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1.
Nat Commun ; 10(1): 1893, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015461

RESUMO

Birthweight is associated with health outcomes across the life course, DNA methylation may be an underlying mechanism. In this meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, we find that DNA methylation in neonatal blood is associated with birthweight at 914 sites, with a difference in birthweight ranging from -183 to 178 grams per 10% increase in methylation (PBonferroni < 1.06 x 10-7). In additional analyses in 7,278 participants, <1.3% of birthweight-associated differential methylation is also observed in childhood and adolescence, but not adulthood. Birthweight-related CpGs overlap with some Bonferroni-significant CpGs that were previously reported to be related to maternal smoking (55/914, p = 6.12 x 10-74) and BMI in pregnancy (3/914, p = 1.13x10-3), but not with those related to folate levels in pregnancy. Whether the associations that we observe are causal or explained by confounding or fetal growth influencing DNA methylation (i.e. reverse causality) requires further research.


Assuntos
Peso ao Nascer/genética , DNA/metabolismo , Epigênese Genética , Genoma Humano , Adolescente , Adulto , Índice de Massa Corporal , Criança , Ilhas de CpG , DNA/genética , Metilação de DNA , Feminino , Desenvolvimento Fetal/genética , Feto , Ácido Fólico/sangue , Estudo de Associação Genômica Ampla , Humanos , Recém-Nascido , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Fumar/efeitos adversos , Fumar/sangue , Fumar/genética
2.
Carcinogenesis ; 2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30698678

RESUMO

p53 is activated by DNA damage and oncogenic stimuli to regulate senescence, apoptosis and cell cycle arrest which are essential to prevent cancer. Here, we utilized UVB radiation, a potent inducer of DNA damage, p53, apoptosis and skin cancer to investigate the mechanism of C/EBPß in regulating p53-mediated apoptosis in keratinocytes and to test whether the deletion of C/EBPß in epidermis can protect mice from UVB-induced skin cancer. UVB-treatment of C/EBPß skin conditional knockout (CKOß) mice increased p53 protein levels in epidermis and enhanced p53-dependent apoptotic activity 3-fold compared to UVB-treated control mice. UVB increased C/EBPß levels through a p53-dependent pathway and stimulated the formation of a C/EBPß-p53 protein complex; knockdown of C/EBPß increased p53 protein stability in keratinocytes. These results suggest a p53-C/EBPß feedback loop, whereby C/EBPß a transcriptional target of a p53 pathway, functions as a survival factor by negatively regulating p53 apoptotic activity in response to DNA damage. RNAseq analysis of UVB-treated CKOß epidermis unexpectedly revealed that type 1 interferon (IFN) pathway was the most highly enriched pathway. Numerous proapoptotic ISGs were up-regulated including some known to enhance p53 apoptosis. Our results indicate that p53 and IFN pathways function together in response to DNA damage to result in the activation of extrinsic apoptosis pathways and caspase 8 cleavage. Lastly, we observed CKOß mice were resistant to UVB-induced skin cancer. Our results suggest C/EBPß represses apoptosis through keratinocyte autonomous suppression of the type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer.

3.
J Allergy Clin Immunol ; 143(6): 2062-2074, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30579849

RESUMO

BACKGROUND: Epigenetic mechanisms, including methylation, can contribute to childhood asthma. Identifying DNA methylation profiles in asthmatic patients can inform disease pathogenesis. OBJECTIVE: We sought to identify differential DNA methylation in newborns and children related to childhood asthma. METHODS: Within the Pregnancy And Childhood Epigenetics consortium, we performed epigenome-wide meta-analyses of school-age asthma in relation to CpG methylation (Illumina450K) in blood measured either in newborns, in prospective analyses, or cross-sectionally in school-aged children. We also identified differentially methylated regions. RESULTS: In newborns (8 cohorts, 668 cases), 9 CpGs (and 35 regions) were differentially methylated (epigenome-wide significance, false discovery rate < 0.05) in relation to asthma development. In a cross-sectional meta-analysis of asthma and methylation in children (9 cohorts, 631 cases), we identified 179 CpGs (false discovery rate < 0.05) and 36 differentially methylated regions. In replication studies of methylation in other tissues, most of the 179 CpGs discovered in blood replicated, despite smaller sample sizes, in studies of nasal respiratory epithelium or eosinophils. Pathway analyses highlighted enrichment for asthma-relevant immune processes and overlap in pathways enriched both in newborns and children. Gene expression correlated with methylation at most loci. Functional annotation supports a regulatory effect on gene expression at many asthma-associated CpGs. Several implicated genes are targets for approved or experimental drugs, including IL5RA and KCNH2. CONCLUSION: Novel loci differentially methylated in newborns represent potential biomarkers of risk of asthma by school age. Cross-sectional associations in children can reflect both risk for and effects of disease. Asthma-related differential methylation in blood in children was substantially replicated in eosinophils and respiratory epithelium.

4.
Cell Death Dis ; 9(11): 1054, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323292

RESUMO

Therapeutic targeting of specific genetic changes in cancer has proven to be an effective therapy and the concept of synthetic lethality has emerged. CCAAT/enhancer-binding protein-ß (C/EBPß), a basic leucine zipper transcription factor, has important roles in cellular processes including differentiation, inflammation, survival, and energy metabolism. Using a genetically engineered mouse model, we report that the deletion C/EBPß in pre-existing oncogenic Ha-Ras mouse skin tumors in vivo resulted in rapid tumor regression. Regressing tumors exhibited elevated levels of apoptosis and p53 protein/activity, while adjacent C/EBPß-deleted skin did not. These results indicate that the deletion of C/EBPß de-represses p53 in oncogenic Ras tumors but not in normal wild-type Ras keratinocytes, and that C/EBPß is essential for survival of oncogenic Ras tumors. Co-deletion of C/EBPß and p53 in oncogenic Ras tumors showed p53 is required for tumor regression and elevated apoptosis. In tumors, loss of a pathway that confers adaptability to a stress phenotype of cancer/tumorigenesis, such as DNA damage, could result in selective tumor cell killing. Our results show that oncogenic Ras tumors display a significant DNA damage/replicative stress phenotype and these tumors have acquired a dependence on C/EBPß for their survival. RNAseq data analysis of regressing tumors deleted of C/EBPß indicates a novel interface between p53, type-1 interferon response, and death receptor pathways, which function in concert to produce activation of extrinsic apoptosis pathways. In summary, the deletion of C/EBPß in oncogenic Ras skin tumors is a synthetic lethal event, making it a promising target for future potential anticancer therapies.

5.
Environ Health Perspect ; 126(3): 037003, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29529597

RESUMO

BACKGROUND: Imprinted genes are defined by their preferential expression from one of the two parental alleles. This unique mode of gene expression is dependent on allele-specific DNA methylation profiles established at regulatory sequences called imprinting control regions (ICRs). These loci have been used as biosensors to study how environmental exposures affect methylation and transcription. However, a critical unanswered question is whether they are more, less, or equally sensitive to environmental stressors as the rest of the genome. OBJECTIVES: Using cadmium exposure in humans as a model, we aimed to determine the relative sensitivity of ICRs to perturbation of methylation compared to similar, nonimprinted loci in the genome. METHODS: We assayed DNA methylation genome-wide using bisulfite sequencing of 19 newborn cord blood and 20 maternal blood samples selected on the basis of maternal blood cadmium levels. Differentially methylated regions (DMRs) associated with cadmium exposure were identified. RESULTS: In newborn cord blood and maternal blood, 641 and 1,945 cadmium-associated DMRs were identified, respectively. DMRs were more common at the 15 maternally methylated ICRs than at similar nonimprinted loci in newborn cord blood (p=5.64×10-8) and maternal blood (p=6.22×10-14), suggesting a higher sensitivity for ICRs to cadmium. Genome-wide, Enrichr analysis indicated that the top three functional categories for genes that overlapped DMRs in maternal blood were body mass index (BMI) (p=2.0×10-5), blood pressure (p=3.8×10-5), and body weight (p=0.0014). In newborn cord blood, the top three functional categories were BMI, atrial fibrillation, and hypertension, although associations were not significant after correction for multiple testing (p=0.098). These findings suggest that epigenetic changes may contribute to the etiology of cadmium-associated diseases. CONCLUSIONS: We analyzed cord blood and maternal blood DNA methylation profiles genome-wide at nucleotide resolution in individuals selected for high and low blood cadmium levels in the first trimester. Our findings suggest that ICRs may be hot spots for perturbation by cadmium, motivating further study of these loci to investigate potential mechanisms of cadmium action. https://doi.org/10.1289/EHP2085.

