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1.
JCI Insight ; 7(20)2022 10 24.
Article in English | MEDLINE | ID: mdl-36278490

ABSTRACT

We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.


Subject(s)
Huntington Disease , Mice , Humans , Animals , Infant , Huntington Disease/drug therapy , Huntington Disease/genetics , Protein Aggregates , Huntingtin Protein/genetics , Huntingtin Protein/cerebrospinal fluid , Disease Models, Animal , Corpus Striatum/metabolism , Disease Progression
2.
Sci Rep ; 9(1): 14863, 2019 10 16.
Article in English | MEDLINE | ID: mdl-31619719

ABSTRACT

We developed a test to predict which patients will achieve pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) and which will have residual disease (RD). Gene expression data from pretreatment biopsies of patients with all breast cancer subtypes were combined into a 519-patient cohort containing 177 TNBC patients. Two RNA classifiers of 16 genes each were sequentially applied to the total cohort, classifying patients into 3 distinct classes. The test performance was further validated in an independent 304-patient cohort. The test accurately identified 70.5% (79/112) of pCR and 83.5% (340/407) of RD patients in the total population, and 75.0% (45/60) of pCR and 75.2% (88/117) of RD patients in the TNBC subset. For the independent cohort, the test identified 91.5% RD patients in the total population and 86.2% RD patients in the TNBC subset. However, the identification of pCR in both total and TNBC population are as low as 21.1% and 30%, respectively. The TNBC RD patients were subdivided by our classifiers, with one class showing significantly higher levels of Ki67 expression and having significantly poorer survival rates than the other classes. This stratification of patients may allow predicted residual disease classes to be assigned an alternative therapy.


Subject(s)
Gene Expression Regulation, Neoplastic , Neoadjuvant Therapy/methods , RNA, Neoplasm/genetics , Receptor, ErbB-2/genetics , Triple Negative Breast Neoplasms/diagnosis , Triple Negative Breast Neoplasms/drug therapy , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Female , Gene Expression Profiling , Humans , Middle Aged , Neoplasm, Residual , Prognosis , RNA, Neoplasm/metabolism , Receptor, ErbB-2/metabolism , Recurrence , Remission Induction , Research Design , Survival Analysis , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/mortality
3.
Int J Mol Sci ; 20(19)2019 Oct 02.
Article in English | MEDLINE | ID: mdl-31581693

ABSTRACT

The aim of this study was to identify genes with higher expression in solid tumor cells by comparing human tumor biopsies with healthy blood samples using both in silico statistical analysis and experimental validations. This approach resulted in a novel panel of 80 RNA biomarkers with high discrimination power to detect circulating tumor cells in blood samples. To identify the 80 RNA biomarkers, Affymetrix HG-U133 plus 2.0 microarrays datasets were used to compare breast tumor tissue biopsies and breast cancer cell lines with blood samples from patients with conditions other than cancer. A total of 859 samples were analyzed at the discovery stage, consisting of 417 mammary tumors, 41 breast lines, and 401 control samples. To confirm this discovery, external datasets of eight types of tumors were used, and experimental validation studies (NanoString n-counter gene expression assay) were performed, totaling 5028 samples analyzed. In these analyses, the 80 biomarkers showed higher expression in all solid tumors analyzed relative to healthy blood samples. Experimental validation studies using NanoString assay confirmed the results were not dependent of the gene expression platform. A panel of 80 RNA biomarkers was described here, with the potential to detect solid tumor cells present in the blood of multiple tumor types.


Subject(s)
Biomarkers, Tumor , Neoplasms/genetics , Transcriptome , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Neoplastic Cells, Circulating/metabolism , Reproducibility of Results
4.
Dis Markers ; 2019: 1940347, 2019.
Article in English | MEDLINE | ID: mdl-31217830

ABSTRACT

BACKGROUND: With the development of new drug combinations and targeted treatments for multiple types of cancer, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during nonmalignant epithelial cell organization. This "cancer in reverse" approach resulted in a panel of biomarkers relevant for at least 7 cancer types, providing gene expression profiles representing key cellular signaling pathways beyond mutations in "driver genes." Objective. To further investigate this biomarker panel, the objective of the current study is to (1) validate the assay reproducibility for the 325 RNA biomarkers and (2) compare gene expression profiles side by side using two technology platforms. METHODS AND RESULTS: We have mapped the 325 RNA transcripts and in a custom NanoString nCounter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-micron thick FFPE tissue over three different days by two different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r = 0.995 for Affymetrix and r = 0.999 for NanoString. Correlation in multiple days and multiple users was for Affymetrix r = (0.962 - 0.999) and for NanoString r = (0.982 - 0.991). CONCLUSION: The 325 RNA biomarkers showed reproducibility in two technology platforms with moderate to high concordance. Future directions include performing clinical validation studies and generating rationale for patient selection in clinical trials using the technically validated assay.


Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , RNA/genetics , Biomarkers, Tumor/standards , Breast Neoplasms/pathology , Female , Humans , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotide Array Sequence Analysis/standards , RNA/standards , Reproducibility of Results
5.
Front Pharmacol ; 10: 445, 2019.
Article in English | MEDLINE | ID: mdl-31133849

ABSTRACT

Chemical genomics aims to comprehensively define, and ultimately predict, the effects of small molecule compounds on biological systems. Chemical activity profiling approaches must consider chemical effects on all pathways operative in mammalian cells. To enable a strategic and maximally efficient chemical profiling of pathway space, we have created the NCATS BioPlanet, a comprehensive integrated pathway resource that incorporates the universe of 1,658 human pathways sourced from publicly available, manually curated sources, which have been subjected to thorough redundancy and consistency cross-evaluation. BioPlanet supports interactive browsing, retrieval, and analysis of pathways, exploration of pathway connections, and pathway search by gene targets, category, and availability of corresponding bioactivity assay, as well as visualization of pathways on a 3-dimensional globe, in which the distance between any two pathways is proportional to their degree of gene component overlap. Using this resource, we propose a strategy to identify a minimal set of 362 biological assays that can interrogate the universe of human pathways. The NCATS BioPlanet is a public resource, which will be continually expanded and updated, for systems biology, toxicology, and chemical genomics, available at http://tripod.nih.gov/bioplanet/.

6.
Leukemia ; 33(1): 159-170, 2019 01.
Article in English | MEDLINE | ID: mdl-29967379

ABSTRACT

Patients with newly diagnosed multiple myeloma (NDMM) with high-risk disease are in need of new treatment strategies to improve the outcomes. Multiple clinical, cytogenetic, or gene expression features have been used to identify high-risk patients, each of which has significant weaknesses. Inclusion of molecular features into risk stratification could resolve the current challenges. In a genome-wide analysis of the largest set of molecular and clinical data established to date from NDMM, as part of the Myeloma Genome Project, we have defined DNA drivers of aggressive clinical behavior. Whole-genome and exome data from 1273 NDMM patients identified genetic factors that contribute significantly to progression free survival (PFS) and overall survival (OS) (cumulative R2 = 18.4% and 25.2%, respectively). Integrating DNA drivers and clinical data into a Cox model using 784 patients with ISS, age, PFS, OS, and genomic data, the model has a cumlative R2 of 34.3% for PFS and 46.5% for OS. A high-risk subgroup was defined by recursive partitioning using either a) bi-allelic TP53 inactivation or b) amplification (≥4 copies) of CKS1B (1q21) on the background of International Staging System III, comprising 6.1% of the population (median PFS = 15.4 months; OS = 20.7 months) that was validated in an independent dataset. Double-Hit patients have a dire prognosis despite modern therapies and should be considered for novel therapeutic approaches.


Subject(s)
Biomarkers, Tumor/genetics , Chromosome Aberrations , Genome, Human , Genomics/methods , High-Throughput Nucleotide Sequencing/methods , Multiple Myeloma/genetics , Humans , Multiple Myeloma/diagnosis , Prognosis , Risk Factors , Survival Rate
7.
Blood ; 132(6): 587-597, 2018 08 09.
Article in English | MEDLINE | ID: mdl-29884741

ABSTRACT

Understanding the profile of oncogene and tumor suppressor gene mutations with their interactions and impact on the prognosis of multiple myeloma (MM) can improve the definition of disease subsets and identify pathways important in disease pathobiology. Using integrated genomics of 1273 newly diagnosed patients with MM, we identified 63 driver genes, some of which are novel, including IDH1, IDH2, HUWE1, KLHL6, and PTPN11 Oncogene mutations are significantly more clonal than tumor suppressor mutations, indicating they may exert a bigger selective pressure. Patients with more driver gene abnormalities are associated with worse outcomes, as are identified mechanisms of genomic instability. Oncogenic dependencies were identified between mutations in driver genes, common regions of copy number change, and primary translocation and hyperdiploidy events. These dependencies included associations with t(4;14) and mutations in FGFR3, DIS3, and PRKD2; t(11;14) with mutations in CCND1 and IRF4; t(14;16) with mutations in MAF, BRAF, DIS3, and ATM; and hyperdiploidy with gain 11q, mutations in FAM46C, and MYC rearrangements. These associations indicate that the genomic landscape of myeloma is predetermined by the primary events upon which further dependencies are built, giving rise to a nonrandom accumulation of genetic hits. Understanding these dependencies may elucidate potential evolutionary patterns and lead to better treatment regimens.


