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1.
Adv Exp Med Biol ; 1412: 491-509, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37378785

RESUMO

This chapter describes the application of genomic, transcriptomic, proteomic, and metabolomic methods in the study of SARS-CoV-2 variants of concern. We also describe the important role of machine learning tools to identify the most significant biomarker signatures and discuss the latest point-of-care devices that can be used to translate these findings to the physician's office or to bedside care. The main emphasis is placed on increasing our diagnostic capacity and predictability of disease outcomes to guide the most appropriate treatment strategies.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Proteômica , COVID-19/diagnóstico , COVID-19/genética , Genômica
2.
Mol Cancer ; 21(1): 183, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36131292

RESUMO

BACKGROUND: Up to 80% of cases of prostate cancer present with multifocal independent tumour lesions leading to the concept of a field effect present in the normal prostate predisposing to cancer development. In the present study we applied Whole Genome DNA Sequencing (WGS) to a group of morphologically normal tissue (n = 51), including benign prostatic hyperplasia (BPH) and non-BPH samples, from men with and men without prostate cancer. We assess whether the observed genetic changes in morphologically normal tissue are linked to the development of cancer in the prostate. RESULTS: Single nucleotide variants (P = 7.0 × 10-03, Wilcoxon rank sum test) and small insertions and deletions (indels, P = 8.7 × 10-06) were significantly higher in morphologically normal samples, including BPH, from men with prostate cancer compared to those without. The presence of subclonal expansions under selective pressure, supported by a high level of mutations, were significantly associated with samples from men with prostate cancer (P = 0.035, Fisher exact test). The clonal cell fraction of normal clones was always higher than the proportion of the prostate estimated as epithelial (P = 5.94 × 10-05, paired Wilcoxon signed rank test) which, along with analysis of primary fibroblasts prepared from BPH specimens, suggests a stromal origin. Constructed phylogenies revealed lineages associated with benign tissue that were completely distinct from adjacent tumour clones, but a common lineage between BPH and non-BPH morphologically normal tissues was often observed. Compared to tumours, normal samples have significantly less single nucleotide variants (P = 3.72 × 10-09, paired Wilcoxon signed rank test), have very few rearrangements and a complete lack of copy number alterations. CONCLUSIONS: Cells within regions of morphologically normal tissue (both BPH and non-BPH) can expand under selective pressure by mechanisms that are distinct from those occurring in adjacent cancer, but that are allied to the presence of cancer. Expansions, which are probably stromal in origin, are characterised by lack of recurrent driver mutations, by almost complete absence of structural variants/copy number alterations, and mutational processes similar to malignant tissue. Our findings have implications for treatment (focal therapy) and early detection approaches.


Assuntos
Hiperplasia Prostática , Neoplasias da Próstata , Células Clonais/patologia , Humanos , Masculino , Nucleotídeos , Próstata/patologia , Hiperplasia Prostática/genética , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia
3.
PLoS Genet ; 13(9): e1007001, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28945760

RESUMO

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.2-p15.1 (15.53%), Chr6q27 (16.50%) and Chr18q12.3 (17.48%). Comprehensive mutation screens of genes, lincRNA encoding sequences, control regions and conserved domains within SCNAs demonstrated that a two-hit genetic model was supported in only a minor proportion of recurrent SCNA losses examined (15/40). We found that recurrent breakpoints and regions of inversion often occur within Knudson model SCNAs, leading to the identification of ZNF292 as a target gene for the deletion at 6q14.3-q15 and NKX3.1 as a two-hit target at 8p21.3-p21.2. The importance of alterations of lincRNA sequences was illustrated by the identification of a novel mutational hotspot at the KCCAT42, FENDRR, CAT1886 and STCAT2 loci at the 16q23.1-q24.3 loss. Our data confirm that the burden of SCNAs is predictive of biochemical recurrence, define nine individual regions that are associated with relapse, and highlight the possible importance of ion channel and G-protein coupled-receptor (GPCR) pathways in cancer development. We concluded that a two-hit genetic model accounts for about one third of SCNA indicating that mechanisms, such haploinsufficiency and epigenetic inactivation, account for the remaining SCNA losses.


