Publications by authors named "Brad T Sherman"

31 Publications

Correction: Hu et al. Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27. 2019, , 6207.

Int J Mol Sci 2021 May 12;22(10). Epub 2021 May 12.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

The authors wish to make the following corrections to this paper [...].
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http://dx.doi.org/10.3390/ijms22105129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151633PMC
May 2021

MicroRNA Profiles in Monocyte-Derived Macrophages Generated by Interleukin-27 and Human Serum: Identification of a Novel HIV-Inhibiting and Autophagy-Inducing MicroRNA.

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

Laboratory of Human Retrovirology and Immunoinformatics, Applied and Development Research Directorate, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.
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http://dx.doi.org/10.3390/ijms22031290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865382PMC
January 2021

Profiles of MicroRNAs in Interleukin-27-Induced HIV-Resistant T Cells: Identification of a Novel Antiviral MicroRNA.

J Acquir Immune Defic Syndr 2021 03;86(3):378-387

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD.

Objectives: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step.

Purpose: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs.

Methods: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-β, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit.

Results: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1.

Conclusion: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.
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http://dx.doi.org/10.1097/QAI.0000000000002565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879852PMC
March 2021

Genome-wide association study of high-sensitivity C-reactive protein, D-dimer, and interleukin-6 levels in multiethnic HIV+ cohorts.

AIDS 2021 02;35(2):193-204

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland.

Objectives: Elevated levels of interleukin-6 (IL-6), D-dimer, and C-reactive protein (hsCRP) are associated with increased incidence of comorbid disease and mortality among people living with HIV (PLWH). Prior studies suggest a genetic basis for these biomarker elevations in the general population. The study objectives are to identify the genetic basis for these biomarkers among PLWH.

Methods: Baseline levels of hsCRP, D-dimer, and IL-6, and single nucleotide polymorphisms (SNPs) were determined for 7768 participants in three HIV treatment trials. Single variant analysis was performed for each biomarker on samples from each of three ethnic groups [African (AFR), Admixed American (AMR), European (EUR)] within each trial including covariates relevant to biomarker levels. For each ethnic group, the results were pooled across trials, then further pooled across ethnicities.

Results: The transethnic analysis identified three, two, and one known loci associated with hsCRP, D-dimer, and IL-6 levels, respectively, and two novel loci, FGB and GCNT1, associated with D-dimer levels. Lead SNPs exhibited similar effects across ethnicities. Additionally, three novel, ethnic-specific loci were identified: CATSPERG associated with D-dimer in AFR and PROX1-AS1 and TRAPPC9 associated with IL-6 in AFR and AMR, respectively.

Conclusion: Eleven loci associated with three biomarker levels were identified in PLWH from the three studies including six loci known in the general population and five novel loci associated with D-dimer and IL-6 levels. These findings support the hypothesis that host genetics may partially contribute to chronic inflammation in PLWH and help to identify potential targets for intervention of serious non-AIDS complications.
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http://dx.doi.org/10.1097/QAD.0000000000002738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789909PMC
February 2021

Defective HIV-1 proviruses produce viral proteins.

Proc Natl Acad Sci U S A 2020 02 6;117(7):3704-3710. Epub 2020 Feb 6.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;

HIV-1 proviruses persist in the CD4 T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as "defective" by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant "graveyard" of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4 T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4 T cells and T cell-cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.
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http://dx.doi.org/10.1073/pnas.1917876117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035625PMC
February 2020

Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27.

Int J Mol Sci 2019 Dec 9;20(24). Epub 2019 Dec 9.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.
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http://dx.doi.org/10.3390/ijms20246207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941108PMC
December 2019

Complete Genome Sequence of Herpes Simplex Virus 1 Strain McKrae.

