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Povzetek: MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.
Ključne besede: CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance
Objavljeno v DiRROS: 17.07.2024; Ogledov: 3; Prenosov: 2
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Ključne besede: medical physics, medical imaging, oncology
Objavljeno v DiRROS: 15.07.2024; Ogledov: 44; Prenosov: 7
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Ključne besede: haemophilia, haemophilic arthropathy, magnetic resonance imaging
Objavljeno v DiRROS: 09.07.2024; Ogledov: 71; Prenosov: 24
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Povzetek: Understanding the neural underpinning of human gait and balance is one of the most pertinent challenges for 21st-century translational neuroscience due to the profound impact that falls and mobility disturbances have on our aging population. Posture and gait control does not happen automatically, as previously believed, but rather requires continuous involvement of central nervous mechanisms. To effectively exert control over the body, the brain must integrate multiple streams of sensory information, including visual, vestibular, and somatosensory signals. The mechanisms which underpin the integration of these multisensory signals are the principal topic of the present work. Existing multisensory integration theories focus on how failure of cognitive processes thought to be involved in multisensory integration leads to falls in older adults. Insufficient emphasis, however, has been placed on specific contributions of individual sensory modalities to multisensory integration processes and cross-modal interactions that occur between the sensory modalities in relation to gait and balance. In the present work, we review the contributions of somatosensory, visual, and vestibular modalities, along with their multisensory intersections to gait and balance in older adults and patients with Parkinson's disease. We also review evidence of vestibular contributions to multisensory temporal binding windows, previously shown to be highly pertinent to fall risk in older adults. Lastly, we relate multisensory vestibular mechanisms to potential neural substrates, both at the level of neurobiology (concerning positron emission tomography imaging) and at the level of electrophysiology (concerning electroencephalography). We hope that this integrative review, drawing influence across multiple subdisciplines of neuroscience, paves the way for novel research directions and therapeutic neuromodulatory approaches, to improve the lives of older adults and patients with neurodegenerative diseases.
Ključne besede: aging, gait, balance, encephalography, functional magnetic resonance imaging, multisensory integration
Objavljeno v DiRROS: 17.06.2024; Ogledov: 110; Prenosov: 107
Celotno besedilo (1,36 MB)
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Povzetek: Modern radiotherapy techniques enable delivery of high doses to the target volume without escalating dose to organs at risk, offering the possibility of better local control while preserving good quality of life. Uncertainties in target volume delineation have been demonstrated for most tumour sites, and various studies indicate that inconsistencies in target volume delineation may be larger than errors in all other steps of the treatment planning and delivery process. The aim of this paper is to summarize the degree of delineation uncertainties for different tumour sites reported in the literature and review the effect of strategies to minimize them. Conclusions. Our review confirmed that interobserver variability in target volume contouring represents the largest uncertainty in the process for most tumour sites, potentially resulting in a systematic error in dose delivery, which could influence local control in individual patients. For most tumour sites the optimal combination of imaging modalities for target delineation still needs to be determined. Strict use of delineation guidelines and protocols is advisable both in every day clinical practice and in clinical studies to diminish interobserver variability. Continuing medical education of radiation oncologists cannot be overemphasized, intensive formal training on interpretation of sectional imaging should be included in the program for radiation oncology residents.
Ključne besede: target volume, interobserver variability, delineation uncertainties, imaging
Objavljeno v DiRROS: 30.04.2024; Ogledov: 432; Prenosov: 412
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