| 1. |
- Banijamali, Elnaz, et al.
(author)
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RNA:RNA interaction in ternary complexes resolved by chemical probing
- 2022
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In: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 29:3, s. 317-329
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Journal article (peer-reviewed)abstract
- RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
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| 2. |
- Baronti, Lorenzo, et al.
(author)
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Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA
- 2020
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 583:7814, s. 139-144
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Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA(1). Here we use R-1 rho relaxation-dispersion nuclear magnetic resonance(2) and molecular simulations(3) to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago(4,5). Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2. Repression of a messenger RNA by a cognate microRNA depends not only on complementary base pairing, but also on the rearrangement of a single base pair, producing a conformation that fits better within the human Ago2 protein.
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| 3. |
- Dethoff, Elizabeth A, et al.
(author)
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Visualizing transient low-populated structures of RNA
- 2012
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 491:7426, s. 724-728
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Journal article (peer-reviewed)abstract
- The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized so far involve long-lived species that can be captured by structure characterization techniques. Here we report the nuclear magnetic resonance visualization of RNA transitions towards 'invisible' excited states (ESs), which exist in too little abundance (2-13%) and for too short a duration (45-250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix-junction-helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signalling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.
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| 4. |
- Grätz, Lukas, et al.
(author)
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NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model
- 2023
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In: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381.
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Journal article (peer-reviewed)abstract
- Background and PurposeWnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer.Experimental ApproachSW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization.Key ResultsWith this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells.Conclusions and ImplicationsBinding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion.
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| 5. |
- Gyllensten, Hanna, 1979, et al.
(author)
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Economic Impact of Adverse Drug Events – A Retrospective Population-Based Cohort Study of 4970 Adults
- 2014
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:3
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Journal article (peer-reviewed)abstract
- Background The aim was to estimate the direct costs caused by ADEs, including costs for dispensed drugs, primary care, other outpatient care, and inpatient care, and to relate the direct costs caused by ADEs to the societal COI (direct and indirect costs), for patients with ADEs and for the entire study population. Methods We conducted a population-based observational retrospective cohort study of ADEs identified from medical records. From a random sample of 5025 adults in a Swedish county council, 4970 were included in the analyses. During a three-month study period in 2008, direct and indirect costs were estimated from resource use identified in the medical records and from register data on costs for resource use. Results Among 596 patients with ADEs, the average direct costs per patient caused by ADEs were USD 444.9 [95% CI: 264.4 to 625.3], corresponding to USD 21 million per 100 000 adult inhabitants per year. Inpatient care accounted for 53.9% of all direct costs caused by ADEs. For patients with ADEs, the average societal cost of illness was USD 6235.0 [5442.8 to 7027.2], of which direct costs were USD 2830.1 [2260.7 to 3399.4] (45%), and indirect costs USD 3404.9 [2899.3 to 3910.4] (55%). The societal cost of illness was higher for patients with ADEs compared to other patients. ADEs caused 9.5% of all direct healthcare costs in the study population. Conclusions Healthcare costs for patients with ADEs are substantial across different settings; in primary care, other outpatient care and inpatient care. Hence the economic impact of ADEs will be underestimated in studies focusing on inpatient ADEs alone. Moreover, the high proportion of indirect costs in the societal COI for patients with ADEs suggests that the observed costs caused by ADEs would be even higher if including indirect costs. Additional studies are needed to identify interventions to prevent and manage ADEs.
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| 6. |
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| 7. |
- Hakkarainen, Katja Marja, et al.
(author)
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Methods for assessing the preventability of adverse drug events : A systematic review
- 2012
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In: Drug Safety. - : Adis. - 0114-5916 .- 1179-1942. ; 35:2, s. 105-126
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Journal article (peer-reviewed)abstract
- Background: Preventable adverse drug events (ADEs) are common in both outpatient and inpatient settings. However, the proportion of preventable ADEs varies considerably in different studies, even when conducted in the same setting, and methods for assessing the preventability of ADEs are diverse. Objective: The aim of this article is to identify and systematically evaluate methods for assessing the preventability of ADEs. Data sources: Seven databases (Cochrane, CINAHL, EMBASE, IPA, MEDLINE, PsycINFO and Web of Science) were searched in September 2010 utilizing the databases' index terms and other common terminology on preventable ADEs. No limits for the years of publication were set. Reference lists of included original articles and relevant review articles were also screened. Study selection: After applying predetermined inclusion and exclusion criteria on 4161 unique citations, 142 (3.4%) original research articles were included in the review. One additional article was included from reference lists. Outcome measures of included studies had to include the frequency of ADEs and the assessment of their preventability. Studies were excluded if they focused on individuals with one specific type of treatment, medical condition, medical procedure or ADE. Data extraction: Measurement instruments for determining the preventability of ADEs in each article were extracted and unique instruments were compared. The process of assessing the preventability of ADEs was described based on reported actions taken to standardize and conduct the assessment, and on information about the reliability and validity of the assessment. Data synthesis: Eighteen unique instruments for determining the preventability of ADEs were identified. They fell under the following four groups: (i) instruments using a definition of preventability only (n = 3); (ii) instruments with a definition of preventability and an assessment scale for determining preventability (n = 5); (iii) instruments with specific criteria for each preventability category (n = 3); and (iv) instruments with an algorithm for determining preventability (n = 7). Of actions to standardize the assessment process, performing a pilot study was reported in 21 (15%), and use of a standardized protocol was reported in 18 (13%), of the included 143 articles. Preventability was assessed by physicians in 86 (60%) articles and by pharmacists in 41 (29%) articles. In 29 (20%) articles, persons conducting the assessment were described as trained for or experienced in preventability assessment. In 94 (66%) articles, more than one person assessed the preventability of each case. Among these 94 articles, assessment was done independently in 73 (51%) articles. Procedures for managing conflicting assessments were diverse. The reliability of the preventability assessment was tested in 39 (27%) articles, and 16 (11%) articles referred to a previous reliability assessment. Reliability ranged from poor to excellent (kappa 0.19-0.98; overall agreement 26-97%). Four (3%) articles mentioned assessing validity, but no sensitivity or specificity analyses or negative or positive predictive values were presented. Conclusions: Instruments for assessing the preventability of ADEs vary from implicit instruments to explicit algorithms. There is limited evidence for the validity of the identified instruments, and instrument reliability varied significantly. The process of assessing the preventability of ADEs is also commonly imprecisely described, which hinders the interpretation and comparison of studies. For measuring the preventability of ADEs more accurately and precisely in future, we believe that existing instruments should be further studied and developed, or that one or more new instruments should be developed, and the validity and reliability of the existing and new instruments be established.