Microbiol Resour Announc 2019 Sep 26;8(39). Epub 2019 Sep 26.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

Herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent and relatively neuroinvasive in animal models compared with other wild-type HSV-1 strains. To identify the genetic determinants that lead to the unique phenotypes of the McKrae strain, we sequenced its genome with PacBio single-molecule real-time (SMRT) technology and resolved the complete sequence.
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http://dx.doi.org/10.1128/MRA.00993-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763650PMC
September 2019

Complete Genome Sequence of Herpes Simplex Virus 1 Strain MacIntyre.

Microbiol Resour Announc 2019 Sep 12;8(37). Epub 2019 Sep 12.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

Herpes simplex virus type 1 (HSV-1) strain MacIntyre has a severe defect in the anterograde spread after replication in the nucleus. To better understand and identify the genetic determinants that lead to the unique phenotypes of the MacIntyre strain, we sequenced its genome with PacBio single-molecule real-time sequencing technology and resolved the complete sequence.
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http://dx.doi.org/10.1128/MRA.00895-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742799PMC
September 2019

Adoptive lymphocyte transfer to an HIV-infected progressor from an elite controller.

JCI Insight 2019 09 19;4(18). Epub 2019 Sep 19.

Critical Care Medicine Department, NIH Clinical Center, NIH, Bethesda, Maryland, USA.

BACKGROUNDHIV-infected patients with poor virologic control and multidrug-resistant virus have limited therapeutic options. The current study was undertaken to evaluate the safety, immunologic effects, and antiviral activity of peripheral lymphocytes transferred from an elite controller, whose immune system is able to control viral replication without antiretroviral medications, to an HLA-B*2705-matched progressor.METHODSApproximately 22 billion cells were collected from an elite controller by lymphapheresis and infused within 6 hours into a recipient with a preinfusion CD4+ T cell count of 10 cells/μL (1%) and HIV plasma viral load of 114,993 copies/mL.RESULTSDonor cells were cleared from the recipient's peripheral blood by day 8. A transient decrease in viral load to 58,421 (day 3) was followed by a rebound to 702,972 (day 6) before returning to baseline values by day 8. The decreased viral load was temporally associated with peak levels of donor T cells, including CD8+ T cells that had high levels of expression of Ki67, perforin, and granzyme B. Notably, recipient CD8+ T cells also showed increased expression of these markers, especially in HIV-specific tetramer-positive cells.CONCLUSIONThese results suggest that the adoptive transfer of lymphocytes from an HIV-infected elite controller to an HIV-infected patient with progressive disease may be able to perturb the immune system of the recipient in both positive and negative ways.TRIAL REGISTRATIONClinicalTrials.gov NCT00559416.FUNDINGIntramural Research Programs of the US NIH Clinical Center and the National Institute of Allergy and Infectious Diseases (NIAID); the National Cancer Institute.
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http://dx.doi.org/10.1172/jci.insight.130664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795294PMC
September 2019

Association Between Single-Nucleotide Polymorphisms in HLA Alleles and Human Immunodeficiency Virus Type 1 Viral Load in Demographically Diverse, Antiretroviral Therapy-Naive Participants From the Strategic Timing of AntiRetroviral Treatment Trial.

J Infect Dis 2019 09;220(8):1325-1334

Centre of Excellence for Health, Immunity and Infections, Department of Infectious Diseases, Rigshospitalet, University of Copenhagen, Denmark.

The impact of variation in host genetics on replication of human immunodeficiency virus type 1 (HIV-1) in demographically diverse populations remains uncertain. In the current study, we performed a genome-wide screen for associations of single-nucleotide polymorphisms (SNPs) to viral load (VL) in antiretroviral therapy-naive participants (n = 2440) with varying demographics from the Strategic Timing of AntiRetroviral Treatment (START) trial. Associations were assessed using genotypic data generated by a customized SNP array, imputed HLA alleles, and multiple linear regression. Genome-wide significant associations between SNPs and VL were observed in the major histocompatibility complex class I region (MHC I), with effect sizes ranging between 0.14 and 0.39 log10 VL (copies/mL). Supporting the SNP findings, we identified several HLA alleles significantly associated with VL, extending prior observations that the (MHC I) is a major host determinant of HIV-1 control with shared genetic variants across diverse populations and underscoring the limitations of genome-wide association studies as being merely a screening tool.
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http://dx.doi.org/10.1093/infdis/jiz294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743845PMC
September 2019

A novel microRNA, hsa-miR-6852 differentially regulated by Interleukin-27 induces necrosis in cervical cancer cells by downregulating the FoxM1 expression.