Assuntos
Variações do Número de Cópias de DNA/genética , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Análise de Sequência de DNA , Alelos , Genoma Humano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Deleção de Sequência
4.
BMC Vet Res ; 12: 208, 2016 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-27640200

RESUMO

BACKGROUND: Previous studies confirmed that classical scrapie can be transmitted via milk in sheep. The current study aimed to investigate whether scrapie can also be transmitted via goat milk using in vivo (new-born lambs fed milk from scrapie-affected goats due to the unavailability of goat kids from guaranteed scrapie-free herds) and in vitro methods (serial protein misfolding cyclic amplification [sPMCA] on milk samples). RESULTS: In an initial pilot study, new-born lambs of two different prion protein gene (PRNP) genotypes (six VRQ/VRQ and five ARQ/ARQ) were orally challenged with 5 g brain homogenate from two scrapie-affected goats to determine susceptibility of sheep to goat scrapie. All sheep challenged with goat scrapie brain became infected based on the immunohistochemical detection of disease-associated PrP (PrP(sc)) in lymphoid tissue, with an ARQ/ARQ sheep being the first to succumb. Subsequent feeding of milk to eight pairs of new-born ARQ/ARQ lambs, with each pair receiving milk from a different scrapie-affected goat, resulted in scrapie in the six pairs that received the largest volume of milk (38-87 litres per lamb), whereas two pairs fed 8-9 litres per lamb, and an environmental control group raised on sheep milk from healthy ewes, did not show evidence of infection when culled at up to 1882 days of age. Infection in those 12 milk recipients occurred regardless of the clinical status, PrP(sc) distribution, caprine arthritis-encephalitis virus infection status and PRNP polymorphisms at codon 142 (II or IM) of the donor goats, but survival time was influenced by PRNP polymorphisms at codon 141. Serial PMCA applied to a total of 32 milk samples (four each from the eight donor goats collected throughout lactation) detected PrP(sc) in one sample each from two goats. CONCLUSIONS: The scrapie agent was present in the milk from infected goats and was able to transmit to susceptible species even at early preclinical stage of infection, when PrP(sc) was undetectable in the brain of the donor goats. Serial PMCA as a PrP(sc) detection method to assess the risk of scrapie transmission via milk in goats proved inefficient compared to the bioassay.


Assuntos
Leite , Scrapie/transmissão , Ovinos , Animais , Animais Recém-Nascidos , Genótipo , Cabras , Proteínas Priônicas/genética , Análise de Sobrevida
5.
Vet Res ; 46: 40, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25889731

RESUMO

Prion diseases are fatal neurological disorders that affect humans and animals. Scrapie of sheep/goats and Chronic Wasting Disease (CWD) of deer/elk are contagious prion diseases where environmental reservoirs have a direct link to the transmission of disease. Using protein misfolding cyclic amplification we demonstrate that scrapie PrP(Sc) can be detected within circulating dusts that are present on a farm that is naturally contaminated with sheep scrapie. The presence of infectious scrapie within airborne dusts may represent a possible route of infection and illustrates the difficulties that may be associated with the effective decontamination of such scrapie affected premises.


Assuntos
Poeira/análise , Proteínas PrPSc/análise , Scrapie/epidemiologia , Doenças dos Ovinos/epidemiologia , Animais , Inglaterra , Scrapie/etiologia , Ovinos , Doenças dos Ovinos/etiologia
6.
Vet Res ; 46: 126, 2015 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-26511838

