Publications

@article{workman_single-molecule_2018b,

title = {Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris},

author = {Rachael E Workman and Alexander M Myrka and William G Wong and Elizabeth Tseng and Kenneth C Welch and Winston Timp},

url = {https://academic.oup.com/gigascience/article/7/3/giy009/4860431},

doi = {10.1093/gigascience/giy009},

year  = {2018},

date = {2018-03-01},

urldate = {2018-03-01},

journal = {GigaScience},

volume = {7},

issue = {3},

pages = {giy009},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.1371/journal.pone.0209408b,

title = {Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria},

author = {Tina Xiong and Dahlia Rohm and Rachael E Workman and Lauren Roundtree and Carl D Novina and Winston Timp and Marc Ostermeier},

url = {https://doi.org/10.1371/journal.pone.0209408},

doi = {10.1371/journal.pone.0209408},

year  = {2018},

date = {2018-01-01},

journal = {PLoS One},

volume = {13},

number = {12},

pages = {1-18},

publisher = {Public Library of Science},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{lee_whole_2017b,

title = {Whole genome DNA methylation sequencing of the chicken retina, cornea and brain},

author = {Isac Lee and Bejan A Rasoul and Ashton S Holub and Alannah Lejeune and Raymond A Enke and Winston Timp},

url = {https://www.nature.com/articles/sdata2017148},

doi = {10.1038/sdata.2017.148},

issn = {2052-4463},

year  = {2017},

date = {2017-10-10},

urldate = {2017-10-10},

journal = {Scientific Data},

volume = {4},

issue = {1},

pages = {1-8},

abstract = {Data Descriptor},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{xiong_targeted_2017b,

title = {Targeted DNA methylation in human cells using engineered dCas9-methyltransferases},

author = {Tina Xiong and Glenna E Meister and Rachael E Workman and Nathaniel C Kato and Michael J Spellberg and Fulya Turker and Winston Timp and Marc Ostermeier and Carl D Novina},

url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532369/},

doi = {10.1038/s41598-017-06757-0},

issn = {2045-2322},

year  = {2017},

date = {2017-07-27},

urldate = {2017-07-27},

journal = {Scientific Reports},

volume = {7},

issue = {1},

pages = {1-14},

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 (dCas9) 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 dCas9-sMTase in mammalian cells led to predictable and efficient (up to textasciitilde70%) 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kandathil_presence_2017b,

title = {Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs},

author = {Abraham J Kandathil and Florian P Breitwieser and Jaiprasath Sachithanandham and Matthew Robinson and Shruti H Mehta and Winston Timp and Steven L Salzberg and David L Thomas and Ashwin Balagopal},

doi = {10.7326/M17-0085},

issn = {1539-3704},

year  = {2017},

date = {2017-06-06},

journal = {Annals of Internal Medicine},

volume = {167},

number = {1},

pages = {1--7},

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: NCT01285050) 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{simpson_detecting_2017b,

title = {Detecting DNA cytosine methylation using nanopore sequencing},

author = {Jared T Simpson and Rachael E Workman and P C Zuzarte and Matei David and L J Dursi and Winston Timp},

url = {http://www.nature.com/nmeth/journal/v14/n4/full/nmeth.4184.html},

doi = {10.1038/nmeth.4184},

issn = {1548-7091},

year  = {2017},

date = {2017-04-01},

journal = {Nature Methods},

volume = {14},

number = {4},

pages = {407--410},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Luog3.300107.2017b,

title = {First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans)},

author = {Ruibang Luo and Aleksey Zimin and Rachael Workman and Yunfan Fan and Geo Pertea and Nina Grossman and Maggie P Wear and Bei Jia and Heather Miller and Arturo Casadevall and Winston Timp and Sean X Zhang and Steven L Salzberg},

doi = {10.1534/g3.117.300107},

year  = {2017},

date = {2017-01-01},

journal = {G3: Genes, Genomes, Genetics},

volume = {7},

number = {11},

pages = {3831--3836},

publisher = {G3: Genes, Genomes, Genetics},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{credle_multiplexed_2017b,

title = {Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization},

author = {Joel J Credle and Christopher Y Itoh and Tiezheng Yuan and Rajni Sharma and Erick R Scott and Rachael E Workman and Yunfan Fan and Franck Housseau and Nicolas J Llosa and Robert W Bell and Heather Miller and Sean X Zhang and Winston Timp and Benjamin H Larman},

doi = {10.1093/nar/gkx471},

year  = {2017},

date = {2017-01-01},

journal = {Nucleic Acids Research},

volume = {45},

number = {14},

pages = {e128--e128},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{norris_nanopore_2016b,

title = {Nanopore sequencing detects structural variants in cancer},

author = {Alexis L Norris and Rachael E Workman and Yunfan Fan and James R Eshleman and Winston Timp},

