| 1. |
- Banijamali, Elnaz, et al.
(författare)
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RNA:RNA interaction in ternary complexes resolved by chemical probing
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 29:3, s. 317-329
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA
- 2020
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 583:7814, s. 139-144
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Visualizing transient low-populated structures of RNA
- 2012
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 491:7426, s. 724-728
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381.
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Economic Impact of Adverse Drug Events – A Retrospective Population-Based Cohort Study of 4970 Adults
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:3
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Methods for assessing the preventability of adverse drug events : A systematic review
- 2012
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Ingår i: Drug Safety. - : Adis. - 0114-5916 .- 1179-1942. ; 35:2, s. 105-126
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 7:3
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Forskningsöversikt (refereegranskat)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.
(författare)
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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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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9
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Tidskriftsartikel (refereegranskat)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-
(författare)
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Inappropriate prescribing, non-adherence to long-term medications and related morbidities : Pharmacoepidemiological aspects
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 11. |
- Hedna, Khedidja, 1978, et al.
(författare)
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Refill adherence and self-reported adverse drug reactions and sub-therapeutic effects: a population-based study.
- 2013
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Ingår i: Pharmacoepidemiology and drug safety. - : Wiley. - 1099-1557 .- 1053-8569. ; 22:12, s. 1317-1325
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Tidskriftsartikel (refereegranskat)abstract
- To assess refill adherence to dispensed oral long-term medications among the adult population and to investigate whether the percentages of self-reported adverse drug reactions (ADRs) and sub-therapeutic effects (STEs) differed for medications with adequate refill adherence, oversupply, and undersupply.
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| 12. |
- Honarparvar, Bahareh, et al.
(författare)
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Pentacycloundecane-diol-Based HIV-1 Protease Inhibitors : Biological Screening, 2D NMR, and Molecular Simulation Studies
- 2012
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Ingår i: ChemMedChem. - : Wiley. - 1860-7179 .- 1860-7187. ; 7:6, s. 1009-1019
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Tidskriftsartikel (refereegranskat)abstract
- Novel compounds incorporating a pentacycloundecane (PCU) diol moiety were designed, synthesized, and evaluated as inhibitors of the wild-type C-South African (C-SA) HIV-1 protease. Seven compounds are reported herein, three of which displayed IC50 values in the 0.50.6 mu M range. The cytotoxicity of PCU cage peptides toward human MT-4 cells appears to be several orders of magnitude less toxic than the current antiviral medications ritonavir and lopinavir. NMR studies based on the observed through-space 1H,1H distances/contacts in the EASY-ROESY spectra of three of the considered PCU peptide inhibitors enabled us to describe their secondary solution structure. Conserved hydrogen bonding interactions were observed between the hydroxy group of the PCU diol inhibitors and the catalytic triad (Asp25, Ile26, Gly27) of HIV protease in docking and molecular dynamics simulations. The biological significance and possible mode of inhibition by PCU-based HIV protease inhibitors discussed herein facilitates a deeper understanding of this family of inhibitors and their potential application to a vast number of alternative diseases related to proteases.
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| 13. |
- Khedidja, Hedna, et al.
(författare)
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Potentially inappropriate prescribing and adverse drug reactions in the elderly: a population-based study
- 2015
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Ingår i: European Journal of Clinical Pharmacology. - : Springer Science and Business Media LLC. - 0031-6970 .- 1432-1041. ; 71:12, s. 1525-1533
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Tidskriftsartikel (refereegranskat)abstract
- Potentially inappropriate prescriptions (PIPs) criteria are widely used for evaluating the quality of prescribing in elderly. However, there is limited evidence on their association with adverse drug reactions (ADRs) across healthcare settings. The study aimed to determine the prevalence of PIPs, defined by the Screening Tool of Older Persons' potentially inappropriate Prescriptions (STOPP) criteria, in the Swedish elderly general population and to investigate the association between PIPs and occurrence of ADRs. Persons a parts per thousand yen65 years old were identified from a random sample of 5025 adults drawn from the Swedish Total Population Register. A retrospective cohort study was conducted among 813 elderly with healthcare encounters in primary and specialised healthcare settings during a 3-month period in 2008. PIPs were identified from the Swedish Prescribed Drug Register, medical records and health administrative data. ADRs were independently identified by expert reviewers in a stepwise manner using the Howard criteria. Multivariable logistic regression examined the association between PIPs and ADRs. Overall, 374 (46.0 %) persons had a parts per thousand yen1 PIPs and 159 (19.5 %) experienced a parts per thousand yen1 ADRs during the study period. In total, 29.8 % of all ADRs was considered caused by PIPs. Persons prescribed with PIPs had more than twofold increased odds of experiencing ADRs (OR 2.47; 95 % CI 1.65-3.69). PIPs were considered the cause of 60 % of ADRs affecting the vascular system, 50 % of ADRs affecting the nervous system and 62.5 % of ADRs resulting in falls. PIPs are common among the Swedish elderly and are associated with increased odds of experiencing ADRs. Thus, interventions to decrease PIPs may contribute to preventing ADRs, in particular ADRs associated with nervous and vascular disorders and falls.