6.
mSphere ; 2(6)2017 Nov-Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29202043

RESUMO

Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in "clumps," and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

7.
Front Genet ; 8: 168, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29163636

RESUMO

Cell-based assays are an attractive option to measure gene expression response to exposure, but the cost of whole-transcriptome RNA sequencing has been a barrier to the use of gene expression profiling for in vitro toxicity screening. In addition, standard RNA sequencing adds variability due to variable transcript length and amplification. Targeted probe-sequencing technologies such as TempO-Seq, with transcriptomic representation that can vary from hundreds of genes to the entire transcriptome, may reduce some components of variation. Analyses of high-throughput toxicogenomics data require renewed attention to read-calling algorithms and simplified dose-response modeling for datasets with relatively few samples. Using data from induced pluripotent stem cell-derived cardiomyocytes treated with chemicals at varying concentrations, we describe here and make available a pipeline for handling expression data generated by TempO-Seq to align reads, clean and normalize raw count data, identify differentially expressed genes, and calculate transcriptomic concentration-response points of departure. The methods are extensible to other forms of concentration-response gene-expression data, and we discuss the utility of the methods for assessing variation in susceptibility and the diseased cellular state.

8.
J Leukoc Biol ; 102(6): 1371-1380, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29021367

RESUMO

The vertebrate immune response comprises multiple molecular and cellular components that interface to provide defense against pathogens. Because of the dynamic complexity of the immune system and its interdependent innate and adaptive functionality, an understanding of the whole-organism response to pathogen exposure remains unresolved. Zebrafish larvae provide a unique model for overcoming this obstacle, because larvae are protected against pathogens while lacking a functional adaptive immune system during the first few weeks of life. Zebrafish larvae were exposed to immune agonists for various lengths of time, and a microarray transcriptome analysis was executed. This strategy identified known immune response genes, as well as genes with unknown immune function, including the E3 ubiquitin ligase tripartite motif-9 (Trim9). Although trim9 expression was originally described as "brain specific," its expression has been reported in stimulated human Mϕs. In this study, we found elevated levels of trim9 transcripts in vivo in zebrafish Mϕs after immune stimulation. Trim9 has been implicated in axonal migration, and we therefore investigated the impact of Trim9 disruption on Mϕ motility and found that Mϕ chemotaxis and cellular architecture are subsequently impaired in vivo. These results demonstrate that Trim9 mediates cellular movement and migration in Mϕs as well as neurons.


Assuntos
Movimento Celular , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Movimento Celular/genética , Forma Celular , Quimiotaxia , Humanos , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas com Motivo Tripartido/genética , Células U937 , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/genética
9.
Appl Environ Microbiol ; 83(21)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28842547

RESUMO

In the foodborne pathogen Listeria monocytogenes, arsenic resistance is encountered primarily in serotype 4b clones considered to have enhanced virulence and is associated with an arsenic resistance gene cluster within a 35-kb chromosomal region, Listeria genomic island 2 (LGI2). LGI2 was first identified in strain Scott A and includes genes putatively involved in arsenic and cadmium resistance, DNA integration, conjugation, and pathogenicity. However, the genomic localization and sequence content of LGI2 remain poorly characterized. Here we investigated 85 arsenic-resistant L. monocytogenes strains, mostly of serotype 4b. All but one of the 70 serotype 4b strains belonged to clonal complex 1 (CC1), CC2, and CC4, three major clones associated with enhanced virulence. PCR analysis suggested that 53 strains (62.4%) harbored an island highly similar to LGI2 of Scott A, frequently (42/53) in the same location as Scott A (LMOf2365_2257 homolog). Random-primed PCR and whole-genome sequencing revealed seven novel insertion sites, mostly internal to chromosomal coding sequences, among strains harboring LGI2 outside the LMOf2365_2257 homolog. Interestingly, many CC1 strains harbored a noticeably diversified LGI2 (LGI2-1) in a unique location (LMOf2365_0902 homolog) and with a novel additional gene. With few exceptions, the tested LGI2 genes were not detected in arsenic-resistant strains of serogroup 1/2, which instead often harbored a Tn554-associated arsenic resistance determinant not encountered in serotype 4b. These findings indicate that in L. monocytogenes, LGI2 has a propensity for certain serotype 4b clones, exhibits content diversity, and is highly promiscuous, suggesting an ability to mobilize various accessory genes into diverse chromosomal loci.IMPORTANCEListeria monocytogenes is widely distributed in the environment and causes listeriosis, a foodborne disease with high mortality and morbidity. Arsenic and other heavy metals can powerfully shape the populations of human pathogens with pronounced environmental lifestyles such as L. monocytogenes Arsenic resistance is encountered primarily in certain serotype 4b clones considered to have enhanced virulence and is associated with a large chromosomal island, Listeria genomic island 2 (LGI2). LGI2 also harbors a cadmium resistance cassette and genes putatively involved in DNA integration, conjugation, and pathogenicity. Our findings indicate that LGI2 exhibits pronounced content plasticity and is capable of transferring various accessory genes into diverse chromosomal locations. LGI2 may serve as a paradigm on how exposure to a potent environmental toxicant such as arsenic may have dynamically selected for arsenic-resistant subpopulations in certain clones of L. monocytogenes which also contribute significantly to disease.


