Functional annotation of human long noncoding RNAs via molecular phenotyping

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.Peer reviewe

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Cellular and molecular mechanisms by which specific GSK3 inhibition affects the processing of the Amyloid Precursor Protein

Alzheimer’s disease (AD) has long been associated with altered activity of the serine/threonine kinases, Glycogen Synthase Kinase-3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. While the molecular links between GS3K and tau pathology are well established, the molecular basis by which GSK3 affects the formation of amyloid-β (Aβ) remains unknown. The aim of this investigation was to identify the underlying mechanisms by which inhibition of GSK3 affects the processing of the amyloid precursor protein (APP). To this end various methods were employed to alter the basal activity of the GSK3 enzyme in N2a cells, a mouse neuroblastoma cell-line, overexpressing the Swedish variant of APP. Specific perturbation of GSK3 activity leads to an alteration in APP processing and Aβ production, something that until now, was seen as controversial. Specific pharmacological inhibition of GSK3 resulted in a decrease in activity, expression and transcription of BACE1, which is the main enzyme responsible for Aβ generation. Activation of the canonical Wnt pathway, which is associated with negative regulation of GSK3, reproduced the previous findings and led to reduction in transcription and expression of BACE1. Furthermore, specific pharmacological GSK3 inhibition and GSK3α/β knockdown enhanced full-length APP degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with the effect of specific GSK3 inhibition on the nuclear translocation of transcription factor EB (TFEB), which is a master regulator of lysosomal biogenesis. All together, GSK3, in addition to being associated with hyperphosphorylation of tau, can also influence Aβ generation through regulating BACE1 expression and the homeostatic turnover of APP. This data reinforces the hypothesis that GSK3 could be a therapeutic target for AD as it is shown to be a central signalling node in the pathogenesis of the disease

Cellular and molecular mechanisms by which specific GSK3 inhibition affects the processing of the Amyloid Precursor Protein

Alzheimer’s disease (AD) has long been associated with altered activity of the serine/threonine kinases, Glycogen Synthase Kinase-3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. While the molecular links between GS3K and tau pathology are well established, the molecular basis by which GSK3 affects the formation of amyloid-β (Aβ) remains unknown. The aim of this investigation was to identify the underlying mechanisms by which inhibition of GSK3 affects the processing of the amyloid precursor protein (APP). To this end various methods were employed to alter the basal activity of the GSK3 enzyme in N2a cells, a mouse neuroblastoma cell-line, overexpressing the Swedish variant of APP. Specific perturbation of GSK3 activity leads to an alteration in APP processing and Aβ production, something that until now, was seen as controversial. Specific pharmacological inhibition of GSK3 resulted in a decrease in activity, expression and transcription of BACE1, which is the main enzyme responsible for Aβ generation. Activation of the canonical Wnt pathway, which is associated with negative regulation of GSK3, reproduced the previous findings and led to reduction in transcription and expression of BACE1. Furthermore, specific pharmacological GSK3 inhibition and GSK3α/β knockdown enhanced full-length APP degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with the effect of specific GSK3 inhibition on the nuclear translocation of transcription factor EB (TFEB), which is a master regulator of lysosomal biogenesis. All together, GSK3, in addition to being associated with hyperphosphorylation of tau, can also influence Aβ generation through regulating BACE1 expression and the homeostatic turnover of APP. This data reinforces the hypothesis that GSK3 could be a therapeutic target for AD as it is shown to be a central signalling node in the pathogenesis of the disease.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Studying the post-COVID-19 condition: research challenges, strategies, and importance of Core Outcome Set development

13 páginasBackground A substantial portion of people with COVID-19 subsequently experience lasting symptoms including fatigue, shortness of breath, and neurological complaints such as cognitive dysfunction many months after acute infection. Emerging evidence suggests that this condition, commonly referred to as long COVID but also known as post-acute sequelae of SARS-CoV-2 infection (PASC) or post-COVID-19 condition, could become a significant global health burden. Main text While the number of studies investigating the post-COVID-19 condition is increasing, there is no agreement on how this new disease should be defined and diagnosed in clinical practice and what relevant outcomes to measure. There is an urgent need to optimise and standardise outcome measures for this important patient group both for clinical services and for research and to allow comparing and pooling of data. Conclusions A Core Outcome Set for post-COVID-19 condition should be developed in the shortest time frame possible, for improvement in data quality, harmonisation, and comparability between different geographical locations. We call for a global initiative, involving all relevant partners, including, but not limited to, healthcare professionals, researchers, methodologists, patients, and caregivers. We urge coordinated actions aiming to develop a Core Outcome Set (COS) for post-COVID-19 condition in both the adult and paediatric populations

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

細胞種に応じてゲノム編集を制御する技術を開発. 京都大学プレスリリース. 2017-05-22.The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information

MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells.

iPS細胞を選択的に識別・分離・除去できるしくみを合成RNAを用いて構築. 京都大学プレスリリース. 2016-09-09.The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information

N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells

mRNAスイッチの性能を大幅に向上させることのできる修飾塩基を発見. 京都大学プレスリリース. 2020-03-06.Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools