Absolute quantification of viruses by TaqMan real-time RT-PCR in grapevines.

The absolute quantification determines the absolute amount of a targeted nucleic acid expressed as a copy number or concentration. The knowledge of virus concentrations in commercial crops possesses high relevance to ensure a reliable diagnosis. The objective of this study was to perform an absolute quantification of five viruses in infected grapevines (Vitis spp.). Different known amounts of the standard sample (cloned viral cDNA or in vitro transcribed viral RNA) were quantified by TaqMan RT-qPCR. Based on these data, standard curves were generated plotting Ct values (threshold cycle) against the log of the standard sample amount. Infected grapevine samples were evaluated to determine virus titers, which were highly variable. This result may contribute to improve virus diagnosis by accurately quantifying virus titre variations in grapevines. Key words: RT-qPCR, GRSPaV, GVA, GVD, GLRaV-3 and -4. RESUMO: A quantificação absoluta determina a quantidade absoluta de um ácido nucleico alvo expressa como número de cópias ou concentração. O conhecimento das concentrações virais em culturas comerciais tem grande relevância para assegurar um diagnóstico confiável. O objetivo deste trabalho foi realizar uma quantificação absoluta de cinco vírus em videiras infectadas (Vitis spp.). Diferentes quantidades conhecidas da amostra padrão (cDNA viral clonado ou RNA viral transcrito in vitro) foram quantificadas por RT-qPCR TaqMan. A partir destes dados, curvas padrão foram geradas plotando-se os valores de Ct (ciclo limiar) contra o log da quantidade da amostra padrão. Amostras de videiras infectadas foram avaliadas visando-se determinar os títulos virais que foram bastante variáveis. Este resultado contribui para melhorar o diagnóstico viral ao quantificar com precisão variações no título viral em videiras. Palavras-chave: RT-qPCR, GRSPaV, GVA, GVD, GLRaV-3 e -4. A quantificação absoluta determina a quantidade absoluta de um ácido nucleico alvo expressa como número de cópias ou concentração. O conhecimento das concentrações virais em culturas comerciais tem grande relevância para assegurar um diagnóstico confiável. O objetivo deste trabalho foi realizar uma quantificação absoluta de cinco vírus em videiras infectadas (Vitis spp.). Diferentes quantidades conhecidas da amostra padrão (cDNA viral clonado ou RNA viral transcrito in vitro) foram quantificadas por RT-qPCR TaqMan. A partir destes dados, curvas padrão foram geradas plotando-se os valores de Ct (ciclo limiar) contra o log da quantidade da amostra padrão. Amostras de videiras infectadas foram avaliadas visando-se determinar os títulos virais que foram bastante variáveis. Este resultado contribui para melhorar o diagnóstico viral ao quantificar com precisão variações no título viral em videiras. Palavras-chave: RT-qPCR, GRSPaV, GVA, GVD, GLRaV-3 e -4

Expressão de proteína capsídica recombinante do Apple stem pitting virus e produção de antissoro policlonal.

O objetivo deste trabalho foi a expressão do gene da proteína capsidíca do ASPV em bactérias e a produção do antissoro policlonal contra a proteína expressada.Resumo

MGMT promoter methylation and IDH1 mutations do not affect [18F]FDOPA uptake in primary brain tumors

The aim of our study was to investigate the effects of methylation of O-6-methylguanine-DNA methyltransferase promoter (MGMTp) and isocitrate dehydrogenase 1 (IDH 1) mutations on amino acid metabolism evaluated with 3,4-dihydroxy-6-[F-18]-fluoro-l-phenylalanine ([F-18] FDOPA) positron emission tomography/computed tomography (PET/CT). Seventy-two patients with primary brain tumors were enrolled in the study (33 women and 39 men; mean age 44 +/- 12 years old). All of them were subjected to PET/CT examination after surgical treatment. Of them, 29 (40.3%) were affected by grade II glioma and 43 (59.7%) by grade III. PET/CT was scored as positive or negative and standardized uptake value ratio (SUVr) was calculated as the ratio between SUVmax of the lesion vs. that of the background. Statistical analysis was performed with the Mann-Whitney U test. Methylation of MGMTp was detectable in 61 out of the 72 patients examinated. Mean SUVr in patients without methylation of MGMTp was 1.44 +/- 0.38 vs. 1.35 +/- 0.48 of patients with methylation (p = 0.15). Data on IDH1 mutations were available for 43 subjects; of them, 31 are IDH-mutant. Mean SUVr was 1.38 +/- 0.51 in patients IDH mutant and 1.46 +/- 0.56 in patients IDH wild type. MGMTp methylation and IDH1 mutations do not affect [F-18] FDOPA uptake in primary brain tumors and therefore cannot be assessed or predicted by radiopharmaceutical uptake parameters

Centrosomal and mitotic abnormalities in cell lines derived from papillary thyroid cancer harboring specific gene alterations

<p>Abstract</p> <p>Background</p> <p>Differentiated thyroid carcinoma offers a good model to investigate the possible correlation between specific gene mutations and chromosome instability. Papillary thyroid neoplasms are characterized by different mutually exclusive genetic alterations, some of which are associated with aneuploidy and aggressive phenotype.</p> <p>Results</p> <p>We investigated the centrosome status and mitotic abnormalities in three thyroid carcinoma-derived cell lines, each maintaining the specific, biologically relevant gene alteration harbored by the parental tumors: <it>RET/PTC1 </it>rearrangement in TPC1; heterozygous and homozygous <it>BRAF^V600E</it>mutation in K1 and in B-CPAP, respectively. B-CPAP cells showed a statistically significant (<it>P </it>< 0.01) higher frequency of abnormal mitotic figures compared to TPC1 and K1 cells.</p> <p>Conclusions</p> <p>Our data indicate that <it>RET/PTC1 </it>oncogenic activity is not related to mitotic chromosome impairment and missegregation whereas, based on the consistent difference in types/frequencies of centrosome and spindle abnormalities observed between K1 and B-CPAP cells, the hetero/homozygous allelic status of <it>BRAF^V600E</it>mutation seems to be not irrelevant in respect to chromosomal instability development.</p

