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1.
BMC Genomics ; 22(1): 643, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488624

RESUMO

BACKGROUND: The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. RESULTS: We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. CONCLUSIONS: We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Cromossomo X , DNA , Feminino , Fungos , Humanos , Masculino , Análise de Sequência de DNA
2.
J Gen Virol ; 102(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33950806

RESUMO

Rotavirus C (RVC) is associated with acute diarrhoea in both children and young animals. Because of its frequent occurrence, additional sequences have recently been generated. In this study, we sequenced 21 complete genomes from porcine diarrhoea samples and analysed them together with all available reference sequences collected from the GenBank database [National Center for Biotechnology Information (NCBI)]. Based on phylogenetic analysis and genetic distance calculation, the number of each segment was identified as 31G, 26P, 13I, 5R, 5C, 5M, 12A, 10 N, 9T, 8E and 4 H for genotypes encoding VP7, VP4, VP6, VP1, VP2, VP3 and NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. From the analysis, genotypes G19-G31, P[22]-P[26], R5, A9-A12, N9-N10, T7-T9 and E6-E8 were defined as newly identified genotypes, and genotype C6 was combined with C5, and M6 was combined with M1, due to their closely related nature. Estimated with the identity frequency ratio between the intergenotype and intragenotype, the nucleotide identity cutoff values for different genotypes were determined as 85, 85, 86, 84, 83, 84, 82, 87, 84, 81 and 79 % for VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5, respectively. Genotyping of the 49 US strains indicated possible segment reassortment in 9 of the 11 segments, with the exceptions being VP1 and NSP5, and the most prevalent genotypes for each segment genes in the USA were G6/G5/G21/G9-P5/P4-I6/I5-R1-C5-M1-A8-N1/N10-T1-E1-H1. Our study updated the genotypes of RVC strains and provided more evidence of RVC strain diversity that may be relevant to better understand genetic diversity, and the distribution and evolution of RVC strains.


Assuntos
Variação Genética , Genoma Viral , Infecções por Rotavirus/veterinária , Rotavirus/classificação , Rotavirus/genética , Doenças dos Suínos/virologia , Animais , Bases de Dados de Ácidos Nucleicos , Diarreia/veterinária , Diarreia/virologia , Evolução Molecular , Genes Virais , Genótipo , Filogenia , Infecções por Rotavirus/virologia , Suínos , Estados Unidos , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genética , Sequenciamento Completo do Genoma
3.
Transbound Emerg Dis ; 68(3): 1414-1423, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32816334

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real-time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real-time PCR reaction. By analysing all available 678 type 2 PRRSV (PRRSV-2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4-ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine-like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 94.7% (54/57) of Ingelvac PRRS® MLV (MLV) strain and the MLV-like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real-time PCR assay. Evaluated with 417 PRRSV-2 positive clinical samples, 95% were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay.


Assuntos
Separação Imunomagnética/veterinária , Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Animais , Sequência de Bases , Separação Imunomagnética/métodos , Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Sus scrofa , Suínos , Estados Unidos , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia
4.
J Microbiol Methods ; 172: 105887, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32165161

RESUMO

Streptococcus equi subsp. equi is a Gram positive bacterial pathogen commonly associated with strangles in horses, a respiratory disease characterized by abscessation of submandibular and retropharyngeal lymph nodes which can lead to obstruction of the airway. Several real-time PCR (qPCR) assays have been developed for detection of S. equi from horses with many targeting conserved regions of the S. equi cell wall-associated M-protein (SeM), a major virulence factor and immunogen of S. equi. Our objective was to develop a nested PCR (nPCR) targeting SeM and an 18S rRNA internal control gene for detection of S. equi from horses with potential improvement in detection sensitivity compared to a qPCR. Primers and probes from the Kansas State Veterinary Diagnostic Laboratory (KSVDL) S. equi clinical testing assay were utilized for all qPCR testing. Primers flanking the SeM qPCR target region were selected for an initial end-point PCR step of the nested assay; PCR product from the end-point reaction then served as template for the qPCR reaction step of the nested assay. Sample nucleic acid was also tested directly with qPCR to allow for assay comparison. Nucleic acid from clinical specimens (n = 188) submitted to KSVDL were tested in parallel with each assay. The nPCR and qPCR assays identified 22.9% (43/188) and 13.3% (25/188) of samples positive for S. equi, respectively. None of the samples positive by qPCR were negative by nPCR. The PCR products from all positive samples were submitted for DNA sequencing. Each of the 25 samples positive by both assays had a high nucleotide identity match (>96%) to the SeM gene. Among the samples positive by nPCR but negative by qPCR, 17 of 18 were sequence confirmed for SeM at greater than 96% nucleotide identity. Based on the nPCR Ct (37.8) of the one sequence un-confirmed case, it is likely that the S. equi bacterial load in this sample was below the necessary concentration for successful sequencing. Limit of detection (LOD) for the nPCR was established at a Ct of 37, and based both on the LOD of the qPCR assay (Ct of 37), as determined by standard curve data, and on the highest nPCR Cts (~37) of clinical samples able to result in SeM sequence-confirmation. As demonstrated by sequencing confirmation, the nPCR assay targeting the SeM gene is highly specific to S. equi. The increased sensitivity of the nPCR, compared to the qPCR, may reduce the number of false negative sample results in clinical testing and provide a superior detection method during low bacterial shedding periods.