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| 8. |
- Hakkarainen, Katja M, et al.
(author)
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Percentage of patients with preventable adverse drug reactions and preventability of adverse drug reactions--a meta-analysis.
- 2012
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In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 7:3
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Research review (peer-reviewed)abstract
- Numerous observational studies suggest that preventable adverse drug reactions are a significant burden in healthcare, but no meta-analysis using a standardised definition for adverse drug reactions exists. The aim of the study was to estimate the percentage of patients with preventable adverse drug reactions and the preventability of adverse drug reactions in adult outpatients and inpatients.
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| 9. |
- Hakkarainen, Katja M, et al.
(author)
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Prevalence and perceived preventability of self-reported adverse drug events--a population-based survey of 7099 adults.
- 2013
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In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9
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Journal article (peer-reviewed)abstract
- Adverse drug events (ADEs) are common and often preventable among inpatients, but self-reported ADEs have not been investigated in a representative sample of the general public. The objectives of this study were to estimate the 1-month prevalence of self-reported ADEs among the adult general public, and the perceived preventability of 2 ADE categories: adverse drug reactions (ADRs) and sub-therapeutic effects (STEs).
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| 10. |
- Hedna, Khedidja, 1978-
(author)
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Inappropriate prescribing, non-adherence to long-term medications and related morbidities : Pharmacoepidemiological aspects
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Background: Inappropriate use of medications (IUM), in particular inappropriate prescribing and non-adherence to prescribed medications, are important causes of drug-related morbidities (DRMs). They are increasing problems with the ageing populations and the growing burden of chronic conditions. However, research is needed on the association of IUMs with DRMs in outpatient settings and in the general population.Aim: The aim of this thesis is to estimate and analyse the burden of potentially inappropriate prescriptions (PIPs) in the elderly and non-adherence to long-term medications among adults across care settings, and to investigate how IUM is associated to DRMs.Methods: A meta-analysis summarised the previous evidence on the percentage of adverse drug reactions (ADRs) associated to IUM across healthcare settings (Study I). From a cohort in the general population, using medical records and register data, the prevalence of PIPs in the elderly and its association with ADRs were estimated retrospectively (Study II). From the same cohort, the factors associated with refill non-adherence to antihypertensive therapy, considering the use of multiple medications, and the association between non-adherence and sub-therapeutic effects (STEs) were investigated (Study III). A survey assessed the refill behaviour to antihypertensive, lipid lowering and oral antidiabetic medications (undersupply, adequate supply and oversupply), and its association with perceived ADRs and STEs (Study IV).Results: IUM was the cause 52% and 45% of ADRs occurring in adult outpatients and inpatients respectively. Across healthcare settings, 46% of the elderly refilled PIPs over a 6-month period; PIPs were considered the cause of 30% of all ADRs; and the elderly who were prescribed PIPs had increased odds to experience ADRs (OR 2.47, 95% CI 1.65-3.69). In total, 35% was nonadherent to the full multidrug therapy and 13% was non-adherent to any medication (complete non-adherence). Sociodemographic factors (working age and lower income) were associated with non-adherence to any medication, while clinical factors (use of specialised care, use of multiple medications, and being a new user) with non-adherence to the full multidrug therapy. STEs were associated with non-adherence to any medication a month prior to a healthcare visit (OR 3.27, 95% CI 1.27-8.49), but not with long-term measures of non-adherence. Among survey respondents, 22% of the medications were oversupplied and 12% were undersupplied. Inadequate refill behaviour was not associated with reporting ADRs or STEs (p<0.05).Conclusions: A large proportion of ADRs occurring in hospital is caused by IUM, but more knowledge is needed in other settings. PIPs are common in the elderly general population and associated with ADRs. Therefore decreasing PIPs could contribute towards ADR prevention. Considering the use of multiple medications may help to better understand the factors associated with non-adherence to a multidrug therapy for tailoring the interventions to patient needs. Monitoring the adherence prior to a healthcare visit may facilitate interpreting STEs. Yet, the absence of an association between long-term measures of refill non-adherence with clinical and perceived DRMs suggest the need to enhance the knowledge of this association in clinical practice. In summary, this thesis shows a significant potential for improvements of medication use and outcomes.
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