Subject(s)
Gene Expression Regulation, Neoplastic , Multiple Myeloma/genetics , Mutagenesis , Oncogenes , Clone Cells , DNA Mutational Analysis , DNA, Neoplasm/genetics , Datasets as Topic , Gene Dosage , Genome-Wide Association Study , Genomic Instability , Genomics , Humans , Loss of Heterozygosity , Multiple Myeloma/pathology , Mutation , Prognosis , Translocation, Genetic , Treatment Outcome , Exome Sequencing
8.
Sci Rep ; 4: 7455, 2014 Dec 12.
Article in English | MEDLINE | ID: mdl-25503687

ABSTRACT

Most influenza pandemics have been caused by H1N1 viruses of purely or partially avian origin. Here, using Cox proportional hazard model, we attempt to identify the genetic variations in the whole genome of wild-type North American avian H1N1 influenza A viruses that are associated with their virulence in mice by residue variations, host origins of virus (Anseriformes-ducks or Charadriiformes-shorebirds), and host-residue interactions. In addition, through structural modeling, we predicted that several polymorphic sites associated with pathogenicity were located in structurally important sites, especially in the polymerase complex and NS genes. Our study introduces a new approach to identify pathogenic variations in wild-type viruses circulating in the natural reservoirs and ultimately to understand their infectious risks to humans as part of risk assessment efforts towards the emergence of future pandemic strains.


Subject(s)
Influenza A Virus, H1N1 Subtype/genetics , Influenza in Birds/virology , Animals , Charadriiformes/virology , Chick Embryo , Disease Reservoirs/virology , Ducks/virology , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza in Birds/mortality , Mice, Inbred DBA , Mutation, Missense , Polymorphism, Genetic , Proportional Hazards Models , Proteome/genetics , Viral Proteins/genetics , Virulence
9.
Nucleic Acids Res ; 42(18): 11363-82, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25249627

ABSTRACT

Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, CBP (CREBBP) and p300 (EP300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. Here we compared global CBP recruitment with gene expression in wild-type and CBP/p300 double-knockout (dKO) fibroblasts. ChIP-seq using CBP-null cells as a control revealed nearby CBP recruitment for 20% of constitutively-expressed genes, but surprisingly, three-quarters of these genes were unaffected or slightly activated in dKO cells. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak near the enhancer-like element were more predictive, with CBP/p300 deletion attenuating expression of 40% of such constitutively-expressed genes. Examining signal-responsive (Hypoxia Inducible Factor) genes showed that 97% were within 50 kilobases of an inducible CBP peak, and 70% of these required CBP/p300 for full induction. Unexpectedly, most inducible CBP peaks occurred near signal-nonresponsive genes. Finally, single-cell expression analysis revealed additional context dependence where some signal-responsive genes were not uniformly dependent on CBP/p300 in individual cells. While CBP/p300 was needed for full induction of some genes in single-cells, for other genes CBP/p300 increased the probability of maximal expression. Thus, target gene context influences the transcriptional requirement for CBP/p300, possibly by multiple mechanisms.


Subject(s)
CREB-Binding Protein/metabolism , Transcriptional Activation , 2,2'-Dipyridyl/pharmacology , Animals , CREB-Binding Protein/genetics , Cells, Cultured , Chromatin Immunoprecipitation , Enhancer Elements, Genetic , Genome , Mice , Promoter Regions, Genetic , Response Elements , Single-Cell Analysis , Transcription Initiation Site , Transcription, Genetic , p300-CBP Transcription Factors/genetics , p300-CBP Transcription Factors/metabolism
10.
J Virol ; 87(21): 11476-86, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23966381

ABSTRACT

In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2(E627K), HA(F128V), HA(F454L), or HA(H300P) variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.


