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Abstract: Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103–106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results.
Keywords: targeted high-throughput sequencing, bacterial microbiome, resistome, bacteria detection, DNA extraction, metrology, diagnostics, repeatability
Published in DiRROS: 30.08.2024; Views: 225; Downloads: 155
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Keywords: varstvo gozdov, morfološke analize, Geosmithia morbida, PCR v realnem času, programi preizkav
Published in DiRROS: 30.08.2024; Views: 194; Downloads: 0
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Keywords: varstvo gozdov, morfološke analize, Geosmithia morbida, bolezen tisočerih rakov, qPCR, Juglans, program preiskav
Published in DiRROS: 30.08.2024; Views: 225; Downloads: 0
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Abstract: The cholinergic system has been implicated in postural deficits, in particular falls, in Parkinson’s disease. Falls and freezing of gait typically occur during dynamic and challenging balance and gait conditions, such as when initiating gait, experiencing postural perturbations, or making turns. However, the precise cholinergic neural substrate underlying dynamic postural and gait changes remains poorly understood. The aim of this study was to investigate whether brain vesicular acetylcholine transporter binding, as measured with [18F]-fluoroethoxybenzovesamicolbinding PET, correlates with dynamic gait and balance impairments in 125 patients with Parkinson’s disease (mean age 66.89±7.71 years) using the abbreviated Balance Evaluation Systems Test total and its four functional domain sub-scores (anticipatory postural control, reactive postural control, dynamic gait, and sensory integration). Whole brain false discoverycorrected (P < 0.05) correlations for total abbreviated Balance Evaluation Systems Test scores included the following bilateral or asymmetric hemispheric regions: gyrus rectus, orbitofrontal cortex, anterior part of the dorsomedial prefrontal cortex, dorsolateral prefrontal cortex, cingulum, frontotemporal opercula, insula, fimbria, right temporal pole, mesiotemporal, parietal and visual cortices, caudate nucleus, lateral and medial geniculate bodies, thalamus, lingual gyrus, cerebellar hemisphere lobule VI, left cerebellar crus I, superior cerebellar peduncles, flocculus, and nodulus. No significant correlations were found for the putamen or anteroventral putamen. The four domain-specific sub-scores demonstrated overlapping cholinergic topography in the metathalamus, fimbria, thalamus proper, and prefrontal cortices but also showed distinct topographic variations. For example, reactive postural control functions involved the right flocculus but not the upper brainstem regions. The anterior cingulum associated with reactive postural control whereas the posterior cingulum correlated with anticipatory control. The spatial extent of associated cholinergic system changes were least for dynamic gait and sensory orientation functional domains compared to the anticipatory and reactive postural control functions. We conclude that specific aspects of dynamic balance and gait deficits in Parkinson’s disease associate with overlapping but also distinct patterns of cerebral cholinergic system changes in numerous brain regions. Our study also presents novel evidence of cholinergic topography involved in dynamic balance and gait in Parkinson’s disease that have not been typically associated with mobility disturbances, such as the right anterior temporal pole, right anterior part of the dorsomedial prefrontal cortex, gyrus rectus, fimbria, lingual gyrus, flocculus, nodulus and right cerebellar hemisphere lobules VI and left crus I.
Keywords: Parkinson’s disease, dynamic balance, cholinergic, PET
Published in DiRROS: 30.08.2024; Views: 248; Downloads: 495
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Abstract: Ethylene response factors (ERFs) have been associated with biotic stress in Arabidopsis, while their function in non-model plants is still poorly understood. Here we investigated the role of potato ERF StPti5 in plant immunity. We show that StPti5 acts as a susceptibility factor. It negatively regulates potato immunity against potato virus Y and Ralstonia solanacearum, pathogens with completely different modes of action, and thereby has a different role than its orthologue in tomato. Remarkably, StPti5 is destabilised in healthy plants via the autophagy pathway and accumulates exclusively in the nucleus upon infection. We demonstrate that StEIN3 and StEIL1 directly bind the StPti5 promoter and activate its expression, while synergistic activity of the ethylene and salicylic acid pathways is required for regulated StPti expression. To gain further insight into the mode of StPti5 action in attenuating potato defence responses, we investigated transcriptional changes in salicylic acid deficient potato lines with silenced StPti5 expression. We show that StPti5 regulates the expression of other ERFs and downregulates the ubiquitin-proteasome pathway as well as several proteases involved in directed proteolysis. This study adds a novel element to the complex puzzle of immune regulation, by deciphering a two-level regulation of ERF transcription factor activity in response to pathogens.
Keywords: ethylene response factor, immune signalling, potato virus Y, Pti5, Ralstonia solanacearum, Solanum tuberosum, susceptibility factor
Published in DiRROS: 29.08.2024; Views: 224; Downloads: 654
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