Sci Rep 2018 01 17;8(1):900. Epub 2018 Jan 17.

Laboratory of Human Retrovirology and Immunoinformatics, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 21702, USA.

We have previously demonstrated that Interleukin-27 differentially regulates the expression of seven novel microRNAs. Here we elucidate the functional significance of these novel microRNAs. Of the seven microRNAs, over expression of miRNA-6852 (miR-SX4) mimic induces cell cycle arrest at G2/M phase and induces necrosis in HEK293 and panel of cervical cancer cells (Human Papilloma Virus (HPV) infected cell lines; HeLa, CaSki and SiHa cells). To define the mechanism of the miR-SX4-mediated G2/M arrest, a microarray gene chip array and western blot analysis were performed. FoxM1, a transcription factor is identified as a key protein down-regulated by miR-SX4, even though the miR-SX4 does not target 3'UTR of FoxM1. Knock down of FoxM1 using si-RNA demonstrate that FoxM1 silenced cell induces G2/M cell cycle arrest and necrosis. Our data demonstrated for the first time that miR-SX4 could be a potent anti-cancer microRNA.
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http://dx.doi.org/10.1038/s41598-018-19259-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772045PMC
January 2018

STING is an essential mediator of the Ku70-mediated production of IFN-λ1 in response to exogenous DNA.

Sci Signal 2017 Jul 18;10(488). Epub 2017 Jul 18.

Laboratory of Human Retrovirology and Immunoinformatics, Applied and Developmental Research Directorate, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-β). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cells produced IFN-λ1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK 293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. Additionally, the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-λ1 in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-λ1 innate immune response to exogenous DNA or DNA virus infection.
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http://dx.doi.org/10.1126/scisignal.aah5054DOI Listing
July 2017

Genome-Wide Analyses of MicroRNA Profiling in Interleukin-27 Treated Monocyte-Derived Human Dendritic Cells Using Deep Sequencing: A Pilot Study.

Int J Mol Sci 2017 Apr 28;18(5). Epub 2017 Apr 28.

Laboratory of Human Retrovirology and Immunoinformatics, Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

MicroRNAs (miRNAs) regulate gene expression and thereby influence cell fate and function. Recent studies suggest that an abundant class of miRNAs play important roles in immune cells, such as T cells, natural killer (NK) cells, B cells, and dendritic cells (DCs). Interleukin (IL)-27 is a member of the IL-12 family of cytokines with broad anti-viral effects. It is a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages, as well as monocyte-derived immature dendritic cells (iDCs). This pilot study compared miRNA profiles between iDCs and IL-27-treated iDCs (27DCs) using deep sequencing methods and identified 46 known miRNAs that were significantly differentially expressed in 27DCs: 36 were upregulated and 10 downregulated by IL-27. Many of the potential target genes of these miRNAs are involved in IL-27 associated pathways, such as JAK/STAT, MAPKs, and PI3K and several were also previously reported to be involved in the regulation of human DC function. This study found that these miRNAs also potentially target several viral genomes and therefore may have antiviral effects. Four of these differential miRNAs (miR-99a-5p, miR-222-3p, miR-138-5p, and miR-125b-5p) were validated using quantitative reverse transcription PCR (RT-qPCR). Twenty-two novel miRNAs were discovered from deep sequencing and confirmed using RT-qPCR. This study furthers the understanding of the role of IL-27 in immunity and lays a foundation for future characterization of the role of specific miRNAs in DCs.
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http://dx.doi.org/10.3390/ijms18050925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454838PMC
April 2017

Interleukin-15 (IL-15) Strongly Correlates with Increasing HIV-1 Viremia and Markers of Inflammation.

