Steven Poon

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source...

Essential features of cancer tissue cellular heterogeneity such as negatively selected genome topologies, sub-clonal mutation patterns and genome replication states can only effectively be studied by sequencing single-cell genomes at scale and high fidelity. Using an amplification-free single-cell genome sequencing approach implemented on commodity...

We have reported telomere attrition in β and α cells of the pancreas in elderly patients with type 2 diabetes, but it has not been explored how the telomere lengths of these islet cells change according to age in normal subjects. To examine the telomere lengths of β and α cells in individuals without diabetes across a wide range of ages, we conduct...

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity an...

Primers Used for RT-PCR. (DOCX)

CABYR Knockdown sensitizes A549 and H460 cells to various concentrations of cisplatin. Cells were seeded and transfected as described in the Methods section for colony formation assays. Different concentrations of cisplatin were added the following day for 24 hours. The cells were then harvested, trypsinized, and seeded at 500 cells/well and incuba...

Differentially expressed genes are enriched in p53 signaling. Hierarchical clustering of the differentially expressed genes reveals pathway enrichment in p53 signaling, ascorbate and aldarate metabolism, and pentose and glucoronate interconversions. (TIF)

GO Enrichment Analysis of Over-Expressed Genes Based on Biological Process Ontology. (DOCX)

siRNA Screen Results for the Nucleotide Excision Repair Pathway. Genes that appeared to be cisplatin-potentiating targets according to our analysis criteria are highlighted in green. Genes that are highlighted in red are those that displayed lethality upon gene silencing via siRNA. (DOCX)

Background: Telomeres play a key role in preventing chromosomal instability. There is a striking link between advanced age and increased incidence of pancreatic cancer, suggesting a combined effect of mutation load, epigenetic regulation, telomere dysfunction, and an altered stromal milieu. Pancreatic intraepithelial neoplasia (PanIN) is a major pr...

IntroductionThe extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. Here we applied an unbiased approach to understanding the functional role of signaling molecules in several models of normal physiological growth and transl...

Along with the increasing need for living-donor liver transplantation (LDLT), the issue of organ shortage has become a serious problem. Therefore, the use of organs from elderly donors has been increasing. While the short-term results of LDLT have greatly improved, problems affecting the long-term outcome of transplant patients remain unsolved. Fur...

Aim Process automation is an important requirement for streamlining and standardizing technical procedures, such as cell isolation, in a routine laboratory. We have previously developed RoboSep™, the first instrument to fully automate the cell labeling and separation steps of an immunomagnetic cell isolation procedure. RoboSep™ uses EasySep™ reagen...

Trisomies 18 and 21 are genetic disorders in which cells possess an extra copy of each of the relevant chromosomes. Individuals with these disorders who survive birth generally have a shortened life expectancy. As telomeres are known to play an important role in the maintenance of genomic integrity by protecting the chromosomal ends, we conducted a...

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clon...

Chromoendoscopy with Lugol iodine staining provides important information on the development of squamous cell carcinoma (SCC). In particular, distinct iodine-unstained lesions (DIULs) larger than 10 mm show a high prevalence in high-grade intraepithelial neoplasia. It has also been reported that inactive ALDH2*1/*2 and less-active ADH1B*1/*1, and s...

Combination of Diameter and Multiplicity of DIULs in NTCRs. (DOCX)

Combination of ALDH2 and ADH1B genotypes in NTCRs. (DOCX)

Purpose: Evaluation of the relationships existing among 3 histologic types of urothelial tumors, chromosomal instability, and telomere length. Patients and methods: We examined 37 consecutive cases of papillary urothelial neoplasm, from which 26 (70.3%) were suitable for karyotype analysis, comprising 7 papillary urothelial neoplasms of low mali...

The human epidermal growth factor receptor 2 (HER2) proto-oncogene plays an important role in the development and progression of breast and gastric cancer. Monitoring of the HER2 status and treatment with trastuzumab was performed initially in breast cancer, and subsequently in gastric cancer. However, the HER2 status of thyroid cancer remains unex...

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater...

Comparison of median telomere length (MTL) in CD45RA+CD20− “Naïve” T lymphocytes and other nucleated blood cell types from the same individual over four distinct age groups. This table complements Figure 2 and Figure S2A. It displays the correlative r values between paired cell population telomere length values. Age-related changes in the relations...