Subject(s)
Adaptation, Biological , Feces/virology , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/isolation & purification , Lung/virology , RNA, Viral/genetics , Animals , DNA Mutational Analysis , Female , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Influenza A Virus, H1N1 Subtype/pathogenicity , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Molecular Sequence Data , Mutation , Sequence Analysis, DNA , Virulence
11.
Nat Methods ; 8(8): 652-4, 2011 Jun 12.
Article in English | MEDLINE | ID: mdl-21666668

ABSTRACT

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.


Subject(s)
Algorithms , DNA, Neoplasm/genetics , Genome/genetics , Neoplasms/genetics , Polymorphism, Single Nucleotide/genetics , Sequence Alignment/methods , Software , Animals , Base Pair Mismatch , Humans , Sequence Analysis, DNA/methods
12.
Immunity ; 31(6): 909-20, 2009 Dec 18.
Article in English | MEDLINE | ID: mdl-20005134

ABSTRACT

The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)(+) regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRalpha retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Peptide Fragments/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Autoantigens/immunology , Autoantigens/metabolism , Forkhead Transcription Factors/immunology , Glycoproteins/immunology , Glycoproteins/pharmacology , Interleukin-2/biosynthesis , Interleukin-2/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments/genetics , Peptide Fragments/pharmacology , Receptors, Antigen, T-Cell, alpha-beta/genetics
13.
J Proteome Res ; 8(1): 211-26, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19067583

ABSTRACT

Intrinsically disordered proteins are predicted to be highly abundant and play broad biological roles in eukaryotic cells. In particular, by virtue of their structural malleability and propensity to interact with multiple binding partners, disordered proteins are thought to be specialized for roles in signaling and regulation. However, these concepts are based on in silico analyses of translated whole genome sequences, not on large-scale analyses of proteins expressed in living cells. Therefore, whether these concepts broadly apply to expressed proteins is currently unknown. Previous studies have shown that heat-treatment of cell extracts lead to partial enrichment of soluble, disordered proteins. On the basis of this observation, we sought to address the current dearth of knowledge about expressed, disordered proteins by performing a large-scale proteomics study of thermostable proteins isolated from mouse fibroblast cells. With the use of novel multidimensional chromatography methods and mass spectrometry, we identified a total of 1320 thermostable proteins from these cells. Further, we used a variety of bioinformatics methods to analyze the structural and biological properties of these proteins. Interestingly, more than 900 of these expressed proteins were predicted to be substantially disordered. These were divided into two categories, with 514 predicted to be predominantly disordered and 395 predicted to exhibit both disordered and ordered/folded features. In addition, 411 of the thermostable proteins were predicted to be folded. Despite the use of heat treatment (60 min at 98 degrees C) to partially enrich for disordered proteins, which might have been expected to select for small proteins, the sequences of these proteins exhibited a wide range of lengths (622 +/- 555 residues (average length +/- standard deviation) for disordered proteins and 569 +/- 598 residues for folded proteins). Computational structural analyses revealed several unexpected features of the thermostable proteins: (1) disordered domains and coiled-coil domains occurred together in a large number of disordered proteins, suggesting functional interplay between these domains; and (2) more than 170 proteins contained lengthy domains (>300 residues) known to be folded. Reference to Gene Ontology Consortium functional annotations revealed that, while disordered proteins play diverse biological roles in mouse fibroblasts, they do exhibit heightened involvement in several functional categories, including, cytoskeletal structure and cell movement, metabolic and biosynthetic processes, organelle structure, cell division, gene transcription, and ribonucleoprotein complexes. We believe that these results reflect the general properties of the mouse intrinsically disordered proteome (IDP-ome) although they also reflect the specialized physiology of fibroblast cells. Large-scale identification of expressed, thermostable proteins from other cell types in the future, grown under varied physiological conditions, will dramatically expand our understanding of the structural and biological properties of disordered eukaryotic proteins.


Subject(s)
Proteomics/methods , Animals , Chromatography, Liquid/methods , Computational Biology/methods , Databases, Protein , Fibroblasts/metabolism , Mass Spectrometry/methods , Mice , NIH 3T3 Cells , Protein Conformation , Protein Folding , Proteome , Software , Temperature , Time Factors
14.
Mol Ther ; 16(3): 525-33, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18195719

ABSTRACT

Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether gamma-globin lentiviral vectors containing enhancer elements from the beta-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine beta-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.