PLoS One 2016 23;11(11):e0167091. Epub 2016 Nov 23.

Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, United States of America.

Objective: IL-15 has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. We sought to measure the level of IL-15 in a large, well characterised cohort of HIV-1 infected patients and correlate this with well known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14.

Design And Methods: IL-15 levels were measured in 501 people (460 patients with HIV-1 infection and 41 uninfected controls). The HIV-1 infected patients were divided into 4 groups based on viral load: <50 copies/ml, 51-10,000 copies/ml, 10,001-100,000 copies/ml and >100,000 copies/ml. The Mann Whitney test (non-parametric) was used to identify significant relationships between different patient groups.

Results: IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 ± 1.53 pg/ml) compared to both uninfected controls (1.69 ± 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 ± 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001).

Conclusions: IL-15 levels are significantly elevated in HIV-1 infected patients with viral loads >100,000 copies/ml compared to uninfected controls, with a significant direct correlation noted between IL-15 and HIV-1 viremia and an inverse correlation between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167091PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120855PMC
June 2017

Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.

BioData Min 2016 5;9:13. Epub 2016 Apr 5.

Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.

Background: Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.

Results: We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.

Conclusions: PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.
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http://dx.doi.org/10.1186/s13040-016-0090-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820869PMC
April 2016

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

Nat Commun 2016 Feb 22;7:10740. Epub 2016 Feb 22.

Genome Sequencing and Analysis Program, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.
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http://dx.doi.org/10.1038/ncomms10740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764891PMC
February 2016

Evolution of the pygmy phenotype: evidence of positive selection fro genome-wide scans in African, Asian, and Melanesian pygmies.

Hum Biol 2013 Feb-Jun;85(1-3):251-84

Department of Anthropology, University College London, London, UK.

Human pygmy populations inhabit different regions of the world, from Africa to Melanesia. In Asia, short-statured populations are often referred to as "negritos." Their short stature has been interpreted as a consequence of thermoregulatory, nutritional, and/or locomotory adaptations to life in tropical forests. A more recent hypothesis proposes that their stature is the outcome of a life history trade-off in high-mortality environments, where early reproduction is favored and, consequently, early sexual maturation and early growth cessation have coevolved. Some serological evidence of deficiencies in the growth hormone/insulin-like growth factor axis have been previously associated with pygmies' short stature. Using genome-wide single-nucleotide polymorphism genotype data, we first tested whether different negrito groups living in the Philippines and Papua New Guinea are closely related and then investigated genomic signals of recent positive selection in African, Asian, and Papuan pygmy populations. We found that negritos in the Philippines and Papua New Guinea are genetically more similar to their nonpygmy neighbors than to one another and have experienced positive selection at different genes. These results indicate that geographically distant pygmy groups are likely to have evolved their short stature independently. We also found that selection on common height variants is unlikely to explain their short stature and that different genes associated with growth, thyroid function, and sexual development are under selection in different pygmy groups.
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http://dx.doi.org/10.3378/027.085.0313DOI Listing
April 2015

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS.

J Data Mining Genomics Proteomics 2013 Jul;4(3)

Laboratory of Immunopathogenesis and Bioinformatics, SAIC-Frederick, Inc., Frederick National Laboratory, MD 21702, USA.

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.
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http://dx.doi.org/10.4172/2153-0602.1000136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811116PMC
July 2013

Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis.

FASEB J 2013 May 7;27(5):1962-72. Epub 2013 Feb 7.

Critical Care Medicine Department, NIH Clinical Center, 10 Center Dr., Bethesda, MD 20892, USA.

Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and order of genes in a linear configuration, whereas P. jirovecii has a circular mtDNA containing nearly the same set of genes but in a different order. Detailed studies of the mtDNA terminal structures of P. murina and P. carinii suggest a unique replication mechanism for linear mtDNA. Phylogenetic analysis supports a close association of Pneumocystis species with Taphrina, Saitoella, and Schizosaccharomyces, and divergence within Pneumocystis species, with P. murina and P. carinii being more closely related to each other than either is to P. jirovecii. Comparative analysis of four complete P. jirovecii mtDNA sequences in this study and previously reported mtDNA sequences for diagnosing and genotyping suggests that the current diagnostic and typing methods can be improved using the complete mtDNA data. The availability of the complete P. jirovecii mtDNA also opens the possibility of identifying new therapeutic targets.
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http://dx.doi.org/10.1096/fj.12-224444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633818PMC
May 2013

DAVID-WS: a stateful web service to facilitate gene/protein list analysis.

Bioinformatics 2012 Jul 27;28(13):1805-6. Epub 2012 Apr 27.

Laboratory of Immunopathogenesis and Bioinformatics, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

Summary: The database for annotation, visualization and integrated discovery (DAVID), which can be freely accessed at http://david.abcc.ncifcrf.gov/, is a web-based online bioinformatics resource that aims to provide tools for the functional interpretation of large lists of genes/proteins. It has been used by researchers from more than 5000 institutes worldwide, with a daily submission rate of ∼1200 gene lists from ∼400 unique researchers, and has been cited by more than 6000 scientific publications. However, the current web interface does not support programmatic access to DAVID, and the uniform resource locator (URL)-based application programming interface (API) has a limit on URL size and is stateless in nature as it uses URL request and response messages to communicate with the server, without keeping any state-related details. DAVID-WS (web service) has been developed to automate user tasks by providing stateful web services to access DAVID programmatically without the need for human interactions.

Availability: The web service and sample clients (written in Java, Perl, Python and Matlab) are made freely available under the DAVID License at http://david.abcc.ncifcrf.gov/content.jsp?file=WS.html.
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http://dx.doi.org/10.1093/bioinformatics/bts251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381967PMC
July 2012

Gene duplications in prokaryotes can be associated with environmental adaptation.

BMC Genomics 2010 Oct 20;11:588. Epub 2010 Oct 20.

Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

Background: Gene duplication is a normal evolutionary process. If there is no selective advantage in keeping the duplicated gene, it is usually reduced to a pseudogene and disappears from the genome. However, some paralogs are retained. These gene products are likely to be beneficial to the organism, e.g. in adaptation to new environmental conditions. The aim of our analysis is to investigate the properties of paralog-forming genes in prokaryotes, and to analyse the role of these retained paralogs by relating gene properties to life style of the corresponding prokaryotes.

Results: Paralogs were identified in a number of prokaryotes, and these paralogs were compared to singletons of persistent orthologs based on functional classification. This showed that the paralogs were associated with for example energy production, cell motility, ion transport, and defence mechanisms. A statistical overrepresentation analysis of gene and protein annotations was based on paralogs of the 200 prokaryotes with the highest fraction of paralog-forming genes. Biclustering of overrepresented gene ontology terms versus species was used to identify clusters of properties associated with clusters of species. The clusters were classified using similarity scores on properties and species to identify interesting clusters, and a subset of clusters were analysed by comparison to literature data. This analysis showed that paralogs often are associated with properties that are important for survival and proliferation of the specific organisms. This includes processes like ion transport, locomotion, chemotaxis and photosynthesis. However, the analysis also showed that the gene ontology terms sometimes were too general, imprecise or even misleading for automatic analysis.

