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1.
Immunity ; 42(4): 679-91, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25902482

RESUMO

Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.


Assuntos
Ilhas de CpG/imunologia , Epigênese Genética , Macrófagos Peritoneais/imunologia , Proteína 2 de Ligação a Metil-CpG/imunologia , Microglia/imunologia , Síndrome de Rett/imunologia , Animais , Receptor 1 de Quimiocina CX3C , Metilação de DNA , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase/imunologia , Humanos , Integrases/genética , Integrases/imunologia , Longevidade/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/patologia , Masculino , Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/patologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Síndrome de Rett/genética , Síndrome de Rett/patologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
2.
Dev Biol ; 481: 14-29, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34543654

RESUMO

Environmental teratogens such as smoking are known risk factors for developmental disorders such as cleft palate. While smoking rates have declined, a new type of smoking, called vaping is on the rise. Vaping is the use of e-cigarettes to vaporize and inhale an e-liquid containing nicotine and food-like flavors. There is the potential that, like smoking, vaping could also pose a danger to the developing human. Rather than waiting for epidemiological and mammalian studies, we have turned to an aquatic developmental model, Xenopus laevis, to more quickly assess whether e-liquids contain teratogens that could lead to craniofacial malformations. Xenopus, like zebrafish, has the benefit of being a well-established developmental model and has also been effective in predicting whether a chemical could be a teratogen. We have determined that embryonic exposure to dessert flavored e-liquids can cause craniofacial abnormalities, including an orofacial cleft in Xenopus. To better understand the underlying mechanisms contributing to these defects, transcriptomic analysis of the facial tissues of embryos exposed to a representative dessert flavored e-liquid vapor extract was performed. Analysis of differentially expressed genes in these embryos revealed several genes associated with retinoic acid metabolism or the signaling pathway. Consistently, retinoic acid receptor inhibition phenocopied the craniofacial defects as those embryos exposed to the vapor extract of the e-liquid. Such malformations also correlated with a group of common differentially expressed genes, two of which are associated with midface birth defects in humans. Further, e-liquid exposure sensitized embryos to forming craniofacial malformations when they already had depressed retinoic acid signaling. Moreover, 13-cis-retinoic acid treatment could significantly reduce the e-liquid induced malformation in the midface. Such results suggest the possibility of an interaction between retinoic acid signaling and e-liquid exposure. One of the most popular and concentrated flavoring chemicals in dessert flavored e-liquids is vanillin. Xenopus embryos exposed to this chemical closely resembled embryos exposed to dessert-like e-liquids and a retinoic acid receptor antagonist. In summary, we determined that e-liquid chemicals, in particular vanillin, can cause craniofacial defects potentially by dysregulating retinoic acid signaling. This work warrants the evaluation of vanillin and other such flavoring additives in e-liquids on mammalian development.


Assuntos
Benzaldeídos/administração & dosagem , Anormalidades Craniofaciais , Embrião não Mamífero/embriologia , Aromatizantes/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Produtos do Tabaco/toxicidade , Tretinoína/metabolismo , Animais , Benzaldeídos/farmacologia , Anormalidades Craniofaciais/induzido quimicamente , Anormalidades Craniofaciais/embriologia , Embrião não Mamífero/patologia , Aromatizantes/farmacologia , Xenopus laevis
3.
Nature ; 535(7612): 425-9, 2016 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-27409813

RESUMO

Immune dysfunction is commonly associated with several neurological and mental disorders. Although the mechanisms by which peripheral immunity may influence neuronal function are largely unknown, recent findings implicate meningeal immunity influencing behaviour, such as spatial learning and memory. Here we show that meningeal immunity is also critical for social behaviour; mice deficient in adaptive immunity exhibit social deficits and hyper-connectivity of fronto-cortical brain regions. Associations between rodent transcriptomes from brain and cellular transcriptomes in response to T-cell-derived cytokines suggest a strong interaction between social behaviour and interferon-γ (IFN-γ)-driven responses. Concordantly, we demonstrate that inhibitory neurons respond to IFN-γ and increase GABAergic (γ-aminobutyric-acid) currents in projection neurons, suggesting that IFN-γ is a molecular link between meningeal immunity and neural circuits recruited for social behaviour. Meta-analysis of the transcriptomes of a range of organisms reveals that rodents, fish, and flies elevate IFN-γ/JAK-STAT-dependent gene signatures in a social context, suggesting that the IFN-γ signalling pathway could mediate a co-evolutionary link between social/aggregation behaviour and an efficient anti-pathogen response. This study implicates adaptive immune dysfunction, in particular IFN-γ, in disorders characterized by social dysfunction and suggests a co-evolutionary link between social behaviour and an anti-pathogen immune response driven by IFN-γ signalling.