Assuntos
Doenças dos Cavalos/diagnóstico , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/veterinária , Streptococcus/genética , Streptococcus/isolamento & purificação , Animais , DNA Bacteriano/análise , Doenças dos Cavalos/microbiologia , Cavalos , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Infecções Estreptocócicas/microbiologia
5.
J Clin Microbiol ; 58(3)2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-31896667

RESUMO

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx 1 and stx 2 and the intimin gene eae, are important foodborne pathogens. They are referred to as the "top 7" Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx 1 were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.


Assuntos
Reação em Cadeia da Polimerase/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Análise de Célula Única/métodos , Fatores de Virulência/genética , Animais , Bovinos , DNA Bacteriano , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Microbiologia de Alimentos , Genes Bacterianos , Carne/microbiologia , Antígenos O/genética , Sorogrupo , Toxina Shiga , Escherichia coli Shiga Toxigênica/genética
6.
Transbound Emerg Dis ; 67(3): 1284-1294, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31886622

RESUMO

In recent years, reports indicated that PCV3 may be involved in porcine dermatitis and nephropathy syndrome (PDNS)-like disease similar to that linked to PCV2. A total of 2,125 porcine samples from 910 cases were collected during 2016-2018 and tested for presence of PCV3 and PCV2 by real-time PCR assays. Results showed high prevalence of PCV3 and PCV2: 28.4% samples from 41.2% cases were PCV3 positive and 16.4% samples from 16.7% cases were PCV2 positive. The overall coinfection rate was 5.4% and 8.4% at the sample and case level, respectively. Temporal analysis indicated that PCV3 positive case rate increased from 31.6% in 2016, 40.9% in 2017, to 55.6% in 2018. Although its prevalence was lower, PCV2-positive case rate in 2018 (28.8%) doubled that in 2017 (14.4%). The coinfection case rate also increased from 3.4% in 2016, 8.0% in 2017 to 16.1% in 2018. The high positive rate of PCV3 (56.9%) and PCV2 (33.8%) in oral fluids, PCV3 in foetuses (57.1%) and PCV2 in tonsils (54.8%) implied viral transmission route and tissue tropism. In phylogenetic analysis, two small PCV3 clusters (1 and 2) were separated but others were clustered with low bootstrapping values indicating overall low genetic diversity. Genotypes, PCV2a-h, were confirmed by analysing 2,944 strains, with a new genotype proposed as PCV2i. In this study, 61 PCV3 unique whole genomes were sequenced; 12 belonged to a separate cluster that were characterized by five consistent amino acid changes in the capsid protein (24V, 27K, 56D, 98R and 168K) and may be associated with potential differences in immunogenicity. Among the 43 unique PCV2 whole genomes sequenced, 31 belonged to PCV2d, 7 to PCV2a and 5 to PCV2b. Thus, our study demonstrates that PCV2d is the predominant genotype and PCV3 is widely circulating in the Midwest of the USA.