doi = {10.1080/15384047.2016.1139236},

issn = {1555-8576},

year  = {2016},

date = {2016-03-01},

journal = {Cancer Biology & Therapy},

volume = {17},

number = {3},

pages = {246--253},

abstract = {Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (textless300 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_think_2014b,

title = {Think Small: Nanopores for Sensing and Synthesis},

author = {Winston Timp and Allison M Nice and E M Nelson and V Kurz and K McKelvey and Gregory Timp},

doi = {10.1109/ACCESS.2014.2369506},

issn = {2169-3536},

year  = {2014},

date = {2014-11-01},

journal = {IEEE Access},

volume = {2},

pages = {1396--1408},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_large_2014b,

title = {Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors},

author = {Winston Timp and Hector Corrada Bravo and Oliver G McDonald and Michael Goggins and Chris Umbricht and Martha Zeiger and Andrew P Feinberg and Rafael A Irizarry},

doi = {10.1186/s13073-014-0061-y},

issn = {1756-994X},

year  = {2014},

date = {2014-08-01},

journal = {Genome Medicine},

volume = {6},

number = {8},

pages = {61},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kurz_epigenetic_2013b,

title = {Epigenetic Memory Emerging from Integrated Transcription Bursts},

author = {Volker Kurz and Edward M Nelson and Nicolas Perry and Winston Timp and Gregory Timp},

url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785891/},

doi = {10.1016/j.bpj.2013.08.010},

issn = {0006-3495},

year  = {2013},

date = {2013-09-01},

journal = {Biophysical Journal},

volume = {105},

number = {6},

pages = {1526--1532},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_cancer_2013b,

title = {Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host},

author = {Winston Timp and Andrew P Feinberg},

doi = {10.1038/nrc3486},

issn = {1474-1768},

year  = {2013},

date = {2013-07-01},

journal = {Nature Reviews Cancer},

volume = {13},

number = {7},

pages = {497--510},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nelson_using_2012b,

title = {Using a Nanopore for Single Molecule Detection and Single Cell Transfection},

author = {Edward M Nelson and Volker Kurz and Jiwook Shim and Winston Timp and Gregory Timp},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384492/},

doi = {10.1039/c2an35571j},

issn = {0003-2654},

year  = {2012},

date = {2012-07-01},

journal = {The Analyst},

volume = {137},

number = {13},

pages = {3020--3027},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_dna_2012b,

title = {DNA base-calling from a nanopore using a Viterbi algorithm},

author = {Winston Timp and Jeffrey Comer and Aleksei Aksimentiev},

doi = {10.1016/j.bpj.2012.04.009},

issn = {1542-0086},

year  = {2012},

date = {2012-05-01},

journal = {Biophysical Journal},

volume = {102},

number = {10},

pages = {L37--39},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mcdonald_genome-scale_2011b,

title = {Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition},

author = {Oliver G McDonald and Hao Wu and Winston Timp and Akiko Doi and Andrew P Feinberg},

doi = {10.1038/nsmb.2084},

issn = {1545-9985},

year  = {2011},

date = {2011-08-01},

journal = {Nature Structural & Molecular Biology},

volume = {18},

number = {8},

pages = {867--874},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{hansen_increased_2011b,

title = {Increased methylation variation in epigenetic domains across cancer types},

author = {Kasper Daniel Hansen and Winston Timp and Héctor Corrada Bravo and Sarven Sabunciyan and Benjamin Langmead and Oliver G McDonald and Bo Wen and Hao Wu and Yun Liu and Dinh Diep and Eirikur Briem and Kun Zhang and Rafael A Irizarry and Andrew P Feinberg},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145050/},

doi = {10.1038/ng.865},

issn = {1061-4036},

year  = {2011},

date = {2011-06-01},

urldate = {2019-04-27},

journal = {Nature Genetics},

volume = {43},

number = {8},

pages = {768--775},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_nanopore_2010b,

title = {Nanopore Sequencing: Electrical Measurements of the Code of Life},

author = {Winston Timp and Utkur M Mirsaidov and Deqiang Wang and Jeff Comer and Aleksei Aksimentiev and Gregory Timp},

doi = {10.1109/TNANO.2010.2044418},

issn = {1536-125X},

year  = {2010},

date = {2010-05-01},

journal = {IEEE Transactions on Nanotechnology},

volume = {9},

number = {3},

pages = {281--294},

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 (textgreater5 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_jamming_2009b,

title = {Jamming prokaryotic cell-to-cell communications in a model biofilm},

author = {Winston Timp and Utkur Mirsaidov and Paul Matsudaira and Gregory Timp},

doi = {10.1039/b810157d},

issn = {1473-0197},

year  = {2009},

date = {2009-04-01},

journal = {Lab on a Chip},

volume = {9},

number = {7},

pages = {925--934},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_new_2009b,