Assuntos
Interferon gama/fisiologia , Vias Neurais , Comportamento Social , Animais , Drosophila melanogaster/genética , Feminino , Neurônios GABAérgicos/metabolismo , Masculino , Meninges/citologia , Meninges/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/metabolismo , Ratos , Transdução de Sinais , Linfócitos T/imunologia , Transcriptoma , Peixe-Zebra/genética
4.
Nature ; 539(7630): 570-574, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27820945

RESUMO

Professional phagocytes (such as macrophages) and non-professional phagocytes (such as epithelial cells) clear billions of apoptotic cells and particles on a daily basis. Although professional and non-professional macrophages reside in proximity in most tissues, whether they communicate with each other during cell clearance, and how this might affect inflammation, is not known. Here we show that macrophages, through the release of a soluble growth factor and microvesicles, alter the type of particles engulfed by non-professional phagocytes and influence their inflammatory response. During phagocytosis of apoptotic cells or in response to inflammation-associated cytokines, macrophages released insulin-like growth factor 1 (IGF-1). The binding of IGF-1 to its receptor on non-professional phagocytes redirected their phagocytosis, such that uptake of larger apoptotic cells was reduced whereas engulfment of microvesicles was increased. IGF-1 did not alter engulfment by macrophages. Macrophages also released microvesicles, whose uptake by epithelial cells was enhanced by IGF-1 and led to decreased inflammatory responses by epithelial cells. Consistent with these observations, deletion of IGF-1 receptor in airway epithelial cells led to exacerbated lung inflammation after allergen exposure. These genetic and functional studies reveal that IGF-1- and microvesicle-dependent communication between macrophages and epithelial cells can critically influence the magnitude of tissue inflammation in vivo.


Assuntos
Células Epiteliais/citologia , Macrófagos/citologia , Fagócitos/citologia , Fagocitose , Pneumonia , Alérgenos/imunologia , Animais , Apoptose , Comunicação Celular , Citocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Fagócitos/imunologia , Fagócitos/metabolismo , Pneumonia/imunologia , Pneumonia/metabolismo , Receptor IGF Tipo 1/deficiência , Receptor IGF Tipo 1/metabolismo , Sistema Respiratório/citologia , Somatomedinas/metabolismo
5.
Cancer Immunol Immunother ; 70(8): 2151-2164, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33454795

RESUMO

The strength and durability of systemic anti-tumor immune responses induced by cancer vaccines depends on adjuvants to support an immunogenic vaccine site microenvironment (VSME). Adjuvants include water-in-oil emulsions with incomplete Freund's adjuvant (IFA) and combinations of toll-like receptor (TLR) agonists, including a preparation containing TLR4 and TLR9 agonists with QS-21 (AS15). IFA-containing vaccines can promote immune cell accumulation at the VSME, whereas effects of AS15 are largely unexplored. Therefore, we assessed innate and adaptive immune cell accumulation and gene expression at the VSME after vaccination with AS15 and compared to effects with IFA. We hypothesized that AS15 would promote less accumulation of innate and adaptive immune cells at the VSME than IFA vaccines. In two clinical trials, patients with resected high-risk melanoma received either a multipeptide vaccine with IFA or a recombinant MAGE-A3 protein vaccine with AS15. Vaccine site biopsies were obtained after one or multiple vaccines. T cells accumulated early after vaccines with AS15, but this was not durable or of the same magnitude as vaccination in IFA. Vaccines with AS15 increased durable expression of DC- and T cell-related genes, as well as PD-L1 and IDO1, suggesting complex activation and regulation of innate and adaptive immune function with AS15. These changes were generally greater with vaccines containing IFA, but IFA induced reduction in myeloid suppressor cells markers. Evidence of tertiary lymphoid structure (TLS) formation was observed with both adjuvants. Our findings highlight adjuvant-dependent changes in immune features at the VSME that may impact systemic immune responses.