RESUMO

Sheep are susceptible to the bovine spongiform encephalopathy (BSE) agent and in the UK they may have been exposed to BSE via contaminated meat and bone meal. An experimental sheep flock was established to determine whether ovine BSE could be naturally transmitted under conditions of intensive husbandry. The flock consisted of 113 sheep of different breeds and susceptible PRNP genotypes orally dosed with BSE, 159 sheep subsequently born to them and 125 unchallenged sentinel controls. BSE was confirmed in 104 (92%) orally dosed sheep and natural transmission was recorded for 14 of 79 (18%) lambs born to BSE infected dams, with rates varying according to PRNP genotype. The likelihood of natural BSE transmission was linked to stage of incubation period of the dam: the attack rate for lambs born within 100 days of the death of BSE infected dams was significantly higher (9/22, 41%) than for the rest (5/57, 9%). Within the group of ewes lambing close to death, those rearing infected progeny (n = 8, for 9/12 infected lambs) showed a significantly greater involvement of lymphoid tissues than those rearing non-infected offspring (n = 8, for 0/10 infected lambs). Horizontal transmission to the progeny of non-infected mothers was recorded only once (1/205, 0.5%). This low rate of lateral transmission was attributed, at least partly, to an almost complete absence of infected placentas. We conclude that, although BSE can be naturally transmitted through dam-lamb close contact, the infection in this study flock would not have persisted due to low-efficiency maternal and lateral transmissions.


Assuntos
Encefalopatia Espongiforme Bovina/transmissão , Transmissão Vertical de Doenças Infecciosas/veterinária , Doenças dos Ovinos/transmissão , Animais , Bovinos , Inglaterra , Feminino , Masculino , Ovinos
7.
Methods Mol Biol ; 2511: 53-65, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35838951

RESUMO

COVID-19 disease caused by the novel SARS-CoV-2 virus represents a new challenge for healthcare systems. The molecular confirmation of infection is crucial to guide public health decision-making. This task could be made more difficult during the next influenza season. Thus, a rapid and user-friendly diagnostic test to discriminate SARS-CoV-2 from influenza viruses is urgently needed. Here, we present a multiplex quantitative polymerase chain reaction (qPCR) assay capable of distinguishing SARS-CoV-2 from influenza A and B cases. This assay benefits from the use of an inhibitor tolerant PCR mix which obviates the need for the rate-limiting extraction step, allowing for a more rapid and accurate analysis.


Assuntos
COVID-19 , Herpesvirus Cercopitecino 1 , Vírus da Influenza A , Influenza Humana , COVID-19/diagnóstico , Testes Diagnósticos de Rotina , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade
8.
Methods Mol Biol ; 2511: 67-78, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35838952

RESUMO

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes which fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here we describe a method using the Inhibitor-Tolerant RT-qPCR kit, developed by Meridian Bioscience kit which allows simultaneous real-time quantitation of the UK, South Africa, and Brazil SARS-CoV-2 variants.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Sensibilidade e Especificidade
9.
J Virol ; 83(18): 9464-73, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19587050

RESUMO

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.


Assuntos
Encéfalo/metabolismo , Encefalopatia Espongiforme Bovina/diagnóstico , Encefalopatia Espongiforme Bovina/genética , Transcrição Gênica , Animais , Bovinos , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos
10.
BMC Vet Res ; 6: 13, 2010 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-20202205

RESUMO

BACKGROUND: As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation. RESULTS: The sensitivity and specificity of the short clinical protocol in detecting a scrapie case in the scrapie-affected herds was 3.9% and 99.6%, respectively, based on the presence of tremor, positive scratch test, extensive hair loss, ataxia and absent menace response. All BSE- and scrapie-affected goats displayed abnormalities in sensation (over-reactivity to external stimuli, startle responses, pruritus, absent menace response) and movement (ataxia, tremor, postural deficits) at an advanced clinical stage but the first detectable sign associated with scrapie or BSE could vary between animals. Signs of pruritus were not always present despite similar prion protein genotypes. Clinical signs of scrapie were also displayed by two scrapie cases that presented with detectable disease-associated prion protein only in lymphoid tissues. CONCLUSIONS: BSE and scrapie may present as pruritic and non-pruritic forms in goats. Signs assessed for the clinical diagnosis of scrapie or BSE in goats should include postural and gait abnormalities, pruritus and visual impairment. However, many scrapie cases will be missed if detection is solely based on the display of clinical signs. PrPd accumulation in the brain appeared to be related to the severity of clinical disease but not to the display of individual neurological signs.


