor risk},
    volume = {104},
    year = {2007}
    }

2006

  • [DOI] W. Timp, N. Watson, A. Sabban, O. Zik, and P. Matsudaira, “Wet electron microscopy with quantum dots,” BioTechniques, vol. 41, iss. 3, p. 295–298, 2006.
    [Bibtex]
    @article{timp_wet_2006,
    abstract = {Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.},
    author = {Timp, Winston and Watson, Nicki and Sabban, Alon and Zik, Ory and Matsudaira, Paul},
    doi = {10.2144/000112239},
    issn = {0736-6205},
    journal = {BioTechniques},
    keywords = {Actins, Animals, Cell Line, Cytoskeleton, Fluorescent Dyes, Image Processing, Computer-Assisted, Macrophages, Mice, Microscopy, Electron, Quantum Dots, Scattering, Radiation, Time Factors},
    language = {eng},
    month = {September},
    number = {3},
    pages = {295--298},
    pmid = {16989089},
    title = {Wet electron microscopy with quantum dots},
    volume = {41},
    year = {2006}
    }
  • [DOI] G. M. Akselrod, W. Timp, U. Mirsaidov, Q. Zhao, C. Li, R. Timp, K. Timp, P. Matsudaira, and G. Timp, “Laser-guided assembly of heterotypic three-dimensional living cell microarrays,” Biophysical Journal, vol. 91, iss. 9, p. 3465–3473, 2006.
    [Bibtex]
    @article{akselrod_laser-guided_2006,
    abstract = {We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation {\textless}400 nm. In particular, we have assembled networks of Swiss 3T3 fibroblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power {\textless}2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.},
    author = {Akselrod, G. M. and Timp, W. and Mirsaidov, U. and Zhao, Q. and Li, C. and Timp, R. and Timp, K. and Matsudaira, P. and Timp, G.},
    doi = {10.1529/biophysj.106.084079},
    issn = {0006-3495},
    journal = {Biophysical Journal},
    keywords = {Biological Assay, Cell Culture Techniques, Cells, Cultured, Equipment Design, Equipment Failure Analysis, Humans, Lasers, Microarray Analysis, Microfluidic Analytical Techniques},
    language = {eng},
    month = {November},
    number = {9},
    pages = {3465--3473},
    pmcid = {PMC1614477},
    pmid = {16891375},
    title = {Laser-guided assembly of heterotypic three-dimensional living cell microarrays},
    volume = {91},
    year = {2006}
    }

1999

  • [DOI] M. L. O’Malley, G. L. Timp, W. Timp, S. V. Moccio, J. P. Garno, and R. N. Kleiman, “Electrical simulation of scanning capacitance microscopy imaging of the pn junction with semiconductor probe tips,” Applied Physics Letters, vol. 74, iss. 24, p. 3672–3674, 1999.
    [Bibtex]
    @article{omalley_electrical_1999,
    author = {O’Malley, M. L. and Timp, G. L. and Timp, W. and Moccio, S. V. and Garno, J. P. and Kleiman, R. N.},
    doi = {10.1063/1.123217},
    issn = {0003-6951},
    journal = {Applied Physics Letters},
    month = {June},
    number = {24},
    pages = {3672--3674},
    title = {Electrical simulation of scanning capacitance microscopy imaging of the pn junction with semiconductor probe tips},
    url = {https://aip.scitation.org/doi/abs/10.1063/1.123217},
    urldate = {2019-04-27},
    volume = {74},
    year = {1999}
    }