Assuntos
Infecções por Circoviridae/virologia , Circovirus/genética , Variação Genética , Doenças dos Suínos/virologia , Animais , Proteínas do Capsídeo/genética , Circovirus/classificação , Coinfecção , Genótipo , Meio-Oeste dos Estados Unidos/epidemiologia , Filogenia , Prevalência , Suínos , Doenças dos Suínos/epidemiologia
7.
Viruses ; 11(8)2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370351

RESUMO

Viruses belonging to the genus Bocaparvovirus (BoV) are a genetically diverse group of DNA viruses known to cause respiratory, enteric, and neurological diseases in animals, including humans. An intestinal sample from an alpaca (Vicugna pacos) herd with reoccurring diarrhea and respiratory disease was submitted for next-generation sequencing, revealing the presence of a BoV strain. The alpaca BoV strain (AlBoV) had a 58.58% whole genome nucleotide percent identity to a camel BoV from Dubai, belonging to a tentative ungulate BoV 8 species (UBoV8). Recombination events were lacking with other UBoV strains. The AlBoV genome was comprised of the NS1, NP1, and VP1 proteins. The NS1 protein had the highest amino acid percent identity range (57.89-67.85%) to the members of UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses. The low NS1 amino acid identity suggests that AlBoV is a tentative new species. The whole genome, NS1, NP1, and VP1 phylogenetic trees illustrated distinct branching of AlBoV, sharing a common ancestor with UBoV8. Walker loop and Phospholipase A2 (PLA2) motifs that are vital for virus infectivity were identified in NS1 and VP1 proteins, respectively. Our study reports a novel BoV strain in an alpaca intestinal sample and highlights the need for additional BoV research.


Assuntos
Bocavirus/classificação , Camelídeos Americanos/virologia , Genoma Viral , Filogenia , Animais , Bocavirus/isolamento & purificação , Diarreia/virologia , Fezes/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Infecções por Parvoviridae/virologia , Infecções Respiratórias/virologia , Análise de Sequência de DNA , Estados Unidos
8.
Artigo em Inglês | MEDLINE | ID: mdl-31035709

RESUMO

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin produced by several Gibberella and Fusarium species. Accumulating evidence has indicated that ZEA strongly stimulates cell proliferation. However the detailed molecular and cellular mechanisms of ZEA-mediated induction of cell proliferation have not yet been completely explained. The aim of this study was to detect the role of miRNAs in ZEA-mediated induction of cell proliferation. The effects of ZEA on cell proliferation were assessed using a cell counting kit assay and xCELLigence system. Micro-RNA sequencing was performed after treatment of TM3 cells with ZEA (0.01 µmol/L) for different time periods (0, 2, 6 and 18 h). Cell function and pathway analysis of the miRNA target genes were performed by Ingenuity Pathway Analysis (IPA). We found that ZEA promotes TM3 cell proliferation at low concentrations. miRNA sequenceing revealed 66 differentially expressed miRNAs in ZEA-treated cells in comparison to the untreated control ( p < 0.05). The miRNA sequencing indicated that compared to control group, there were 66 miRNAs significant change (p < 0.05) in ZEA-treated groups. IPA analysis showed that the predicated miRNAs target gene involved in cell Bio-functions including cell cycle, growth and proliferation, and in signaling pathways including MAPK and RAS-RAF-MEK-ERK pathways. Results from flow cytometry and Western Blot analysis validated the predictions that ZEA can affect cell cycle, and the MAPK signaling pathway. Taking these together, the cell proliferation induced ZEA is regulated by miRNAs. The results shed light on the molecular and cellular mechanisms for the mediation of ZEA to induce proliferation.


Assuntos
Proliferação de Células/efeitos dos fármacos , MicroRNAs/metabolismo , Zearalenona/toxicidade , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Estrogênios Conjugados (USP) , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , MicroRNAs/genética , Transdução de Sinais/efeitos dos fármacos
9.
Diagn Microbiol Infect Dis ; 95(1): 59-66, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31130238

RESUMO

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.


Assuntos
Doenças dos Bovinos/diagnóstico , Infecções por Orthomyxoviridae/veterinária , Orthomyxoviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Doenças dos Suínos/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/virologia , Diagnóstico Diferencial , Genes Virais/genética , Orthomyxoviridae/classificação , Infecções por Orthomyxoviridae/virologia , RNA Viral/genética , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologia
10.
J Microbiol Methods ; 160: 87-92, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30930057

RESUMO

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R2) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye.


Assuntos
Doenças dos Bovinos/microbiologia , Herpesvirus Bovino 1/isolamento & purificação , Ceratoconjuntivite , Moraxella bovis/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Mycoplasma bovis/isolamento & purificação , Animais , Bovinos , Ceratoconjuntivite/microbiologia , Ceratoconjuntivite/veterinária , Infecções por Moraxellaceae/microbiologia , Infecções por Mycoplasma/microbiologia
11.
Food Chem Toxicol ; 126: 262-276, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30825585

RESUMO

Zearalenone (ZEA), a non-steroidal estrogen mycotoxin produced by several species of Fusarium fungi, can be metabolized into many other derivatives by microorganisms, plants, animals and humans. It can affect mammalian reproductive capability by impacting the synthesis and secretion of sex hormones, including testosterone, estradiol and progesterone. This review summarizes the mechanisms in which ZEA and its derivatives disturb the synthesis and secretion of sex steroid hormones. Because of its structural analogy to estrogen, ZEA and its derivatives can exert a variety of estrogen-like effects and engage in estrogen negative feedback regulation, which can result in mediating the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland. ZEA and its derivatives can ultimately reduce the number of Leydig cells and granulosa cells by inducing oxidative stress, endoplasmic reticulum (ER) stress, cell cycle arrest, cell apoptosis, and cell regeneration delay. Additionally, they can disrupt the mitochondrial structure and influence mitochondrial functions through overproduction of reactive oxygen species (ROS) and aberrant autophagy signaling ways. Finally, ZEA and its derivatives can disturb the expressions and activities of the related steroidogenic enzymes through cross talking between membrane and nuclear estrogen receptors.


Assuntos
Hormônios Esteroides Gonadais/biossíntese , Mamíferos/fisiologia , Zearalenona/química , Zearalenona/toxicidade , Animais , Feminino , Hormônios Esteroides Gonadais/metabolismo , Humanos , Masculino , Reprodução/efeitos dos fármacos
12.
Artigo em Inglês | MEDLINE | ID: mdl-30533603

RESUMO

Influenza C virus (ICV) has been identified for the first time from bovine respiratory disease complex (BRDC) samples in the United States. Here, we report the complete genome sequence of the strain C/bovine/Montana/12/2016, identified from a nasal swab sample collected from a sick calf with clinical signs of respiratory disease in Montana.

13.
Emerg Infect Dis ; 24(10): 1926-1929, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30226175

RESUMO

We identified influenza C virus (ICV) in samples from US cattle with bovine respiratory disease through real-time PCR testing and sequencing. Bovine ICV isolates had high nucleotide identities (≈98%) with each other and were closely related to human ICV strains (≈95%). Further research is needed to determine bovine ICV's zoonotic potential.


Assuntos
Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/virologia , Influenzavirus C/classificação , Influenzavirus C/genética , Infecções por Orthomyxoviridae/veterinária , Infecções Respiratórias/veterinária , Animais , Bovinos , Doenças dos Bovinos/história , História do Século XXI , Filogenia , Estados Unidos/epidemiologia , Proteínas da Matriz Viral/genética
14.
Front Immunol ; 9: 611, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29643853

RESUMO

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease resulting from abnormal interactions between T and B cells. The acquisition of SLE is linked to genetic susceptibility, and diverse environmental agents can trigger disease onset in genetically susceptible individuals. However, the strongest risk factor for developing SLE is being female (9:1 female to male ratio). The female sex steroid, estradiol, working through its receptors, contributes to the gender bias in SLE although the mechanisms remain enigmatic. In a small clinical trial, monthly administration of the estrogen receptor (ERα) antagonist, ICI182,780 (fulvestrant), significantly reduced disease indicators in SLE patients. In order to identify changes that could account for improved disease status, the present study utilized fulvestrant (Faslodex) to block ERα action in cultured SLE T cells that were purified from blood samples collected from SLE patients (n = 18, median age 42 years) and healthy control females (n = 25, median age 46 years). The effects of ERα antagonism on estradiol-dependent gene expression and canonical signaling pathways were analyzed. Pathways that were significantly altered by addition of Faslodex included T helper (Th) cell differentiation, steroid receptor signaling [glucocorticoid receptor (GR), ESR1 (ERα)], ubiquitination, and sumoylation pathways. ERα protein expression was significantly lower (p < 0.018) in freshly isolated, resting SLE T cells suggesting ERα turnover is inherently faster in SLE T cells. In contrast, ERα/ERß mRNA and ERß protein levels were not significantly different between SLE and normal control T cell samples. Plasma estradiol levels did not differ (p > 0.05) between SLE patients and controls. A previously undetected interaction between GR and ERα signaling pathways suggests posttranslational modification of steroid receptors in SLE T cells may alter ERα/GR actions and contribute to the strong gender bias of this autoimmune disorder.