Assuntos
Arsênico/metabolismo , Ilhas Genômicas , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Variação Genética , Humanos , Listeria monocytogenes/metabolismo , Virulência
10.
Genome Announc ; 5(19)2017 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-28495780

RESUMO

Listeria monocytogenes frequently exhibits resistance to arsenic. We report here the draft genome sequences of eight genetically diverse arsenic-resistant L. monocytogenes strains from human listeriosis and food-associated environments. The availability of these genomes will help elucidate the role of heavy-metal resistance in the ecology of L. monocytogenes.

11.
Mol Cancer Res ; 15(2): 165-178, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27856957

RESUMO

Lipolysis-stimulated lipoprotein receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens. We found LSR within the membrane, cytoplasm, and the nucleus of breast cancer cells representing multiple intrinsic subtypes. Chromatin immunoprecipitation (ChIP) showed direct binding of LSR to DNA, and sequence analysis identified putative functional motifs and post-translational modifications of the LSR protein. While neither overexpression of transcript variants, nor pharmacologic manipulation of post-translational modification significantly altered localization, inhibition of nuclear export enhanced nuclear localization, suggesting a mechanism for nuclear retention. Coimmunoprecipitation and proximal ligation assays indicated LSR-pericentrin interactions, presenting potential mechanisms for nuclear-localized LSR. The clinical significance of LSR was evaluated using data from over 1,100 primary breast tumors, which showed high LSR levels in basal-like tumors and tumors from African-Americans. In tumors histosections, nuclear localization was significantly associated with poor outcomes. Finally, in vivo xenograft studies revealed that basal-like breast cancer cells that overexpress LSR exhibited both membrane and nuclear localization, and developed tumors with 100% penetrance, while control cells lacking LSR developed no tumors. These results show that nuclear LSR alters gene expression and may promote aggressive cancer phenotypes. IMPLICATIONS: LSR functions in the promotion of aggressive breast cancer phenotypes and poor patient outcome via differential subcellular localization to alter cell signaling, bioenergetics, and gene expression. Mol Cancer Res; 15(2); 165-78. ©2016 AACR.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/metabolismo , Receptores de LDL/metabolismo , Animais , Neoplasias da Mama/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Xenoenxertos , Humanos , Camundongos , Receptores de LDL/biossíntese , Receptores de LDL/genética
12.
Int J Syst Evol Microbiol ; 66(11): 4512-4517, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27506201

RESUMO

A novel rickettsial agent, 'Candidatus Rickettsia asembonensis' strain NMRCiiT, was isolated from cat fleas, Ctenocephalides felis, from Kenya. Genotypic characterization of the new isolate based on sequence analysis of five rickettsial genes, rrs, gltA, ompA, ompB and sca4, indicated that this isolate clustered with Rickettsia felis URRWXCal2. The degree of nucleotide similarity demonstrated that isolate NMRCiiT belongs within the genus Rickettsia and fulfils the criteria for classification as a representative of a novel species. The name Rickettsia asembonensis sp. nov. is proposed, with NMRCiiT (=DSM 100172T=CDC CRIRC RAS001T=ATCC VR-1827T) as the type strain.