Assuntos
Imunidade Adaptativa/imunologia , Vacinas Anticâncer/imunologia , Imunidade Inata/imunologia , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Neoplasias/imunologia , Feminino , Adjuvante de Freund/imunologia , Humanos , Lipídeos/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Vacinação/métodos , Vacinas de Subunidades Antigênicas/imunologia
6.
PLoS Genet ; 14(5): e1007409, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29782499

RESUMO

Although treatment options for localized prostate cancer (CaP) are initially effective, the five-year survival for metastatic CaP is below 30%. Mutation or deletion of the PTEN tumor suppressor is a frequent event in metastatic CaP, and inactivation of the transforming growth factor (TGF) ß signaling pathway is associated with more advanced disease. We previously demonstrated that mouse models of CaP based on inactivation of Pten and the TGFß type II receptor (Tgfbr2) rapidly become invasive and metastatic. Here we show that mouse prostate tumors lacking Pten and Tgfbr2 have higher expression of stem cell markers and genes indicative of basal epithelial cells, and that basal cell proliferation is increased compared to Pten mutants. To better model the primarily luminal phenotype of human CaP we mutated Pten and Tgfbr2 specifically in luminal cells, and found that these tumors also progress to invasive and metastatic cancer. Accompanying the transition to invasive cancer we observed de-differentiation of luminal tumor cells to an intermediate cell type with both basal and luminal markers, as well as differentiation to basal cells. Proliferation rates in these de-differentiated cells were lower than in either basal or luminal cells. However, de-differentiated cells account for the majority of cells in micro-metastases consistent with a preferential contribution to metastasis. We suggest that active TGFß signaling limits lineage plasticity in prostate luminal cells, and that de-differentiation of luminal tumor cells can drive progression to metastatic disease.


Assuntos
Linhagem da Célula/genética , Neoplasias da Próstata/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Animais , Proliferação de Células/genética , Progressão da Doença , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Análise de Sobrevida , Fator de Crescimento Transformador beta/metabolismo
7.
Neurobiol Dis ; 140: 104836, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32179177

RESUMO

Children with malformations of cortical development (MCD) are at risk for epilepsy, developmental delays, behavioral disorders, and intellectual disabilities. For a subset of these children, antiseizure medications or epilepsy surgery may result in seizure freedom. However, there are limited options for treating or curing the other conditions, and epilepsy surgery is not an option in all cases of pharmacoresistant epilepsy. Understanding the genetic and neurobiological mechanisms underlying MCD is a necessary step in elucidating novel therapeutic targets. The tish (telencephalic internal structural heterotopia) rat is a unique model of MCD with spontaneous seizures, but the underlying genetic mutation(s) have remained unknown. DNA and RNA-sequencing revealed that a deletion encompassing a previously unannotated first exon markedly diminished Eml1 transcript and protein abundance in the tish brain. Developmental electrographic characterization of the tish rat revealed early-onset of spontaneous spike-wave discharge (SWD) bursts beginning at postnatal day (P) 17. A dihybrid cross demonstrated that the mutant Eml1 allele segregates with the observed dysplastic cortex and the early-onset SWD bursts in monogenic autosomal recessive frequencies. Our data link the development of the bilateral, heterotopic dysplastic cortex of the tish rat to a deletion in Eml1.


Assuntos
Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/genética , Malformações do Desenvolvimento Cortical do Grupo II/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Córtex Cerebral , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia/genética , Feminino , Masculino , Ratos , Convulsões/genética
8.
BMC Med Genet ; 20(1): 21, 2019 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-30660178

RESUMO

BACKGROUND: The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. METHODS: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. RESULTS: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. CONCLUSIONS: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .


Assuntos
Desaminase APOBEC-3G/genética , Aminoidrolases/genética , Citidina Desaminase/genética , Citosina Desaminase/genética , Infecções por HIV/genética , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Éxons , Feminino , Frequência do Gene , Testes Genéticos , Infecções por HIV/etnologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , África do Sul/etnologia , Adulto Jovem
9.
J Neurosci ; 37(48): 11744-11757, 2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29066557