Conclusions: Properties described by gene ontology terms identified in the overrepresentation analysis are often consistent with individual prokaryote lifestyles and are likely to give a competitive advantage to the organism. Paralogs and singletons dominate different categories of functional classification, where paralogs in particular seem to be associated with processes involving interaction with the environment.
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http://dx.doi.org/10.1186/1471-2164-11-588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091735PMC
October 2010

Pre-gastrula expression of zebrafish extraembryonic genes.

BMC Dev Biol 2010 Apr 27;10:42. Epub 2010 Apr 27.

Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Background: Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL).

Results: We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent.

Conclusion: We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.
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http://dx.doi.org/10.1186/1471-213X-10-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873407PMC
April 2010

Extracting biological meaning from large gene lists with DAVID.

Curr Protoc Bioinformatics 2009 Sep;Chapter 13:Unit 13.11

National Cancer Institute at Frederick, Frederick, Maryland, USA.

High-throughput genomics screening studies, such as microarray, proteomics, etc., often result in large, "interesting" gene lists, ranging in size from hundreds to thousands of genes. Given the challenges of functionally interpreting such large gene lists, it is necessary to incorporate bioinformatics tools in the analysis. DAVID is a Web-based application that provides a high-throughput and integrative gene functional annotation environment to systematically extract biological themes behind large gene lists. High-throughput gene functional analysis with DAVID will provide important insights that allow investigators to understand the biological themes within their given genomic study. This unit will describe step-by-step procedures to use DAVID tools, as well as a brief rationale and key parameters in the DAVID analysis.
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http://dx.doi.org/10.1002/0471250953.bi1311s27DOI Listing
September 2009

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.

Nat Protoc 2009 ;4(1):44-57

Laboratory of Immunopathogenesis and Bioinformatics, Clinical Services Program, SAIC-Frederick Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.

DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
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http://dx.doi.org/10.1038/nprot.2008.211DOI Listing
March 2009

Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.

Nucleic Acids Res 2009 Jan 25;37(1):1-13. Epub 2008 Nov 25.

Laboratory of Immunopathogenesis and Bioinformatics, Clinical Services Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702, USA.

Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
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http://dx.doi.org/10.1093/nar/gkn923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615629PMC
January 2009

DAVID gene ID conversion tool.

Bioinformation 2008 Jul 30;2(10):428-30. Epub 2008 Jul 30.

Unlabelled: Our current biological knowledge is spread over many independent bioinformatics databases where many different types of gene and protein identifiers are used. The heterogeneous and redundant nature of these identifiers limits data analysis across different bioinformatics resources. It is an even more serious bottleneck of data analysis for larger datasets, such as gene lists derived from microarray and proteomic experiments. The DAVID Gene ID Conversion Tool (DICT), a web-based application, is able to convert user's input gene or gene product identifiers from one type to another in a more comprehensive and high-throughput manner with a uniquely enhanced ID-ID mapping database.

Availability: http://david.abcc.ncifcrf.gov/conversion.jsp.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2561161PMC
http://dx.doi.org/10.6026/97320630002428DOI Listing
July 2008

DAVID Knowledgebase: a gene-centered database integrating heterogeneous gene annotation resources to facilitate high-throughput gene functional analysis.

BMC Bioinformatics 2007 Nov 2;8:426. Epub 2007 Nov 2.

Laboratory of Immunopathogenesis and Bioinformatics, Clinical Services Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702, USA.

Background: Due to the complex and distributed nature of biological research, our current biological knowledge is spread over many redundant annotation databases maintained by many independent groups. Analysts usually need to visit many of these bioinformatics databases in order to integrate comprehensive annotation information for their genes, which becomes one of the bottlenecks, particularly for the analytic task associated with a large gene list. Thus, a highly centralized and ready-to-use gene-annotation knowledgebase is in demand for high throughput gene functional analysis.