Assuntos
Ctenocephalides/microbiologia , Filogenia , Rickettsia/classificação , Animais , Técnicas de Tipagem Bacteriana , Gatos , DNA Bacteriano/genética , Genes Bacterianos , Quênia , RNA Ribossômico 16S/genética , Rickettsia/genética , Rickettsia/isolamento & purificação , Análise de Sequência de DNA
13.
Genome Announc ; 4(1)2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26823588

RESUMO

Here, we present the genome sequences of one mesonivirus and four novel arboviruses observed in Culex bitaeniorhynchus and Culex pipiens, captured in and near the demilitarized zone, Republic of Korea. Results suggest the ubiquitous presence of mesoniviruses and the discovery of a potentially new species of arboviruses in field-captured mosquitoes.

14.
Environ Epigenet ; 2(1)2016.
Artigo em Inglês | MEDLINE | ID: mdl-28123784

RESUMO

Prenatal exposure to lead (Pb) is known to decrease fetal growth; but its effects on postnatal growth and mechanistic insights linking Pb to growth are not clearly defined. Genomically imprinted genes are powerful regulators of growth and energy utilization, and may be particularly vulnerable to environmental Pb exposure. Because imprinting is established early and maintained via DNA methylation, we hypothesized that prenatal Pb exposure alters DNA methylation of imprinted genes resulting in lower birth weight and rapid growth. Pb was measured by inductively coupled plasma mass spectrometry (ICP-MS) in peripheral blood of 321 women of the Newborn Epigenetic STudy (NEST) obtained at gestation ~12 weeks. Linear and logistic regression models were used to evaluate associations between maternal Pb levels, methylation of differentially methylated regions (DMRs) regulating H19, MEG3, PEG3, and PLAGL1, measured by pyrosequencing, birth weight, and weight-for-height z score gains between birth and age 1yr, ages 1-2yrs, and 2-3yrs. Children born to women with Pb levels in the upper tertile had higher methylation of the regulatory region of the MEG3 DMR imprinted domain (ß= 1.57, se= 0.82, p= 0.06). Pb levels were also associated with lower birth weight (ß= -0.41, se= 0.15, p= 0.01) and rapid gains in adiposity (OR= 12.32, 95%CI=1.25-121.30, p= 0.03) by age 2-3 years. These data provide early human evidence for Pb associations with hypermethylation at the MEG3 DMR regulatory region and rapid adiposity gain-a risk factor for childhood obesity and cardiometabolic diseases in adulthood.

15.
BMC Bioinformatics ; 16: 416, 2015 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-26714571

RESUMO

BACKGROUND: The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. RESULTS: The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. CONCLUSIONS: By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available.


Assuntos
Interface Usuário-Computador , Algoritmos , Animais , Arenavirus/genética , Arenavirus/isolamento & purificação , Biologia Computacional , Coronavirus/genética , Coronavirus/isolamento & purificação , Bases de Dados Factuais , Flavivirus/genética , Flavivirus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Internet , RNA Viral/química , RNA Viral/metabolismo , Análise de Sequência de RNA
16.
BMC Genomics ; 16: 952, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26573221

RESUMO

BACKGROUND: Human erythrocytes are terminally differentiated, anucleate cells long thought to lack RNAs. However, previous studies have shown the persistence of many small-sized RNAs in erythrocytes. To comprehensively define the erythrocyte transcriptome, we used high-throughput sequencing to identify both short (18-24 nt) and long (>200 nt) RNAs in mature erythrocytes. RESULTS: Analysis of the short RNA transcriptome with miRDeep identified 287 known and 72 putative novel microRNAs. Unexpectedly, we also uncover an extensive repertoire of long erythrocyte RNAs that encode many proteins critical for erythrocyte differentiation and function. Additionally, the erythrocyte long RNA transcriptome is significantly enriched in the erythroid progenitor transcriptome. Joint analysis of both short and long RNAs identified several loci with co-expression of both microRNAs and long RNAs spanning microRNA precursor regions. Within the miR-144/451 locus previously implicated in erythroid development, we observed unique co-expression of several primate-specific noncoding RNAs, including a lncRNA, and miR-4732-5p/-3p. We show that miR-4732-3p targets both SMAD2 and SMAD4, two critical components of the TGF-ß pathway implicated in erythropoiesis. Furthermore, miR-4732-3p represses SMAD2/4-dependent TGF-ß signaling, thereby promoting cell proliferation during erythroid differentiation. CONCLUSIONS: Our study presents the most extensive profiling of erythrocyte RNAs to date, and describes primate-specific interactions between the key modulator miR-4732-3p and TGF-ß signaling during human erythropoiesis.