RESUMO

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed in situ hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, Neuromedin B mRNA (Nmb). Most (∼75%) RTN neurons express low-to-moderate levels of Nmb and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, TASK-2 and Gpr4. These Nmb-low RTN neurons also express varying levels of transcripts for Gal, Penk, and Adcyap1, and receptors for substance P, orexin, serotonin, and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of Nmb mRNA. These Nmb-high RTN neurons do not express Fos after hypercapnia and have low-to-undetectable levels of Kcnk5 or Gpr4 transcripts; they also express Adcyap1, but are essentially devoid of Penk and Gal transcripts. In male rats, Nmb is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, Nmb is a selective marker of the RTN in rodents; Nmb-low neurons, the vast majority, are central respiratory chemoreceptors, whereas Nmb-high neurons likely have other functions.SIGNIFICANCE STATEMENT Central respiratory chemoreceptors regulate arterial PCO2 by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed in situ hybridization, we show here that a single marker, Neuromedin B mRNA (Nmb), identifies RTN neurons in rodents. We also suggest that >75% of these Nmb neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors (Kcnk5 and Gpr4). The other RTN neurons express very high levels of Nmb, but low levels of Kcnk5/Gpr4/pre-pro-galanin/pre-pro-enkephalin, and do not respond to hypercapnia. Their function is unknown.


Assuntos
Bulbo/metabolismo , Neurocinina B/análogos & derivados , Animais , Feminino , Expressão Gênica , Hipóxia/genética , Hipóxia/metabolismo , Masculino , Bulbo/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurocinina B/análise , Neurocinina B/biossíntese , Neurocinina B/genética , Neurônios/química , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
10.
BMC Genomics ; 19(1): 795, 2018 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-30390632

RESUMO

BACKGROUND: Development of the face and mouth is orchestrated by a large number of transcription factors, signaling pathways and epigenetic regulators. While we know many of these regulators, our understanding of how they interact with each other and implement changes in gene expression during orofacial development is still in its infancy. Therefore, this study focuses on uncovering potential cooperation between transcriptional regulators and one important signaling pathway, retinoic acid, during development of the midface. RESULTS: Transcriptome analyses was performed on facial tissues deficient for retinoic acid receptor function at two time points in development; early (35 hpf) just after the neural crest migrates and facial tissues are specified and later (60 hpf) when the mouth has formed and facial structures begin to differentiate. Functional and network analyses revealed that retinoic acid signaling could cooperate with novel epigenetic factors and calcium-NFAT signaling during early orofacial development. At the later stage, retinoic acid may work with WNT and BMP and regulate homeobox containing transcription factors. Finally, there is an overlap in genes dysregulated in Xenopus embryos with median clefts with human genes associated with similar orofacial defects. CONCLUSIONS: This study uncovers novel signaling pathways required for orofacial development as well as pathways that could interact with retinoic acid signaling during the formation of the face. We show that frog faces are an important tool for studying orofacial development and birth defects.


Assuntos
Perfilação da Expressão Gênica , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Transcriptoma , Xenopus/genética , Xenopus/metabolismo , Animais , Biologia Computacional/métodos , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Humanos , Especificidade de Órgãos/genética , Organogênese/genética , Fenótipo , Receptores do Ácido Retinoico/antagonistas & inibidores , Transdução de Sinais , Tretinoína/metabolismo , Xenopus/embriologia
11.
Biol Reprod ; 99(3): 536-545, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29590317

RESUMO

A fully functional initial segment, the most proximal region of the epididymis, is important for male fertility. Our previous study generated a mouse model to investigate the importance of initial segment function in male fertility. In that model, phosphatase and tensin homolog (Pten) was conditionally removed from the initial segment epithelium, which resulted in epithelial de-differentiation. When spermatozoa progressed through the de-differentiated epithelial duct, they developed angled flagella, suggesting compromised sperm maturation, which eventually resulted in male infertility. To understand the molecular mechanisms, by which PTEN regulates epididymal sperm maturation, we compared the transcriptome profile of the initial segment between controls and initial segment-specific Pten knockouts and revealed that water, ion, and organic solute transporter activities were one of the top molecular and cellular functions altered following loss of Pten. Alteration in protein levels and localization of several transporters following loss of Pten were also observed by immunofluorescence analysis. Epithelial cells of the initial segment from knockouts were more permeable to fluorescein isothiocyanate-dextran (4000 Da) compared to controls. Interestingly, conditional deletion of Pten from other organs also resulted in changes in transporter activity, suggesting a common role of PTEN in regulation of transporter activity. Taken together, our data support the hypothesis that loss of Pten from the initial segment epithelium results in changes in the transporting and permeability characteristics of the epithelium, which in turn altered the luminal fluid microenvironment that is so important for sperm maturation and male fertility.