Comparison between leukocyte telomere length data sets and between statistical models over the study period. A. In the first study period, median lymphocyte and granulocyte telomere length measurements determined by flow FISH in 391 healthy individuals from birth (umbilical cord blood) to 102 years of age (blue dots) with reference curves derived f...

Leukocyte telomere lengths of non-carrier siblings or parents of telomerase deficient individuals. The telomere length distribution in healthy individuals (Figure 2) was used to plot the median telomere length values obtained with cells from: leukocytes from 8 non-carrier siblings (cyan), 7 non-carrier parents (orange) of telomerase-deficient patie...

Gender differences in telomere length between leukocyte subpopulations. Median telomere length values in leukocyte subpopulations from 835 healthy females and males ranging from newborns (umbilical cord blood) to hundred years of age determined by flow FISH. The following white blood cell subtypes were tested: lymphocytes, granulocytes, B lymphocyt...

Complete telomere length data sets from healthy individuals. Leukocyte median telomere length data (MTL, kb) were collected over an eight year period and analyzed on two occasions (first analysis after 391 samples and the present analysis after another 445 samples). For the first set of data, freshly isolated nucleated blood cells were used whereas...

Telomere length cell subset correlations at different age ranges. This table complements Figure 2 and Figure S2. It displays the correlative r values between paired cell population telomere length values. The cell population chosen as a reference has a set value of 1. (DOC)

ANOVA results summary table on the effect of gender on cord blood telomere length measurements. Effect of gender test of cord blood samples, in reference to Figure 3A; One way ANOVA with Tukey's multiple comparison test (Table S4). (DOC)

ANOVA post test results summary table on the effect of gender on cord blood telomere length measurements. One way ANOVA with Tukey's multiple comparison test results of the effect of gender on cord blood telomere length measurements. (DOC)

ANOVA results summary table on the effects of gender on telomere length measurements. This table summarizes the results comparing the two linear models (an estimate of the entire population) and an estimate of where factor “X” (gender for example) was taken into consideration and showed a significant difference. The following factors were tested: e...

Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduc...

Liver transplantation for biliary atresia is indicated whenever a Kasai portoenterostomy is considered unfeasible. However, the timing of liver transplantation in biliary atresia has not been precisely defined. Excessive shortening of hepatocellular telomeres may occur in patients with biliary atresia, and therefore, telomere length could be a pred...

A precancerous condition is a lesion that, if left untreated, leads to cancer or can be induced to become malignant. In the oral region, leukoplakia is a lesion that has been regarded as precancerous. In cases of oral carcinoma, we have frequently noticed that a type of leukoplakia histologically demonstrating hyper-orthokeratosis and mild atypia (...

The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated...

Supplemental Figure 1. Volcano plots from the Affymetrix Exon 1.0 ST Array. Genes that were considered for further analysis are represented by yellow squares. The x-axis is the fold change for comparison groups that are indicated on the left side of the figures. The y-axis is the p-value among biological replicates.

Supplemental Table 1. The characteristics and probe locations of the outliers from the volcano plots representing the Affymetrix results.

Supplemental Table 4. Characteristics of the hormonally treated mice. Lists the characteristics of the mice used in the T response cohort.

Supplemental Figure 2. GKO hypothalamic transcription networks merged. The top three networks of GKO hypothalamic transcription merged.

Supplemental Figure 4. GKO and KKO hypothalamic transcription networks. The top three networks for both GKO and KKO hypothalamic transcription shown separately.

Supplemental Figure 3. KKO hypothalamic transcription networks merged. The top three networks of KKO hypothalamic transcription merged.

Supplemental Table 2. Genes carried forward for QPCR validation from initial array analysis. Lists the 95 genes assessed by QPCR that were chosen from the initial array analysis.

Supplemental Tables 5-12. These tables list the complete model results for the four classes of variation for each genotype: (i) purely hormone-dependent transcription (ii) purely genotype-dependent transcription (iii) hormone and genotype-dependent transcription but with no interaction between these variables and (iv) hormone and genotype-dependent...

Supplemental Table 3. Genes carried forward into the T response QPCR analysis. Lists the 48 genes assessed by QPCR using the T response cohort of mice.