Assuntos
Estradiol/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores Sexuais , Linfócitos T Auxiliares-Indutores/fisiologia , Adulto , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Interação Gene-Ambiente , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Glucocorticoides/metabolismo , Fatores de Risco , Transdução de Sinais , Sumoilação , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Ubiquitinação , Adulto Jovem
15.
Chemosphere ; 194: 745-754, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29247934

RESUMO

We developed a high-resolution expression microarray based on 2456 unique transcripts from a cDNA library of the aquatic midge (Chironomus tentans). By using the microarray, we detected that 146, 434 and 243 genes were differentially expressed after C. tentans larvae were exposed to chlorpyrifos (organophosphate insecticide) at 0.1 and 0.5 µg/L, and atrazine (triazine herbicide) at 1000 µg/L, respectively, for 48 h. The number of differentially expressed genes in the larvae exposed to chlorpyrifos at 0.5 µg/L was three times of that in the larvae exposed to chlorpyrifos at 0.1 µg/L. Among the differentially expressed genes in response to chlorpyrifos exposures, 76 genes showed significant Blast hits, and among them 42 were in common between the chlorpyrifos and atrazine exposures. In 19 differentially expressed xenobiotic detoxification genes, 16 were significantly up-regulated in the larvae exposed to chlorpyrifos and/or atrazine. Two cytochrome P450 genes (CtCYP6EV1 and CtCYP4DG2) were specifically up-regulated by chlorpyrifos, whereas three cytochrome P450 genes (CtCYP4DG1, CtCYP6EX3 and CtCYP6EV3) were specifically up-regulated by atrazine. Our results showed that chlorpyrifos exposures even at low concentrations can lead to significant changes in gene expression. The significant transcriptional responses are likely attributed to larval intoxication by the insecticide. These results not only support our previous studies in which candidate gene approaches were used, but also can potentially help develop specific molecular markers for monitoring pesticide exposures in non-target organisms in aquatic systems.


Assuntos
Chironomidae/genética , Larva/genética , Praguicidas/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Atrazina/farmacologia , Clorpirifos/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Herbicidas/metabolismo , Inseticidas/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Praguicidas/metabolismo , Regulação para Cima/efeitos dos fármacos , Poluentes Químicos da Água/farmacologia
16.
Sci Rep ; 7(1): 1067, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28432327

RESUMO

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.


Assuntos
Brucella canis/classificação , Brucelose/veterinária , Doenças do Cão/microbiologia , Genótipo , Técnicas de Genotipagem/métodos , Repetições Minissatélites , Tipagem Molecular/métodos , Animais , Brucella canis/genética , Brucelose/microbiologia , Análise por Conglomerados , Cães , Estados Unidos
17.
Int J Mol Sci ; 18(2)2017 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-28146087

RESUMO

A microarray developed on the basis of 2895 unique transcripts from larval gut was used to compare gut gene expression profiles between a laboratory-selected Cry1Ab-resistant (R) strain and its isoline susceptible (S) strain of the European corn borer (Ostrinia nubilalis) after the larvae were fed the leaves of transgenic corn (MON810) expressing Cry1Ab or its non-transgenic isoline for 6 h. We revealed 398 gut genes differentially expressed (i.e., either up- or down-regulated genes with expression ratio ≥2.0) in S-strain, but only 264 gut genes differentially expressed in R-strain after being fed transgenic corn leaves. Although the percentages of down-regulated genes among the total number of differentially expressed genes (50% in S-strain and 45% in R-strain) were similar between the R- and S-strains, the expression ratios of down-regulated genes were much higher in S-strain than in R-strain. We revealed that 17 and 9 significantly up- or down-regulated gut genes from S and R-strain, respectively, including serine proteases and aminopeptidases. These genes may be associated with Cry1Ab toxicity by degradation, binding, and cellular defense. Overall, our study suggests enhanced adaptation of Cry1Ab-resistant larvae on transgenic Cry1Ab corn as revealed by lower number and lower ratios of differentially expressed genes in R-strain than in S-strain of O. nubilalis.