Subject(s)
Erythrocytes/metabolism , Genetic Vectors/genetics , Lentivirus/genetics , beta-Thalassemia/blood , Animals , Cells, Cultured , Erythrocytes/cytology , Gene Expression , Globins/genetics , Globins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mutagenesis, Insertional , Oligonucleotide Array Sequence Analysis , beta-Thalassemia/pathology
15.
PLoS Pathog ; 3(11): e167, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17997603

ABSTRACT

Migratory waterfowl of the world are the natural reservoirs of influenza viruses of all known subtypes. However, it is unknown whether these waterfowl perpetuate highly pathogenic (HP) H5 and H7 avian influenza viruses. Here we report influenza virus surveillance from 2001 to 2006 in wild ducks in Alberta, Canada, and in shorebirds and gulls at Delaware Bay (New Jersey), United States, and examine the frequency of exchange of influenza viruses between the Eurasian and American virus clades, or superfamilies. Influenza viruses belonging to each of the subtypes H1 through H13 and N1 through N9 were detected in these waterfowl, but H14 and H15 were not found. Viruses of the HP Asian H5N1 subtypes were not detected, and serologic studies in adult mallard ducks provided no evidence of their circulation. The recently described H16 subtype of influenza viruses was detected in American shorebirds and gulls but not in ducks. We also found an unusual cluster of H7N3 influenza viruses in shorebirds and gulls that was able to replicate well in chickens and kill chicken embryos. Genetic analysis of 6,767 avian influenza gene segments and 248 complete avian influenza viruses supported the notion that the exchange of entire influenza viruses between the Eurasian and American clades does not occur frequently. Overall, the available evidence does not support the perpetuation of HP H5N1 influenza in migratory birds and suggests that the introduction of HP Asian H5N1 to the Americas by migratory birds is likely to be a rare event.


Subject(s)
Animal Migration , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza in Birds/transmission , Influenza in Birds/virology , Animals , Anseriformes , Asia/epidemiology , Bird Diseases/epidemiology , Bird Diseases/virology , Canada/epidemiology , Europe/epidemiology , Genes, Viral , Influenza in Birds/epidemiology , Molecular Sequence Data , Phylogeny , United States/epidemiology
16.
Hum Mol Genet ; 16(19): 2261-71, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17616514

ABSTRACT

Although cure rate of childhood acute lymphoblastic leukemia (ALL) has surpassed 80%, drug resistance remains a major cause of treatment failure. We previously identified a panel of 33 genes differentially expressed in prednisolone sensitive versus resistant ALL cells from newly diagnosed children. Here we used bioinformatics to identify resistance genes most likely to contain single nucleotide polymorphisms (SNPs) in their promoter region. The highest priority gene was SMARCB1, a core member of the SWI/SNF complex which promotes glucocorticoid effects through nucleosome remodeling. We identified several SNPs in the SMARCB1 promoter in lymphoblastoid cells from 90 individuals in the Centre d'Etude du Polymorphisme Humain (CEPH) panel. Among these SNPs, the -228G>T SNP (allele frequency 9.4%) was the only one that significantly increased reporter activity in human ALL cell lines. Furthermore, we identified nuclear protein poly (ADP-ribose) polymerase family, member 1 (PARP1) as a nuclear protein binding to the SMARCB1 promoter and showed that the -228 SNP significantly altered PARP1 binding affinity. The -228G>T SNP altered SMARCB1 mRNA and protein levels and a positive association was found between the SMARCB1 mRNA level and both the -228 genotype and prednisolone sensitivity in CEPH cell lines. Finally, knockdown experiments performed in human ALL cell lines confirmed that lower SMARCB1 expression increased prednisolone resistance. In summary, we provide functional evidence that SMARCB1 is involved in prednisolone resistance and identified a promoter SNP that alters the level of SMARCB1 mRNA and protein expression and the binding of PARP1 to the SMARCB1 promoter.


Subject(s)
Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Gene Expression/drug effects , Poly(ADP-ribose) Polymerases/genetics , Steroids/pharmacology , Transcription Factors/genetics , Amino Acid Sequence , Blotting, Western , Cell Line , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Mapping , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Gene Frequency , Genotype , Humans , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Polymorphism, Single Nucleotide , Prednisolone/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , SMARCB1 Protein , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transcription Factors/metabolism
17.
J Virol ; 81(19): 10292-9, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17652405

ABSTRACT

Avian influenza viruses have adapted to human hosts, causing pandemics in humans. The key host-specific amino acid mutations required for an avian influenza virus to function in humans are unknown. Through multiple-sequence alignment and statistical testing of each aligned amino acid, we identified markers that discriminate human influenza viruses from avian influenza viruses. We applied strict thresholds to select only markers which are highly preserved in human influenza virus isolates over time. We found that a subset of these persistent host markers exist in all human pandemic influenza virus sequences from 1918, 1957, and 1968, while others are acquired as the virus becomes a seasonal influenza virus. We also show that human H5N1 influenza viruses are significantly more likely to contain the amino acid predominant in human strains for a few persistent host markers than avian H5N1 influenza viruses. This sporadic enrichment of amino acids present in human-hosted viruses may indicate that some H5N1 viruses have made modest adaptations to their new hosts in the recent past. The markers reported here should be useful in monitoring potential pandemic influenza viruses.