Supplemental Figure 5. Tmem144 in situ hybridization in the hypothalamus of WT and KKO male mice. Tmem144 in situ hybridization in the ARC of the hypothalamus of WT (top) and KKO (bottom) intact male mice (A). The sense probe is used as a negative control and is shown on the left while the antisense probe stains as a dark brown precipitate and is s...

Supplemental Figure 6. TMEM144 protein content in the hypothalamus of WT and KKO male mice. Immunoblotting of anterior hypothalamus (A) or posterior hypothalamus (B) protein lysates showed bands at 39 kDa for TMEM144 and 50 kDa for β-tubulin. Bargraphs represent the TMEM144 protein levels in the anterior (A) and the posterior hypothalamus (B), and...

Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and q...

Telomeres are repetitive G-rich DNA sequences located at the ends of chromosomes. Chromosomal and genomic instability due to telomere dysfunction plays an important role in carcinogenesis. To study telomere shortening in the oesophageal epithelium of alcoholics, we measured the telomere lengths of basal and parabasal cells in comparison with those...

We monitored the telomere lengths and chromosomal instability characteristics of fibroblasts at different population doubling levels (PDLs) to gain further insight into the role of telomere shortening in chromosomal instability. We used 7 normal diploid human fibroblast strains (TIG-1, 3, 7, 103, 104, 112, and 114) and a quantitative fluorescence i...

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere dysfunction in the surrounding background epithelium of squamous cell carcinoma in situ (CIS) of the oesophagus, we measured telomere lengths of basal and parabasal cells of epithelia with and without CIS using qua...

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, cou...

Aging appears to be intrinsically related to carcinogenesis. Genomic instability due to telomere shortening plays an important role in carcinoma development. In order to clarify telomere dysfunction in carcinoma development, we examined the uninvolved epithelium adjacent to carcinoma in situ (CIS), i.e. background of CIS, and CIS itself, compared t...

#101 Stromal-epithelial interactions have been implicated in all stages of mammary gland development and malignancy. Although stromal composition is dynamic over time, fibroblasts account for a large proportion of the cellular compartment. The relationship between the fibroblasts and the mammary epithelial cells hitherto remains ill defined. A stri...

Critically shortened, dysfunctional telomeres may play a role in the genetic instabilities commonly found in cancer. We analyzed 30 surgical specimens of invasive breast carcinoma from women aged 34 to 91 years and estimated telomere lengths as telomere-to-centromere ratio values in the 5 different cell types comprising breast tissue in order to cl...

High-content microscopic screening systems are powerful tools for extracting quantitative multiparameter measures from large number of cells under numerous conditions. These systems perform well in applications that monitor the presence of objects, but lack in their ability to accurately estimate object intensities and summarize these findings due...

We investigated the telomere lengths of individual cell types in lingual mucosa using an improved tissue quantitative fluorescence in situ hybridization (Q-FISH) method. Our tissue Q-FISH method compensates for partially cut nuclei in a tissue section by using the telomere:centromere ratio (TCR). We normalized our TCR measurements (NTCR) using a se...

Critically shortened telomeres make chromosomes susceptible to the instability and widespread cytogenetic alterations that characterize most human cancers. We hypothesized that the very rapid cell proliferation observed in esophageal squamous cell carcinomas might accelerate telomere shortening and chromosomal instability associated with carcinogen...

We developed a novel method for evaluating telomere length in 6 cell types of noncancerous and cancerous mucosal tissues from 11 cases of gastric neoplasm using the quantitative fluorescence in situ hybridization method with telomere and centromere peptide nucleic acid probes. Our telomere length estimates were determined from the background-correc...

One of the most aggressive human malignancies, anaplastic thyroid carcinoma (ATC), has an extremely poor prognosis that may be explained by its genomic instability. We hypothesized that the very rapid cell turnover observed in ATC might accelerate telomere shortening and chromosomal instability associated with tumor cell malignancy. To compare and...

We have developed a novel method for evaluating telomere length in four different cell types in non-cancerous and cancerous mucosal tissue from 15 cases of squamous cell carcinoma of the esophagus using tissue quantitative fluorescence in situ hybridization (Q-FISH). We hypothesized that the very rapid cell proliferation observed in esophageal squa...

Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivate...

This unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide "mimetic" which ha...