Assuntos
Proteínas de Bactérias/genética , Resistência à Doença , Endotoxinas/genética , Proteínas Hemolisinas/genética , Interações Hospedeiro-Parasita , Larva/genética , Mariposas/genética , Transcriptoma , Zea mays/parasitologia , Animais , Animais Geneticamente Modificados , Toxinas de Bacillus thuringiensis , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Perfilação da Expressão Gênica , Mariposas/microbiologia , Folhas de Planta , Plantas Geneticamente Modificadas , Zea mays/genética , Zea mays/metabolismo
18.
BMC Genomics ; 16: 734, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26416786

RESUMO

BACKGROUND: Genome assembly remains an unsolved problem. Assembly projects face a range of hurdles that confound assembly. Thus a variety of tools and approaches are needed to improve draft genomes. RESULTS: We used a custom assembly workflow to optimize consensus genome map assembly, resulting in an assembly equal to the estimated length of the Tribolium castaneum genome and with an N50 of more than 1 Mb. We used this map for super scaffolding the T. castaneum sequence assembly, more than tripling its N50 with the program Stitch. CONCLUSIONS: In this article we present software that leverages consensus genome maps assembled from extremely long single molecule maps to increase the contiguity of sequence assemblies. We report the results of applying these tools to validate and improve a 7x Sanger draft of the T. castaneum genome.


Assuntos
Genoma , Software , Tribolium/genética , Animais , Genômica/métodos , Análise de Sequência de DNA
19.
J Plant Physiol ; 171(14): 1289-98, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25014264

RESUMO

Hard red winter wheat crops on the U.S. Southern Great Plains often experience moderate to severe drought stress, especially during the grain filling stage, resulting in significant yield losses. Cultivars TAM 111 and TAM 112 are widely cultivated in the region, share parentage and showed superior but distinct adaption mechanisms under water-deficit (WD) conditions. Nevertheless, the physiological and molecular basis of their adaptation remains unknown. A greenhouse study was conducted to understand the differences in the physiological and transcriptomic responses of TAM 111 and TAM 112 to WD stress. Whole-plant data indicated that TAM 112 used more water, produced more biomass and grain yield under WD compared to TAM 111. Leaf-level data at the grain filling stage indicated that TAM 112 had elevated abscisic acid (ABA) content and reduced stomatal conductance and photosynthesis as compared to TAM 111. Sustained WD during the grain filling stage also resulted in greater flag leaf transcriptome changes in TAM 112 than TAM 111. Transcripts associated with photosynthesis, carbohydrate metabolism, phytohormone metabolism, and other dehydration responses were uniquely regulated between cultivars. These results suggested a differential role for ABA in regulating physiological and transcriptomic changes associated with WD stress and potential involvement in the superior adaptation and yield of TAM 112.


Assuntos
Ácido Abscísico/metabolismo , Regulação da Expressão Gênica de Plantas , Fotossíntese , Proteínas de Plantas/genética , Estresse Fisiológico , Transcriptoma , Triticum/genética , Adaptação Biológica , Secas , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da Espécie , Triticum/metabolismo , Água/metabolismo
20.
Toxins (Basel) ; 6(4): 1274-94, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24704690

RESUMO

We developed a microarray based on 2895 unique transcripts assembled from 15,000 cDNA sequences from the European corn borer (Ostrinia nubilalis) larval gut. This microarray was used to monitor gene expression in early third-instar larvae of Bacillus thuringiensis (Bt)-susceptible O. nubilalis after 6 h feeding on diet, with or without the Bt Cry1Ab protoxin. We identified 174 transcripts, for which the expression was changed more than two-fold in the gut of the larvae fed Cry1Ab protoxin (p < 0.05), representing 80 down-regulated and 94 up-regulated transcripts. Among 174 differentially expressed transcripts, 13 transcripts putatively encode proteins that are potentially involved in Bt toxicity, and these transcripts include eight serine proteases, three aminopeptidases, one alkaline phosphatase, and one cadherin. The expressions of trypsin-like protease and three aminopeptidase transcripts were variable, but two potential Bt-binding proteins, alkaline phosphatase and cadherin were consistently up-regulated in larvae fed Cry1Ab protoxin. The significantly up and down-regulated transcripts may be involved in Cry1Ab toxicity by activation, degradation, toxin binding, and other related cellular responses. This study is a preliminary survey of Cry1Ab protoxin-induced transcriptional responses in O. nubilalis gut and our results are expected to help with further studies on Bt toxin-insect interactions at the molecular level.


Assuntos
Proteínas de Bactérias/farmacologia , Agentes de Controle Biológico , Endotoxinas/farmacologia , Trato Gastrointestinal/efeitos dos fármacos , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Animais , Toxinas de Bacillus thuringiensis , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Mariposas/embriologia , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Genética/efeitos dos fármacos
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