Description: The DAVID Knowledgebase is built around the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of gene/protein identifiers from a variety of public genomic resources into DAVID gene clusters. The grouping of such identifiers improves the cross-reference capability, particularly across NCBI and UniProt systems, enabling more than 40 publicly available functional annotation sources to be comprehensively integrated and centralized by the DAVID gene clusters. The simple, pair-wise, text format files which make up the DAVID Knowledgebase are freely downloadable for various data analysis uses. In addition, a well organized web interface allows users to query different types of heterogeneous annotations in a high-throughput manner.

Conclusion: The DAVID Knowledgebase is designed to facilitate high throughput gene functional analysis. For a given gene list, it not only provides the quick accessibility to a wide range of heterogeneous annotation data in a centralized location, but also enriches the level of biological information for an individual gene. Moreover, the entire DAVID Knowledgebase is freely downloadable or searchable at http://david.abcc.ncifcrf.gov/knowledgebase/.
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http://dx.doi.org/10.1186/1471-2105-8-426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2186358PMC
November 2007

The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists.

Genome Biol 2007 ;8(9):R183

Laboratory of Immunopathogenesis and Bioinformatics, Clinical Services Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702, USA.

The DAVID Gene Functional Classification Tool http://david.abcc.ncifcrf.gov uses a novel agglomeration algorithm to condense a list of genes or associated biological terms into organized classes of related genes or biology, called biological modules. This organization is accomplished by mining the complex biological co-occurrences found in multiple sources of functional annotation. It is a powerful method to group functionally related genes and terms into a manageable number of biological modules for efficient interpretation of gene lists in a network context.
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http://dx.doi.org/10.1186/gb-2007-8-9-r183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2375021PMC
May 2008

DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists.

Nucleic Acids Res 2007 Jul 18;35(Web Server issue):W169-75. Epub 2007 Jun 18.

Laboratory of Immunopathogenesis and Bioinformatics, SAIC-Frederick, Inc., National Cancer Institute at Frederick, MD 21702, USA.

All tools in the DAVID Bioinformatics Resources aim to provide functional interpretation of large lists of genes derived from genomic studies. The newly updated DAVID Bioinformatics Resources consists of the DAVID Knowledgebase and five integrated, web-based functional annotation tool suites: the DAVID Gene Functional Classification Tool, the DAVID Functional Annotation Tool, the DAVID Gene ID Conversion Tool, the DAVID Gene Name Viewer and the DAVID NIAID Pathogen Genome Browser. The expanded DAVID Knowledgebase now integrates almost all major and well-known public bioinformatics resources centralized by the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of diverse gene/protein identifiers and annotation terms from a variety of public bioinformatics databases. For any uploaded gene list, the DAVID Resources now provides not only the typical gene-term enrichment analysis, but also new tools and functions that allow users to condense large gene lists into gene functional groups, convert between gene/protein identifiers, visualize many-genes-to-many-terms relationships, cluster redundant and heterogeneous terms into groups, search for interesting and related genes or terms, dynamically view genes from their lists on bio-pathways and more. With DAVID (http://david.niaid.nih.gov), investigators gain more power to interpret the biological mechanisms associated with large gene lists.
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http://dx.doi.org/10.1093/nar/gkm415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933169PMC
July 2007

Identifying biological themes within lists of genes with EASE.

Genome Biol 2003 11;4(10):R70. Epub 2003 Sep 11.

Laboratory of Immunopathogenesis and Bioinformatics, PO Box B, SAIC-Frederick, Inc, Frederick, MD 21702, USA.

EASE is a customizable software application for rapid biological interpretation of gene lists that result from the analysis of microarray, proteomics, SAGE and other high-throughput genomic data. The biological themes returned by EASE recapitulate manually determined themes in previously published gene lists and are robust to varying methods of normalization, intensity calculation and statistical selection of genes. EASE is a powerful tool for rapidly converting the results of functional genomics studies from 'genes' to 'themes'.
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http://dx.doi.org/10.1186/gb-2003-4-10-r70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC328459PMC
August 2004
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