Assuntos
Eritrócitos/metabolismo , Perfilação da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Sequência de Bases , Diferenciação Celular/genética , Eritrócitos/citologia , Loci Gênicos/genética , Humanos , Transdução de Sinais/genética , Proteína Smad2/biossíntese , Proteína Smad4/biossíntese , Fator de Crescimento Transformador beta/metabolismo
17.
Genome Announc ; 3(2)2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25767219

RESUMO

Herein we present the draft genome sequence and annotation of "Candidatus Rickettsia asemboensis" strain NMRCii. "Ca. Rickettsia asemboensis" is phylogenetically related to but distinct from the flea-borne spotted fever pathogen Rickettsia felis. "Ca. Rickettsia asemboensis" was initially identified in and subsequently isolated from Ctenocephalides cat and dog fleas from Kenya.

18.
Clin Cancer Res ; 19(5): 1106-15, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23300274

RESUMO

PURPOSE: The phosphoinositide 3-kinase (PI3K) pathway is known to play an active role in many malignancies. The role of PI3K inhibition in the treatment of lymphomas has not been fully delineated. We sought to identify a role for therapeutic PI3K inhibition across a range of B-cell lymphomas. EXPERIMENTAL DESIGN: We selected three small molecule inhibitors to test in a panel of 60 cell lines that comprised diverse lymphoma types. We tested the selective PI3K inhibitor BKM120 and the dual PI3K/mTOR inhibitors BEZ235 and BGT226 in these cell lines. We applied gene expression profiling to better understand the molecular mechanisms associated with responsiveness to these drugs. RESULTS: We found that higher expression of the PAK1 gene was significantly associated with resistance to all three PI3K inhibitors. Through RNA-interference-mediated knockdown of the PAK1 gene, we showed a dramatic increase in the sensitivity to PI3K inhibition. We further tested a small-molecule inhibitor of PAK1 and found significant synergy between PI3K and PAK1 inhibition. CONCLUSION: Thus, we show that PI3K inhibition is broadly effective in lymphomas and PAK1 is a key modulator of resistance to PI3K inhibition.


Assuntos
Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Linfoma/tratamento farmacológico , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinases Ativadas por p21/metabolismo , Aminopiridinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Linfoma/genética , Linfoma/metabolismo , Morfolinas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Bibliotecas de Moléculas Pequenas , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética
19.
Blood ; 116(23): e118-27, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-20733160

RESUMO

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-ß pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.


Assuntos
Linfócitos B , Perfilação da Expressão Gênica/métodos , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/genética , Sequência de Bases , Imunoprecipitação da Cromatina , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/análise , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
20.
Mol Immunol ; 46(7): 1505-16, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19200601

RESUMO

A recessive nonsense mutation in the zebrafish recombination activating gene 1 (rag1) gene results in defective V(D)J recombination; however, animals homozygous for this mutation (rag1(-/-)) are reportedly viable and fertile in standard, nonsterile aquarium conditions but display increased mortality after intraperitoneal injection with mycobacteria. Based on their survival in nonsterile environments, we hypothesized that the rag1(-/-) zebrafish may possess an "enhanced" innate immune response to compensate for the lack of an adaptive immune system. To test this hypothesis, microarray analyses were used to compare the expression profiles of the intestines and hematopoietic kidneys of rag1 deficient zebrafish to the expression profiles of control (heterozygous) siblings. The expression levels of 12 genes were significantly altered in the rag1(-/-) kidney including the up regulation of a putative interferon stimulated gene, and the down regulation of genes encoding fatty acid binding protein 10, keratin 5 and multiple heat shock proteins. The expression levels of 87 genes were shown to be significantly altered in the rag1(-/-) intestine; the majority of these differences reflect increased expression of innate immune genes, including those of the coagulation and complement pathways. Subsequent analyses of orthologous coagulation and complement genes in Rag1(-/-) mice indicate increased transcription of the complement C4 gene in the Rag1(-/-) intestine.


Assuntos
Fatores de Coagulação Sanguínea/genética , Proteínas do Sistema Complemento/genética , Imunidade/fisiologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Animais , Animais Geneticamente Modificados , Fatores de Coagulação Sanguínea/metabolismo , Complemento C4/genética , Complemento C4/metabolismo , Proteínas do Sistema Complemento/metabolismo , Feminino , Perfilação da Expressão Gênica , Genes RAG-1/fisiologia , Imunidade/genética , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Genética , Regulação para Cima , Peixe-Zebra
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