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| 14. |
- Kosek, David M, et al.
(författare)
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Efficient 3′-pairing renders microRNA targeting less sensitive to mRNA seed accessibility
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:20, s. 11162-11177
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2–g8 in the miRNA), but pairing beyond the seed (3′-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3′-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3′-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3′-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3′-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3′-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3′-pairing. We propose that these results could refine predictions of effective target sites.
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| 15. |
- Makatini, Maya M., et al.
(författare)
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Pentacycloundecane-based inhibitors of wild-type C-South African HIV-protease
- 2011
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Ingår i: Bioorganic & Medicinal Chemistry Letters. - : Elsevier BV. - 0960-894X .- 1464-3405. ; 21:8, s. 2274-2277
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we present the first account of pentacycloundecane (PCU) peptide based HIV-protease inhibitors. The inhibitor exhibiting the highest activity made use of a natural HIV-protease substrate peptide sequence, that is, attached to the cage (PCU-EAIS). This compound showed nanomolar IC50 activity against the resistance-prone wild type C-South African HIV-protease (C-SA) catalytic site via a norstatine type functional group of the PCU hydroxy lactam. NMR was employed to determine a logical correlation between the inhibitory concentration (IC50) results and the 3D structure of the corresponding inhibitors in solution. NMR investigations indicated that the activity is related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. The results from docking experiments coincided with the experimental observed activities. These findings open up useful applications for this family of cage peptide inhibitors, considering the vast number of alternative disease related proteases that exist.
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| 16. |
- Makatini, Maya M., et al.
(författare)
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Synthesis, 2D-NMR and molecular modelling studies of pentacycloundecane lactam-peptides and peptoids as potential HIV-1 wild type C-SA protease inhibitors
- 2013
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Ingår i: Journal of enzyme inhibition and medicinal chemistry (Print). - : Informa UK Limited. - 1475-6366 .- 1475-6374. ; 28:1, s. 78-88
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Tidskriftsartikel (refereegranskat)abstract
- In this study, eight non-natural peptides and peptoids incorporating the pentacycloundecane (PCU) lactam were designed and synthesized as potential inhibitors of the wild type C-SA HIV-protease. Five of these inhibitors gave IC50 values ranging from 0.5 up to 0.75 mu M against the resistance-prone wild type C-South African HIV-protease. NMR EASY-ROESY studies enabled us to describe the secondary structure of three of these compounds in solution. The 3D structures of the selected cage peptides were also modelled in solution using QM/MM/MD simulations. Satisfactory agreement between the NMR observations and the low energy calculated structures exists. Only one of these inhibitors (11 peptoid), which showed the best IC50 (0.5 mu M), exhibited a definable 3-D structure in solution. Autodock4 and AutodockVina were used to model the potential interaction between these inhibitors and the HIV-PR. It appears that the docking results are too crude to be correlated with the relative narrow range of experimental IC50 values (0.5-10 mu M). The PCU-peptides and peptoides were several orders less toxic (145 mu M for 11 and 102 mu M for 11 peptoid) to human MT-4 cells than lopinavir (0.025 mu M). This is the first example of a polycyclic cage framework to be employed as an HIV-PR transition state analogue inhibitor and can potentially be utilized for other diseases related proteases.
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| 17. |
- Makatini, Maya M., et al.