Assuntos
Epididimo/fisiopatologia , Infertilidade Masculina/fisiopatologia , Proteínas de Membrana Transportadoras/fisiologia , PTEN Fosfo-Hidrolase/deficiência , Animais , Diferenciação Celular , Permeabilidade da Membrana Celular/fisiologia , Células Epiteliais/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Imunofluorescência , Masculino , Proteínas de Membrana Transportadoras/análise , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Maturação do Esperma/fisiologia
12.
Dev Dyn ; 246(2): 100-115, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28032936

RESUMO

BACKGROUND: The buccopharyngeal membrane is a thin layer of cells covering the embryonic mouth. The perforation of this structure creates an opening connecting the external and the digestive tube which is essential for oral cavity formation. In humans, persistence of the buccopharyngeal membrane can lead to orofacial defects such as choanal atresia, oral synechiaes, and cleft palate. Little is known about the causes of a persistent buccopharyngeal membrane and, importantly, how this structure ruptures. RESULTS: We have determined, using antisense and pharmacological approaches, that Xenopus embryos deficient c-Jun N-terminal kinase (JNK) signaling have a persistent buccopharyngeal membrane. JNK deficient embryos have decreased cell division and increased cellular stress and apoptosis. However, altering these processes independently of JNK did not affect buccopharyngeal membrane perforation. JNK deficient embryos also have increased intercellular adhesion and defects in e-cadherin localization. Conversely, embryos with overactive JNK have epidermal fragility, increased E-cadherin internalization, and increased membrane localized clathrin. In the buccopharyngeal membrane, clathrin is colocalized with active JNK. Furthermore, inhibition of endocytosis results in a persistent buccopharyngeal membrane, mimicking the JNK deficient phenotype. CONCLUSIONS: The results of this study suggest that JNK has a role in the disassembly adherens junctions by means of endocytosis that is required during buccopharyngeal membrane perforation. Developmental Dynamics 246:100-115, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Membranas Intracelulares/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Boca/embriologia , Xenopus laevis/embriologia , Junções Aderentes , Animais , Caderinas/metabolismo , Bochecha , Endocitose , Boca/crescimento & desenvolvimento , Faringe
13.
Clin Microbiol Rev ; 28(3): 541-63, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25876885

RESUMO

In addition to the ever-present concern of medical professionals about epidemics of infectious diseases, the relative ease of access and low cost of obtaining, producing, and disseminating pathogenic organisms or biological toxins mean that bioterrorism activity should also be considered when facing a disease outbreak. Utilization of whole-genome sequencing (WGS) in outbreak analysis facilitates the rapid and accurate identification of virulence factors of the pathogen and can be used to identify the path of disease transmission within a population and provide information on the probable source. Molecular tools such as WGS are being refined and advanced at a rapid pace to provide robust and higher-resolution methods for identifying, comparing, and classifying pathogenic organisms. If these methods of pathogen characterization are properly applied, they will enable an improved public health response whether a disease outbreak was initiated by natural events or by accidental or deliberate human activity. The current application of next-generation sequencing (NGS) technology to microbial WGS and microbial forensics is reviewed.


Assuntos
Infecções Bacterianas/epidemiologia , Surtos de Doenças , Métodos Epidemiológicos , Genoma Microbiano/genética , Surtos de Doenças/legislação & jurisprudência , Monitoramento Epidemiológico
14.
Antimicrob Agents Chemother ; 59(3): 1656-63, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25561339

RESUMO

The global emergence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination.


Assuntos
Proteínas de Bactérias/genética , Infecção Hospitalar/epidemiologia , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Surtos de Doenças , Hospitais , Humanos , Infecções por Klebsiella/microbiologia , Epidemiologia Molecular , Plasmídeos/genética
15.
J Sex Med ; 12(3): 621-30, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25533598

