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  • 1
    Online Resource
    Online Resource
    Oxford University Press (OUP) ; 2016
    In:  Human Molecular Genetics Vol. 25, No. R2 ( 2016-10-01), p. R190-R197
    In: Human Molecular Genetics, Oxford University Press (OUP), Vol. 25, No. R2 ( 2016-10-01), p. R190-R197
    Type of Medium: Online Resource
    ISSN: 0964-6906 , 1460-2083
    RVK:
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2016
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  • 2
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 25, No. 10 ( 2015-10), p. 1581-1589
    Abstract: Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used c lustered r egularly i nterspaced s hort p alindromic r epeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR e pitope t agging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2015
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  • 3
    In: Cell, Elsevier BV, Vol. 186, No. 7 ( 2023-03), p. 1493-1511.e40
    Type of Medium: Online Resource
    ISSN: 0092-8674
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    Language: English
    Publisher: Elsevier BV
    Publication Date: 2023
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  • 4
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 41, No. 16_suppl ( 2023-06-01), p. 524-524
    Abstract: 524 Background: Alliance A011202 is evaluating the efficacy of axillary lymph node dissection (ALND) compared to regional nodal irradiation (RNI) for patients with cN1 breast cancer who receive neoadjuvant chemotherapy (NCTX) that becomes ycN0 but remains pN+. In the absence of its publication, level I guidance does not exist regarding the optimal axillary surgery for patients receiving NCTX. We sought to evaluate outcomes across two national contemporary clinical NCTX trials based on axillary and breast response to NCTX, particularly if more aggressive surgery was associated with more favorable outcomes. Methods: With IRB approval (IRB201802781), we obtained data from NCTX trials NSABP B40 and B41. B40 enrolled women with HER2- disease, and B41 enrolled those with HER2+ disease. RNI and axillary surgery were selected at physician discretion and not protocolled. Patients received sentinel lymph node biopsy (SLNB), SLNB+ALND (S+ALND), or ALND. We examined outcomes of locoregional recurrence (LRR), distant recurrence (DR), disease free survival (DFS), and overall survival (OS). Univariable and multivariable analyses of B40 and B41 data were performed to evaluate the associations of axillary surgery with the outcomes above, adjusting for age, tumor subtype, mastectomy or not, breast pathologic complete response (pCR), axillary pCR, tumor subtype, regional nodal irradiation, and grade. Kaplan-Meier estimation was used for OS and DFS, with cumulative incidence function for LRR and DR. Results: Median follow-up for studies B40 and B41 were 4.5 and 5.1 years, respectively, including 1154 and 504 patients for analysis. A total of 786 (47%) patients were cN+, and of those, 377 had a pcR (48%). 440 (27%), 505 (31%), and 663 (41%) patients had SLNB, S+ALND, and ALND respectively. 855 (52%) and 803 (48%) patients had mastectomy and lumpectomy, respectively. 783 (51%) received RNI. For the 518 ypN+ patients on B40, 7/25 (SLNB), 69/234 (S+ALND), and 107/259 (ALND) experienced an event. DFS at 5 years was 71%, 68%, and 56% for the SLNB, S+ALND, and ALND groups respectively with ypN+ on B40. For the 112 ypN+ patients on B41, 3/6, 15/43, and 27/63 experienced an event. DFS at 5 years was 50%, 64%, and 55% for the SLNB, S+ALND, and ALND groups, respectively, with ypN+ on B41. In multivariable analyses for the combined population of B40 and B41 for LRR, DR, DFS, and OS, SLNB was never associated with a higher chance of relapse or inferior survival (HR 〉 1) compared to S+ALND or ALND. Conclusions: Among women prospectively treated on national trials with NCTX and axillary surgery and RNI selected at physician discretion, receipt of SLNB alone was not associated with a higher likelihood of recurrence compared to S+ALND or ALND. Anticipated results of A011202 will provide level I guidance on axillary surgery for patients with cN1 disease converting to cN0 post-NCTX.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
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    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2023
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  • 5
    In: European Archives of Oto-Rhino-Laryngology, Springer Science and Business Media LLC, Vol. 270, No. 5 ( 2013-5), p. 1569-1580
    Type of Medium: Online Resource
    ISSN: 0937-4477 , 1434-4726
    RVK:
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2013
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  • 6
    In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 136-136