Subject(s)
Birds/virology , Disease Outbreaks , Influenza A Virus, H5N1 Subtype/genetics , Influenza in Birds/virology , Influenza, Human/virology , Amino Acid Substitution/genetics , Animals , Evolution, Molecular , Genetic Markers , Humans , Influenza A Virus, H5N1 Subtype/isolation & purification , Influenza in Birds/epidemiology , Influenza, Human/epidemiology , Mutagenesis , Seasons , Sequence Alignment , Sequence Analysis, Protein , Sequence Analysis, RNA , Viral Proteins/genetics
18.
J Proteome Res ; 5(10): 2839-48, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17022655

ABSTRACT

Intrinsically unstructured proteins (IUPs) represent an important class of proteins primarily involved in cellular signaling and regulation. The aim of this study was to develop methodology for the enrichment and identification of IUPs. We show that heat treatment of NIH3T3 mouse fibroblast cell extracts at 98 degrees C selects for IUPs. The majority of these IUPs were cytosolic or nuclear proteins involved in cell signaling or regulation. These studies represent the first large-scale experimental investigation of the intrinsically unstructured mammalian proteome.


Subject(s)
Proteins/analysis , Proteome/analysis , Proteomics/methods , Signal Transduction , Animals , Cell Nucleus/chemistry , Cytosol/chemistry , Electrophoresis, Gel, Two-Dimensional , Hot Temperature , Hydrophobic and Hydrophilic Interactions , Mice , NIH 3T3 Cells , Nuclear Proteins/analysis , Protein Conformation , Protein Denaturation
19.
Science ; 311(5767): 1576-80, 2006 Mar 17.
Article in English | MEDLINE | ID: mdl-16439620

ABSTRACT

The spread of H5N1 avian influenza viruses (AIVs) from China to Europe has raised global concern about their potential to infect humans and cause a pandemic. In spite of their substantial threat to human health, remarkably little AIV whole-genome information is available. We report here a preliminary analysis of the first large-scale sequencing of AIVs, including 2196 AIV genes and 169 complete genomes. We combine this new information with public AIV data to identify new gene alleles, persistent genotypes, compensatory mutations, and a potential virulence determinant.


Subject(s)
Genes, Viral , Influenza A Virus, H5N1 Subtype/genetics , Influenza A virus/genetics , Viral Nonstructural Proteins/chemistry , Virulence Factors/chemistry , Animals , Birds/virology , Computational Biology , Genome, Viral , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H2N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N8 Subtype/genetics , Influenza A Virus, H5N1 Subtype/chemistry , Influenza A Virus, H5N1 Subtype/pathogenicity , Influenza A Virus, H5N2 Subtype/genetics , Influenza A Virus, H7N7 Subtype/genetics , Influenza A Virus, H9N2 Subtype/genetics , Influenza A virus/chemistry , Influenza A virus/isolation & purification , Influenza A virus/pathogenicity , Influenza in Birds/virology , Influenza, Human/virology , Molecular Sequence Data , Mutation , Phylogeny , RNA, Viral/genetics , Reassortant Viruses/genetics , Sequence Analysis, DNA , Viral Nonstructural Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Virulence Factors/genetics
20.
J Med Chem ; 48(8): 2906-15, 2005 Apr 21.
Article in English | MEDLINE | ID: mdl-15828829

ABSTRACT

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.


Subject(s)
Carboxylesterase/antagonists & inhibitors , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/chemistry , Acetylcholinesterase/chemistry , Animals , Butyrylcholinesterase/chemistry , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/chemistry , Cholinesterase Inhibitors/chemistry , Databases, Factual , Humans , Intestines/enzymology , Models, Molecular , Phenylglyoxal/chemical synthesis , Quantitative Structure-Activity Relationship , Rats , Structure-Activity Relationship , Umbelliferones/chemistry
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