Assuntos
Encefalopatia Espongiforme Bovina/patologia , Doenças das Cabras/patologia , Scrapie/patologia , Animais , Sintomas Comportamentais , Encéfalo/metabolismo , Bovinos , Encefalopatia Espongiforme Bovina/genética , Feminino , Predisposição Genética para Doença , Genótipo , Doenças das Cabras/genética , Cabras , Masculino , Movimento , Postura , Scrapie/genética
11.
BMC Vet Res ; 6: 53, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21143919

RESUMO

BACKGROUND: Various clinical protocols have been developed to aid in the clinical diagnosis of classical bovine spongiform encephalopathy (BSE), which is confirmed by postmortem examinations based on vacuolation and accumulation of disease-associated prion protein (PrPd) in the brain. The present study investigated the occurrence and progression of sixty selected clinical signs and behaviour combinations in 513 experimentally exposed cattle subsequently categorised postmortem as confirmed or unconfirmed BSE cases. Appropriate undosed or saline inoculated controls were examined similarly and the data analysed to explore the possible occurrence of BSE-specific clinical expression in animals unconfirmed by postmortem examinations. RESULTS: Based on the display of selected behavioural, sensory and locomotor changes, 20 (67%) orally dosed and 17 (77%) intracerebrally inoculated pathologically confirmed BSE cases and 21 (13%) orally dosed and 18 (6%) intracerebrally inoculated but unconfirmed cases were considered clinical BSE suspects. None of 103 controls showed significant signs and were all negative on diagnostic postmortem examinations. Signs indicative of BSE suspects, particularly over-reactivity and ataxia, were more frequently displayed in confirmed cases with vacuolar changes in the brain. The display of several BSE-associated signs over time, including repeated startle responses and nervousness, was significantly more frequent in confirmed BSE cases compared to controls, but these two signs were also significantly more frequent in orally dosed cattle unconfirmed by postmortem examinations. CONCLUSIONS: The findings confirm that in experimentally infected cattle clinical abnormalities indicative of BSE are accompanied by vacuolar changes and PrPd accumulation in the brainstem. The presence of more frequently expressed signs in cases with vacuolar changes is consistent with this pathology representing a more advanced stage of disease. That BSE-like signs or sign combinations occur in inoculated animals that were not confirmed as BSE cases by postmortem examinations requires further study to investigate the potential causal relationship with prion disease.


Assuntos
Encefalopatia Espongiforme Bovina/patologia , Animais , Comportamento Animal/fisiologia , Bovinos , Diagnóstico , Encefalopatia Espongiforme Bovina/diagnóstico , Feminino , Masculino , Fatores de Tempo
12.
BMC Vet Res ; 5: 35, 2009 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-19765298

RESUMO

BACKGROUND: Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. RESULTS: None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. CONCLUSION: The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.


Assuntos
Encéfalo/patologia , Encefalopatia Espongiforme Bovina/transmissão , Doenças dos Suínos/patologia , Doenças dos Suínos/transmissão , Animais , Western Blotting , Transplante de Tecido Encefálico/veterinária , Bovinos , Encefalopatia Espongiforme Bovina/patologia , Feminino , Imuno-Histoquímica , Masculino , Príons/isolamento & purificação , Distribuição Aleatória , Suínos
13.
Vet Rec ; 184(3): 97, 2019 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-30602491

RESUMO

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA). A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay. Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3. The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.


Assuntos
Descontaminação/normas , Fazendas , Príons/isolamento & purificação , Scrapie/transmissão , Animais , Animais Recém-Nascidos , Bioensaio/veterinária , Poeira , Monitoramento Ambiental , Genótipo , Príons/genética , Scrapie/epidemiologia , Ovinos , Reino Unido/epidemiologia
14.
Methods Mol Biol ; 1735: 343-350, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29380326

RESUMO

The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods. Although these methods are technically simple, they do not allow exact quantification as can be achieved using a real-time quantitative PCR (qPCR) approach. A qPCR protocol for DNA quantification with applications in NGS library preparation studies is presented here. This can be applied in various fields of study such as medical disorders resulting from nutritional programming disturbances.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase em Tempo Real
15.
Nat Genet ; 50(5): 682-692, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29662167

RESUMO

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials.