    Abstract: Self-renewal and lineage differentiation of hematopoietic stem cells (HSC) is likely regulated by a combination of intrinsic and extrinsic signals. At present these signals are poorly understood. Recently, numerous groups have identified the expressed gene profile of HSC in an attempt to identify novel genes that regulate HSC fate decisions. Likewise, our group compared the expressed gene profiles of CD34+CD33−CD38−Rholoc-kit+ (Rholo) cells, enriched in primitive progenitors and presumed human HSC, compared to CD34+CD33−CD38−Rhohi (Rhohi) cells, depleted of such cells, from umbilical cord blood (UCB) and bone marrow (BM) to identify conserved signaling pathways active in these ontogenically distinct populations using the Afftymetrix™ HG-U133 GeneChip® set. We identified a putative molecular signature for human HSC containing 286 genes, expressed more highly in Rholo or Rhohi cells from both cell sources using a p & lt;0.05 and fold change of 1.5 cutoff. To assess the role of this series of genes in a high-throughput fashion, we developed an in vivo functional genomics screen in the zebrafish that allows for the determination of hematopoietic function of the differentially expressed genes. Candidate gene expression was knocked down by injecting morpholino antisense oligonucleotides (MO) into 1–4 cell embryos from GATA1:DsRed transgenic zebrafish that have red fluorescent GATA1+ blood cells and blood production was scored by fluorescence microscopy at 30 and 48 hours post-fertilization. Of the 286 differentially expressed genes, 128 were deemed too universal or likely non-specific for hematopoiesis, and were not targeted. These included histones, HLA-antigens, hemoglobin genes, and genes encoding proteins involved in general cellular metabolism. Of the remaining 158, MO were designed for 70 (44%), no zebrafish ortholog could be identified for 72 (46%), inadequate sequence information was available to design morpholinos for 9 (6%) and multiple zebrafish orthologs were identified for 7 (4%). Fluorescence microscopy of GATA1:DsRed fish has revealed a reproducible reduction in GATA1+ blood cell number for 13 of 55 MO-targeted genes analyzed to date, giving a 24% frequency for hematopoietic phenotypes in the screen, that compares very favorably with the noted 0.5–1% frequency of hematopoietic genes identified in ENU mutagenesis screens that mutate genes in a near random fashion. The functionally-validated genes identified thus far include known genes that lack a known hematopoietic function such as SPRY1, CRTAP, IRAK3 and UCP2, as well as genes that currently lack a functional annotation such as C12orf2, DKFZp564D137, MGC15875 and FLJ21269. Hematopoietic phenotypes are being further characterized by whole-mount in situ hybridization for hematopoietic genes and rescue of hematopoietic phenotypes by co-injection of overexpression vectors in the zebrafish. In addition, we are confirming the role of the genes in mammalian hematopoiesis using overexpression and knockdown studies in murine and human repopulating HSC. This is the first description of a high-throughput functional genomics screen in the zebrafish to functionally validate differentially expressed genes, an essential step in obtaining meaningful functional data from global gene profiling studies.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2004
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  • 7
    Online Resource
    Online Resource
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 1715-1715
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 1715-1715
    Abstract: Hematopoietic stem cells (HSCs) are functionally defined by their capacity to home to the bone marrow microenvironment, proliferate and differentiate to restore normal hematopoiesis in a myeloablated recipient; however, the molecular determinants of these processes are not well understood. By comparing the gene expression profiles of highly purified human HSC-enriched and HSC-depleted cell populations, and subsequently validating the hematopoietic function of a subset of these differentially expressed genes using zebrafish (Danio rerio), we previously identified human Sprouty 1 (SPRY1), an evolutionarily conserved antagonist of RTK signaling, as a potential regulator of mammalian HSC development and/or cell-fate decisions. To directly assess the role of SPRY1 in mammalian HSC and hematopoietic progenitor cell (HPC) function, we constructed a dual-promoter lentiviral vector to co-express SPRY1 and green fluorescent protein (GFP) (SPRY1-LV) and a control lentiviral vector to express GFP alone (GFP-LV) in the CD34+ fraction of human umbilical cord blood (UCB). While the enforced expression of SPRY1 in CD34+ UCB cells had no effect on the frequency or morphology of colonies generated in short-term in vitro colony-forming cell (CFC) assays (SPRY1-LV = 27.6 ± 12.3% and GFP-LV = 28.4 ± 20.0%; n = 3), it profoundly inhibited the capacity of UCB CD34+ cells to engraft in the bone marrow NOD-SCID mice in vivo. The estimated frequency of week 11 SCID-repopulating cells (SRC) (± 1 standard error) for SPRY1-LV (n = 13 mice) and GFP-LV (n = 15 mice) cells was 1 in 12,678 cells (6,167 – 26,064) and 1 in 3,412 cells (2,272 – 5,124), respectively, as determined using limiting dilution conditions and Poisson statistics. Furthermore, in 14 day cultures designed for the ex vivo expansion and/or maintenance of primitive hematopoietic cells, ectopic expression of SPRY1 in CD34+ UCB cells dramatically inhibited the expansion of total nucleated cells (SPRY1-LV = 37.9 ± 10.1 fold; GFP-LV = 71.1 ± 5.8 fold; n = 3; p & lt;0.05) and CFCs (SPRY1-LV = 5.7 ± 1.2 fold; GFP-LV = 20.8 ± 14.9 fold; n = 3), although it had no effect on expansion of CD34+CD38− cells. We are currently investigating potential mechanisms for the observed affects of SPRY1 on primitive hematopoietic cells, paying particular attention to the possible effects of SPRY1 expression on “early-acting” hematopoietic cytokines and growth factors that activate RTKs - including FGF1, FGF2, VEGF, SCF, FLT3L, and ANGPT1. In conclusion, enforced expression of SPRY1 negatively regulates primitive hematopoietic cell engraftment in vivo and expansion in vitro, thereby presenting the first example of a role for a Sprouty family member, SPRY1, in primitive human hematopoietic cell function. Moreover, this data further validates the use of model organisms, such as zebrafish, for evaluating the functional roles of transcripts identified in large-scale gene expression profiling experiments in mice and humans.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