The physical ends of chromosomes or telomeres are in most species composed of G-rich repeat sequences and associated proteins. The DNA of telomeres consists of specific repetitive sequences (TTAGGG in all vertebrates) that are synthesized from an RNA template by a specialized reverse transcriptase called telomerase. Conventionally, Southern analysi...

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantit...

The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with; age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative...

We have developed a simple and effective filter for edge detection, called the rank difference filter. This filter is simple, employing only integer operations to implement and generates results comparable to or better than more complex edge detectors such as the Laplacian of Gaussian and the Canny (1986). For each pixel, we first apply two differe...

The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not prov...

The very end of every chromosome is a region called the telomere. Telomeres are nucleo-protein complexes containing specific DNA repeat sequences whose lengths are strongly believed to give indications to aging and tumor progression. In order to study the role these repeat sequences play in the cell, we developed a fluorescence microscopy imaging s...

Human ovarian surface epithelial (HOSE) cells transfected with the E6 and E7 oncogenes of the human papilloma virus (PV) do not express measurable telomerase activity. Relative to untransfected control cells, HOSE-PV cells have an extended in vitro lifespan characterized by a very high frequency of telomeric associations (TAs) of chromosomes. In or...

In previous studies, we showed that the number of TTAGGG repeat sequences at the end of metaphase chromosomes can üe measured indirectly using quantitative fluorescence in situ hybridization (Q-FISH) This approach has revealed that telomere length is not randomly distributed among chromosomes and that certain telomeres, such as 17p, are relatively...

Telomeres are essential genetic elements composed of repetitive DNA and associated proteins. Programmed loss of telomere repeats beyond a critical length has been implicated in replicative ageing. Furthermore, defective telomere maintenance in mammalian cells has been shown to contribute to the acquired genetic instability that is characteristic fo...

Human chromosomes terminate in a series of T2AG3 repeats, which, together with associated proteins, are essential for chromosome stability. In somatic cells, these sequences are known to be gradually lost through successive cells divisions; however, information about changes on specific chromosomes is not available. Individual telomeres could media...

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very hetero...

The limited life span of most blood cells requires the continuous production of cells, which in adults exceeds 1012 cells/day. This impressive production of cells (∼ 4 × 1016 cells over a lifetime) is achieved by the proliferation and differentiation of committed progenitor cells, which themselves are derived from a population of pluri-potent stem...

A digital camera has been designed based on a 1317 by 1035 pixel, full frame, CCD which offers the large area, high density sampling but with the options of programmable sub-area scanning and multiple independent x and y binning and programmable gain and offset. The sub-area scanning and binning can be used to increase the effective frame rate of t...

To reconstruct the object from its observed images, the characteristics of the imaging system must first be obtained. In a microscope imaging system, the characteristics vary not only in the imaging plane, but also vary as a function of the focus in which the image is taken. Thus, a three dimensional system response or point spread function (PSF) n...

A new generation, large frame (1317 X 1035 pel) digital camera, based on the MicroImager 1400 (MI1400), has been developed. The new MicroImager 1400-12 (MI1400- 12) uses improved noise reduction circuits to allow for 12 bit digitization up to 8 Mpel/s. Methods of correlated double sampling were investigated and a clamp-and-sample design was chosen....

Different methods are investigated in selecting and generating the appropriate microscope images for analysis of three-dimensional objects in quantitative microscopy. Traditionally, the ‘best’ focused image from a set is used for quantitative analysis. Such an objectively determined image is optimal for the extraction of some features, but may not...

In bright field microscopy, quantitative analysis of acquired images is customarily performed using the `best' image. Since an image with sufficient detail and clarity is required for consistent classification and discrimination of objects in the image, the image with higher magnification is commonly chosen for the analysis, which generally corresp...

A simple technique which automatically detects and then segments nucleated cells in Wright's giemsa-stained blood smears is presented. Our method differs from others in (1) the simplicity of our algorithms; (2) inclusion of touching (as well as nontouching) cells; and (3) use of these algorithms to segment as well as to detect nucleated cells emplo...

The acquisition of high resolution, large area, microscope images is a crucial aspect of image cytometry. A unique quantitative visible light microscope was developed for this purpose, by positioning a high density, charge-coupled device (CCD) in the primary image plane of a chromatic aberration free, flat field objective lens. The CCD clocking and...

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and reco...