title = {A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction},

author = {Winston Timp and Andre Levchenko and Andrew P Feinberg},

doi = {10.4161/cc.8.3.7542},

issn = {1551-4005},

year  = {2009},

date = {2009-02-01},

journal = {Cell Cycle (Georgetown, Tex.)},

volume = {8},

number = {3},

pages = {383--390},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mirsaidov_nanoelectromechanics_2009b,

title = {Nanoelectromechanics of methylated DNA in a synthetic nanopore},

author = {U Mirsaidov and W Timp and X Zou and V Dimitrov and K Schulten and A P Feinberg and G Timp},

doi = {10.1016/j.bpj.2008.12.3760},

issn = {1542-0086},

year  = {2009},

date = {2009-02-01},

journal = {Biophysical Journal},

volume = {96},

number = {4},

pages = {L32--34},

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 textless2 nm diameter pore, which we attribute to the stretching transition; this can differ by textgreater1 V/20 nm depending on the methylation level, but not the DNA sequence.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mirsaidov_live_2008b,

title = {Live cell lithography: using optical tweezers to create synthetic tissue},

author = {Utkur Mirsaidov and Jan Scrimgeour and Winston Timp and Kaethe Beck and Mustafa Mir and Paul Matsudaira and Gregory Timp},

doi = {10.1039/b807987k},

issn = {1473-0197},

year  = {2008},

date = {2008-12-01},

journal = {Lab on a Chip},

volume = {8},

number = {12},

pages = {2174--2181},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mirsaidov_optimal_2008b,

title = {Optimal optical trap for bacterial viability},

author = {Utkur Mirsaidov and Winston Timp and Kaethe Timp and Mustafa Mir and Paul Matsudaira and Gregory Timp},

doi = {10.1103/PhysRevE.78.021910},

issn = {1539-3755},

year  = {2008},

date = {2008-08-01},

journal = {Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics},

volume = {78},

number = {2 Pt 1},

pages = {021910},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_chapter_2008b,

title = {Chapter 14: Electron microscopy of hydrated samples},

author = {Winston Timp and Paul Matsudaira},

doi = {10.1016/S0091-679X(08)00614-6},

issn = {0091-679X},

year  = {2008},

date = {2008-01-01},

journal = {Methods in Cell Biology},

volume = {89},

pages = {391--407},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kaneda_enhanced_2007b,

title = {Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk},

author = {Atsushi Kaneda and Chiaochun J Wang and Raymond Cheong and Winston Timp and Patrick Onyango and Bo Wen and Christine A Iacobuzio-Donahue and Rolf Ohlsson and Rita Andraos and Mark A Pearson and Alexei A Sharov and Dan L Longo and Minoru S H Ko and Andre Levchenko and Andrew P Feinberg},

doi = {10.1073/pnas.0710359105},

issn = {1091-6490},

year  = {2007},

date = {2007-12-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {104},

number = {52},

pages = {20926--20931},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{zeskind_nucleic_2007b,

title = {Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy},

author = {Benjamin J Zeskind and Caroline D Jordan and Winston Timp and Linda Trapani and Guichy Waller and Victor Horodincu and Daniel J Ehrlich and Paul Matsudaira},

doi = {10.1038/nmeth1053},

issn = {1548-7091},

year  = {2007},

date = {2007-07-01},

journal = {Nature Methods},

volume = {4},

number = {7},

pages = {567--569},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{akselrod_laser-guided_2006b,

title = {Laser-guided assembly of heterotypic three-dimensional living cell microarrays},

author = {G M Akselrod and W Timp and U Mirsaidov and Q Zhao and C Li and R Timp and K Timp and P Matsudaira and G Timp},

doi = {10.1529/biophysj.106.084079},

issn = {0006-3495},

year  = {2006},

date = {2006-11-01},

journal = {Biophysical Journal},

volume = {91},

number = {9},

pages = {3465--3473},

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 textless400 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 textless2 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_wet_2006b,

title = {Wet electron microscopy with quantum dots},

author = {Winston Timp and Nicki Watson and Alon Sabban and Ory Zik and Paul Matsudaira},

doi = {10.2144/000112239},

issn = {0736-6205},

year  = {2006},

date = {2006-09-01},

journal = {BioTechniques},

volume = {41},

number = {3},

pages = {295--298},

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{omalley_electrical_1999b,

title = {Electrical simulation of scanning capacitance microscopy imaging of the pn junction with semiconductor probe tips},

author = {M L O’Malley and G L Timp and W Timp and S V Moccio and J P Garno and R N Kleiman},

url = {https://aip.scitation.org/doi/abs/10.1063/1.123217},

doi = {10.1063/1.123217},

issn = {0003-6951},

year  = {1999},

date = {1999-06-01},

urldate = {2019-04-27},

journal = {Applied Physics Letters},

volume = {74},

number = {24},

pages = {3672--3674},

keywords = {},

pubstate = {published},

tppubtype = {article}

}