RESUMO

INTRODUCTION: MicroRNAs (miRs) are noncoding, endogenous RNA molecules that regulate gene expression and play roles in response to vascular injury. AIM: The aim of this study was to identify miRs expressed in corporal tissue (CT) and to determine whether miRs demonstrate differential expression in a mouse model of diet-induced erectile dysfunction (ED). METHODS: RNA was isolated from the CT from control mice and mice with diet-induced ED. A quantifiable miR profiling technique (NanoString) was used to determine the expression of over 600 miRs. MAIN OUTCOME MEASURES: Differential expression analysis was performed using a negative binomial regression model for count-based data. Mean expression levels, fold change, and false discovery-corrected P values were determined. Candidate miRs were validated via quantitative polymerase chain reaction (Q-PCR). RESULTS: In control mice, NanoString analysis revealed that 181 miRs were expressed above background levels and 5 miRs were expressed at high levels. Diet-induced ED resulted in the up-regulation of 6 miRs and the down-regulation of 65 miRs in the CT compared with mice on control diet. Focusing on the upregulated miRs, we chose five for Q-PCR validation. Of these five, two (miR-151-5p and miR-1937c) demonstrated significance via Q-PCR, whereas the other three (miR-720, miR-1937a, miR-205) trended in the correct direction. CONCLUSIONS: MiRs may play a significant role in mRNA regulation in CT and specific miRs may be involved in diet-induced vasculogenic ED. Future studies are aimed at determining the mRNA targets of these miRs.


Assuntos
Impotência Vasculogênica/genética , RNA Mensageiro/biossíntese , Animais , Dieta , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Camundongos , MicroRNAs , Pênis/fisiopatologia , Reação em Cadeia da Polimerase , Regulação para Cima
16.
J Immunol ; 191(9): 4709-19, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24068668

RESUMO

MHC class I D(k) and Ly49G2 (G2) inhibitory receptor-expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without D(k), G2(+) NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, D(k) licenses G2(+) NK cells for effector activity. These data suggested that D(k)-licensed G2(+) NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/My × C57L offspring segregating D(k) were examined before infection and postinfection, including Ly49(+) NK subsets, receptor expression features, and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using the Pearson correlation, followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that G2 expression on naive blood NK cells was predictive of MCMV resistance. However, rapid G2(+) NK cell expansion following viral exposure occurred selectively in D(k) offspring; this response was more highly correlated with MCMV control than all other immune cell features. We infer that D(k)-licensed G2(+) NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. Therefore, MHC polymorphism regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.


Assuntos
Infecções por Herpesviridae/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Animais , Antígenos Ly/metabolismo , Genes MHC Classe I/genética , Genótipo , Infecções por Herpesviridae/virologia , Antígenos de Histocompatibilidade Classe I/genética , Camundongos , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo
17.
Mol Vis ; 20: 1281-95, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25352737

RESUMO

PURPOSE: Cataract is the leading cause of blindness in the world, and in the United States accounts for approximately 60% of Medicare costs related to vision. The purpose of this study was to identify genetic markers for age-related cataract through a genome-wide association study (GWAS). METHODS: In the electronic medical records and genomics (eMERGE) network, we ran an electronic phenotyping algorithm on individuals in each of five sites with electronic medical records linked to DNA biobanks. We performed a GWAS using 530,101 SNPs from the Illumina 660W-Quad in a total of 7,397 individuals (5,503 cases and 1,894 controls). We also performed an age-at-diagnosis case-only analysis. RESULTS: We identified several statistically significant associations with age-related cataract (45 SNPs) as well as age at diagnosis (44 SNPs). The 45 SNPs associated with cataract at p<1×10(-5) are in several interesting genes, including ALDOB, MAP3K1, and MEF2C. All have potential biologic relationships with cataracts. CONCLUSIONS: This is the first genome-wide association study of age-related cataract, and several regions of interest have been identified. The eMERGE network has pioneered the exploration of genomic associations in biobanks linked to electronic health records, and this study is another example of the utility of such resources. Explorations of age-related cataract including validation and replication of the association results identified herein are needed in future studies.


Assuntos
Catarata/genética , Registros Eletrônicos de Saúde/estatística & dados numéricos , Frutose-Bifosfato Aldolase/genética , Predisposição Genética para Doença , MAP Quinase Quinase Quinase 1/genética , Polimorfismo de Nucleotídeo Único , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Catarata/patologia , Bases de Dados de Ácidos Nucleicos , Feminino , Marcadores Genéticos , Genoma Humano , Estudo de Associação Genômica Ampla , Custos de Cuidados de Saúde , Humanos , Fatores de Transcrição MEF2/genética , Masculino , Pessoa de Meia-Idade , Locos de Características Quantitativas , Estados Unidos
18.
Genet Epidemiol ; 35(8): 887-98, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22125226