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  • 8
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 33, No. 11 ( 2023-11), p. 1879-1892
    Abstract: Transcription factors (TFs) are trans -acting proteins that bind cis -regulatory elements (CREs) in DNA to control gene expression. Here, we analyzed the genomic localization profiles of 529 sequence-specific TFs and 151 cofactors and chromatin regulators in the human cancer cell line HepG2, for a total of 680 broadly termed DNA-associated proteins (DAPs). We used this deep collection to model each TF's impact on gene expression, and identified a cohort of 26 candidate transcriptional repressors. We examine high occupancy target (HOT) sites in the context of three-dimensional genome organization and show biased motif placement in distal-promoter connections involving HOT sites. We also found a substantial number of closed chromatin regions with multiple DAPs bound, and explored their properties, finding that a MAFF/MAFK TF pair correlates with transcriptional repression. Altogether, these analyses provide novel insights into the regulatory logic of the human cell line HepG2 genome and show the usefulness of large genomic analyses for elucidation of individual TF functions.
    Type of Medium: Online Resource
    ISSN: 1088-9051 , 1549-5469
    RVK:
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2023
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  • 9
    In: Human Molecular Genetics, Oxford University Press (OUP), Vol. 24, No. 9 ( 2015-05-01), p. 2442-2457
    Type of Medium: Online Resource
    ISSN: 1460-2083 , 0964-6906
    RVK:
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2015
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  • 10
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 42, No. 16_suppl ( 2024-06-01), p. 10065-10065
    Abstract: 10065 Background: Radiotherapy (RT) causes cognitive deficits in pediatric brain tumor survivors (PBTS). This has traditionally been measured using exams such as serial IQ tests administered after diagnosis. Scholastic data provides pre-diagnostic measurements and is practical for patients and families. In our state, testing in reading and mathematics is mandated annually for grades 3-11. We sought to evaluate scholastic achievement in PBTS pre- and post-RT treatment. Methods: With IRB approval, we retrospectively analyzed scholastic achievement in children ( 〈 21) with primary brain tumors treated with RT at our institution. Eligible children all resided within the state of the institution and were treated 2007-2020. The state’s Department of Education (DOE) provided test scores, school grades, and accommodations, which were matched to institutional clinical data. The other scholastic outcome of interest was Achievement Level (range 1-5 where 5 reflects highest achievement) from state-mandated standardized assessments in Mathematics and Reading, tested annually in grades 3-11. The DOE also provided scholastic data on healthy children matched 3:1 to treated patients by year, district, age, and whether the child was eligible for free or reduced lunch. A general linear mixed-effects model was performed with the above dependent variables and independent variables grade, time (a binary value being pre- or post-time of RT), and treatment (a child being treated or healthy). The interaction term of time*treatment was the outcome of interest, with α=0.1 for this pilot. Odds ratios (ORs) are reported. Results: A total of 200 students were available for analysis: 50 recruited patients and 150 matched controls identified by the DOE with median age 11.6 years at treatment and 7 years median follow-up. Fifty-two percent were eligible for free or reduced lunch. Fifty-six percent received craniospinal irradiation. Thirty-seven (60%) children had post-treatment scholastic data with 179 annual observations available. Of those, there were 10 (6%) physical education waivers, 32 (18%) testing accommodations, and 6 (3%) academic retentions. Compared to matched controls, treated patients were significantly more likely to receive accommodations (OR=31; p=0.001) and significantly less likely to receive a standard grade promotion (OR=0.57; p=0.005) or passing Mathematics scores (OR=0.47; p=0.05). Conclusions: We present the first reporting of scholastic data for United States PBTS. We demonstrate a novel method using existing state mandated school performance testing to evaluate academic performance in PBTS receiving RT. Compared to matched healthy children, we noted significantly increased needs and worse scholastic performance for PBTS. Evaluating scholastic success is an unmet need for PBTS, and we demonstrate the feasibility of using school performance as a novel patient-centered metric.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    RVK:
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    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2024
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