Alzheimer\u27s Disease Microbiome Is Associated with Dysregulation of the Anti-Inflammatory P-Glycoprotein Pathway

The microbiota-gut-brain axis is a bidirectional communication system that is poorly understood. Alzheimer\u27s disease (AD), the most common cause of dementia, has long been associated with bacterial infections and inflammation-causing immunosenescence. Recent studies examining the intestinal microbiota of AD patients revealed that their microbiome differs from that of subjects without dementia. In this work, we prospectively enrolled 108 nursing home elders and followed each for up to 5 months, collecting longitudinal stool samples from which we performed metagenomic sequencing and in vitro T84 intestinal epithelial cell functional assays for P-glycoprotein (P-gp) expression, a critical mediator of intestinal homeostasis. Our analysis identified clinical parameters as well as numerous microbial taxa and functional genes that act as predictors of AD dementia in comparison to elders without dementia or with other dementia types. We further demonstrate that stool samples from elders with AD can induce lower P-gp expression levels in vitro those samples from elders without dementia or with other dementia types. We also paired functional studies with machine learning approaches to identify bacterial species differentiating the microbiome of AD elders from that of elders without dementia, which in turn are accurate predictors of the loss of dysregulation of the P-gp pathway. We observed that the microbiome of AD elders shows a lower proportion and prevalence of bacteria with the potential to synthesize butyrate, as well as higher abundances of taxa that are known to cause proinflammatory states. Therefore, a potential nexus between the intestinal microbiome and AD is the modulation of intestinal homeostasis by increases in inflammatory, and decreases in anti-inflammatory, microbial metabolism.IMPORTANCE Studies of the intestinal microbiome and AD have demonstrated associations with microbiome composition at the genus level among matched cohorts. We move this body of literature forward by more deeply investigating microbiome composition via metagenomics and by comparing AD patients against those without dementia and with other dementia types. We also exploit machine learning approaches that combine both metagenomic and clinical data. Finally, our functional studies using stool samples from elders demonstrate how the c microbiome of AD elders can affect intestinal health via dysregulation of the P-glycoprotein pathway. P-glycoprotein dysregulation contributes directly to inflammatory disorders of the intestine. Since AD has been long thought to be linked to chronic bacterial infections as a possible etiology, our findings therefore fill a gap in knowledge in the field of AD research by identifying a nexus between the microbiome, loss of intestinal homeostasis, and inflammation that may underlie this neurodegenerative disorder

Implementação da técnica de RT-PCR em tempo real para diagnóstico viral no Laboratório de Virologia da Embrapa Uva e Vinho.

Resumo

fluoroquinolone resistance and molecular characterization of gyra and parc quinolone resistance determining regions in escherichia coli isolated from poultry

Abstract Escherichia coli are a common inhabitant of the gastrointestinal tract of mammals and birds; nevertheless, they may be associated with a variety of severe and invasive infections. Whereas fluoroquinolones (FQ) have been banned in the United States for use in poultry production, the use of these antimicrobials in poultry husbandry is still possible in the European Union, although with some restrictions. The aim of this study was to investigate the FQ resistance of 235 E. coli isolates recovered from chickens and turkeys. Minimum inhibitory concentrations were determined by a microdilution method, whereas mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected by a PCR-based method. High resistance rates (>60%) were observed for nalidixic acid, flumequine, and difloxacin, whereas resistance to ciprofloxacin, danofloxacin, enrofloxacin, marbofloxacin, and sarafloxacin was less frequently reported

Trace Elements in Chromian Spinels from Four Siberian Kimberlites

We analysed the major, minor and trace elements chemistry of forty-two Cr-spinels from four Siberian kimberlites. They showed a wide range in Mg# (Mg/(Mg + Fe2+); 0.42–0.78) and Cr# (Cr/(Cr + Al); 0.32–0.92) and a common trend of increasing Cr# with decreasing Mg#. The major element classification schemes suggested that there were spinels deriving from a peridotitic source (Xen) and spinels crystallised from kimberlitic melts (Chr). Laser-Ablation Inductively Coupled Plasma Mass Spectrometry on both groups showed that the trace elements with the highest abundance were Mn (985–3390 ppm), Ni (531–3162 ppm), V (694–2510 ppm) and Zn (475–2230 ppm). Testing the effectiveness of trace elements in determining the source for Cr-spinels, we found out that Cr-spinels crystallised directly from a kimberlitic melt usually showed higher Mn, Ni, Sc and V concentrations with respect to those of peridotitic origin. In addition, using the available partitioning models, we found that the correlations between major elements and Ni, Co, Sc and Ga in the Xen group could be explained by subsolidus equilibration between spinel, olivine and clinopyroxene at 800–1000 °C, thus supporting a peridotitic source for this group. Finally, we calculated the composition of the possible melts in equilibrium with the Cr-spinels of the Chr group, using a selected set of partition coefficients. Calculated abundances of Cu, Ga and Zr were comparable to those of the kimberlite, while V was never close to the kimberlite composition. This simulation highlighted the need for new data on the trace elements partition coefficients between kimberlitic melts and Cr-spinel