Assuntos
Neoplasias da Próstata/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA2/genética , Progressão da Doença , Fator 3-alfa Nuclear de Hepatócito/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oncogenes , Neoplasias da Próstata/patologia
16.
Methods Mol Biol ; 1546: 125-133, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27896761

RESUMO

Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well. The availability to multiplex therefore allows the measurement of the expression levels of several targets or genes of interest quickly. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.


Assuntos
Biomarcadores , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase em Tempo Real , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Methods Mol Biol ; 1546: 135-142, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27896762

RESUMO

DNA microarrays contain microscopic DNA spots attached to a solid surface. Each spot contains picomolar levels of a specific DNA probe sequence and hybridization to the corresponding gene products can be detected and quantitated through the use of fluorescently labeled target DNA. In this format, DNA microarrays can be used to measure the expression level of thousands of genes in a single experiment. Here, we present a method to detect the mRNA transcriptional changes in neuronal precursor cells following differentiation using high density cDNA microarrays.


Assuntos
DNA Complementar , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transcriptoma , Humanos
18.
Methods Mol Biol ; 1546: 143-148, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27896763

RESUMO

Quantitative polymerase chain reaction (qPCR) is a routinely used method for detection and quantitation of gene expression in real time. This is achieved through the incorporation and measurement of fluorescent reporter probes in the amplified cDNA strands, since the fluorescent signals increase as the reaction progresses. The availability of multiple probes that fluoresce at different wavelengths allows for multiplexing as this gives rise to amplicons with unique fluorescent signatures. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.


Assuntos
Reação em Cadeia da Polimerase Multiplex , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Alelos , Biologia Computacional/métodos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Software , Estatística como Assunto
19.
BMC Vet Res ; 2: 31, 2006 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-17044917

RESUMO

BACKGROUND: Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods. RESULTS: Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group. CONCLUSION: The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.


Assuntos
Doenças dos Bovinos/patologia , Doenças dos Bovinos/transmissão , Encefalopatia Espongiforme Bovina/diagnóstico , Proteínas PrPSc/isolamento & purificação , Proteínas PrPSc/patogenicidade , Doenças Priônicas/veterinária , Doenças dos Ovinos/transmissão , Animais , Encéfalo/patologia , Bovinos , Doenças dos Bovinos/diagnóstico , Análise por Conglomerados , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/patologia , Camundongos , Fenótipo , Proteínas PrPSc/classificação , Doenças Priônicas/diagnóstico , Doenças Priônicas/patologia , Doenças Priônicas/transmissão , Ovinos , Fatores de Tempo , Reino Unido/epidemiologia
20.
PLoS One ; 11(3): e0151440, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26968011

RESUMO

The minimum dose required to cause infection of Romney and Suffolk sheep of the ARQ/ARQ or ARQ/ARR prion protein gene genotypes following oral inoculation with Romney or Suffolk a sheep Bovine spongiform encephalopathy (BSE)-derived or cattle BSE-derived agent was investigated using doses ranging from 0.0005g to 5g. ARQ/ARQ sheep which were methionine (M) / threonine (T) heterozygous or T/T homozygous at codon 112 of the Prnp gene, dosed ARQ/ARR sheep and undosed controls did not show any evidence of infection. Within groups of susceptible sheep, the minimum effective oral dose of BSE was found to be 0.05g, with higher attack rates following inoculation with the 5g dose. Surprisingly, this study found no effect of dose on survival time suggesting a possible lack of homogeneity within the inoculum. All clinical BSE cases showed PrPd accumulation in brain; however, following cattle BSE inoculation, LRS involvement within Romney recipients was found to be significantly lower than within the Suffolk sheep inoculated group which is in agreement with previous reports.


Assuntos
Encefalopatia Espongiforme Bovina/genética , Príons/genética , Príons/metabolismo , Administração Oral , Animais , Bovinos , Feminino , Genótipo , Masculino , Transporte Proteico , Ovinos , Taxa de Sobrevida
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