RESUMO

Genome-wide association studies (GWAS) are a useful approach in the study of the genetic components of complex phenotypes. Aside from large cohorts, GWAS have generally been limited to the study of one or a few diseases or traits. The emergence of biobanks linked to electronic medical records (EMRs) allows the efficient reuse of genetic data to yield meaningful genotype-phenotype associations for multiple phenotypes or traits. Phase I of the electronic MEdical Records and GEnomics (eMERGE-I) Network is a National Human Genome Research Institute-supported consortium composed of five sites to perform various genetic association studies using DNA repositories and EMR systems. Each eMERGE site has developed EMR-based algorithms to comprise a core set of 14 phenotypes for extraction of study samples from each site's DNA repository. Each eMERGE site selected samples for a specific phenotype, and these samples were genotyped at either the Broad Institute or at the Center for Inherited Disease Research using the Illumina Infinium BeadChip technology. In all, approximately 17,000 samples from across the five sites were genotyped. A unified quality control (QC) pipeline was developed by the eMERGE Genomics Working Group and used to ensure thorough cleaning of the data. This process includes examination of sample and marker quality and various batch effects. Upon completion of the genotyping and QC analyses for each site's primary study, eMERGE Coordinating Center merged the datasets from all five sites. This larger merged dataset reentered the established eMERGE QC pipeline. Based on lessons learned during the process, additional analyses and QC checkpoints were added to the pipeline to ensure proper merging. Here, we explore the challenges associated with combining datasets from different genotyping centers and describe the expansion to eMERGE QC pipeline for merged datasets. These additional steps will be useful as the eMERGE project expands to include additional sites in eMERGE-II, and also serve as a starting point for investigators merging multiple genotype datasets accessible through the National Center for Biotechnology Information in the database of Genotypes and Phenotypes. Our experience demonstrates that merging multiple datasets after additional QC can be an efficient use of genotype data despite new challenges that appear in the process.


Assuntos
Registros Eletrônicos de Saúde , Estudo de Associação Genômica Ampla/normas , Controle de Qualidade , Algoritmos , Genótipo , Humanos , National Human Genome Research Institute (U.S.) , Fenótipo , Estados Unidos
19.
Data Brief ; 45: 108688, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36425971

RESUMO

Here we describe a publicly available environmental DNA (eDNA) sequence dataset, consisting of samples collected from a National Oceanic and Atmospheric Administration (NOAA) Great Lakes Environmental Research Laboratory (GLERL) on Lake Erie. We sequenced samples drawn from before, during, and after a 2019 Microcystis harmful algal bloom (HAB) using 3rd generation sequencing with the Oxford Nanopore MinION device. We classified the eDNA reads taxonomically, and estimated the abundances of all taxa in each sample. While the taxonomic data showed evidence of significant human and E. coli contamination, we found abundant Mycrocystis, especially in the samples drawn from bloom environments. The raw sequence data are available in the Sequence Read Archive (SRA) under accession number PRJNA812770. HABs pose a significant and increasing risk, both to human health and to the Blue Economy, and genomic approaches to early detection promise to help mitigate these risks. As such, this dataset could be of interest to freshwater ecology research teams, or any stakeholders interested in the detection and mitigation of HABs.

20.
Front Genet ; 13: 882268, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35846115

RESUMO

Technological advances in sequencing and single nucleotide polymorphism (SNP) genotyping microarray technology have facilitated advances in forensic analysis beyond short tandem repeat (STR) profiling, enabling the identification of unknown DNA samples and distant relationships. Forensic genetic genealogy (FGG) has facilitated the identification of distant relatives of both unidentified remains and unknown donors of crime scene DNA, invigorating the use of biological samples to resolve open cases. Forensic samples are often degraded or contain only trace amounts of DNA. In this study, the accuracy of genome-wide relatedness methods and identity by descent (IBD) segment approaches was evaluated in the presence of challenges commonly encountered with forensic data: missing data and genotyping error. Pedigree whole-genome simulations were used to estimate the genotypes of thousands of individuals with known relationships using multiple populations with different biogeographic ancestral origins. Simulations were also performed with varying error rates and types. Using these data, the performance of different methods for quantifying relatedness was benchmarked across these scenarios. When the genotyping error was low (<1%), IBD segment methods outperformed genome-wide relatedness methods for close relationships and are more accurate at distant relationship inference. However, with an increasing genotyping error (1-5%), methods that do not rely on IBD segment detection are more robust and outperform IBD segment methods. The reduced call rate had little impact on either class of methods. These results have implications for the use of dense SNP data in forensic genomics for distant kinship analysis and FGG, especially when the sample quality is low.

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