(författare)
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Synthesis and structural studies of pentacycloundecane-based HIV-1 PR inhibitors : A hybrid 2D NMR and docking/QM/MM/MD approach
- 2011
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 46:9, s. 3976-3985
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Tidskriftsartikel (refereegranskat)abstract
- Pentacycloundecane (PCU) lactam-peptide based HIV protease inhibitors were synthesized and nanomolar activity against the resistance-prone wild type C-South African HIV protease is reported. NMR investigations indicated that the activity is related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. EASY-ROESY NMR experiments gave information about the 3D structure of the cage peptides and 3D solution structure could be linked to the experimental IC(50) activity profile of the considered inhibitors. QM/MM/MD simulations of the inhibitors in solution confirmed the NMR observed conformations. Docking experiments and QM/MM/MD simulations of the inhibitor-HIV PR complexes were also performed. These computational results complimented the experimental inhibition activities and enabled us to report a unique binding mode for PCU-based inhibitors at the active site of HIV-protease enzyme. A conserved hydrogen bonding pattern between the norstatine type functional group of the PCU hydroxylactam and active site residues, ASP25/ASP25', was observed in all active compounds. The biological significance and possible mode of inhibition by PCU-based HIV PR inhibitors discussed herein provide us with a deeper understanding of the mode of action of these novel inhibitors. The PCU-peptides are between 6000 and 8500 time less toxic to human MT-4 cells than Lopinavir. This potentially creates new application avenues for these putative inhibitors to be investigated against a vast number of other disease-related proteases.
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| 18. |
- Makatini, Maya M., et al.
(författare)
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Synthesis, screening and computational investigation of pentacycloundecane-peptoids as potent CSA-HIV PR inhibitors
- 2012
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 57, s. 459-467
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Tidskriftsartikel (refereegranskat)abstract
- Herein, we present the first pentacycloundecane (PCU) diol peptoid derived HIV protease inhibitors with IC50 values ranging from 6.5 to 0.075 mu M. Five derivatives were synthesized in an attempt to understand the structure activity relationship of this class of compounds for HIV protease inhibition. NMR spectroscopy (new Efficient Adiabatic Symmetrized Rotating Overhauser Effect Spectroscopy, EASY-ROESY) was employed to determine the predominant conformation of the active compound. In this study docking studies and MD simulations provided insight into the binding theme of this class of peptoid inhibitors to the CSA-HIV PR active site. Conserved and stable hydrogen bonding between the hydroxyl groups of the inhibitors and the active site Asp25/Asp25' residues were observed from the docking and along the MD trajectories.
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| 19. |
- Naicker, Tricia, et al.
(författare)
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Novel tetrahydroisoquinoline based organocatalysts for asymmetric Diels–Alder reactions : insight into the catalytic mode using ROESY NMR and DFT studies
- 2010
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Ingår i: Tetrahedron. - : Elsevier BV. - 0957-4166 .- 1362-511X. ; 21:23, s. 2859-2867
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Tidskriftsartikel (refereegranskat)abstract
- For the first time an organocatalyst bearing a secondary nitrogen within a cyclohexane ring has been evaluated in the asymmetric Diels–Alder reaction. This organocatalyst is also the first of its kind based on a (1R,3S)-6,7-dimethoxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline backbone. These catalysts were tested over a range of dienes and dienophiles and displayed promising chemical conversions of up to 100% with up to 64% ee with triflic acid as the cocatalyst. Density functional theory computational studies and 2D NMR spectroscopy were used to determine the structure of the intermediate iminium ion formed between the most efficient catalyst and cinnamaldehyde. The reaction profile for each of the four possibilities in this reaction were calculated and it was found that the iminium intermediate leading to the major product is higher in energy but kinetically preferred. The activation energies of all possible reaction paths were calculated and the results correlated with the observed products. These experiments revealed that the presence of both (E)- and (Z)-isomers of the cinnamaldehyde were contributing factors for the low enantioselectivity of the reaction products.
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| 20. |
- Natanaelsson, Jennie, et al.
(författare)
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Direct and indirect costs for adverse drug events identified in medical records across care levels, and their distribution among payers
- 2017
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Ingår i: Research in Social and Administrative Pharmacy. - : ELSEVIER SCIENCE INC. - 1551-7411 .- 1934-8150. ; 13:6, s. 1151-1158
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Tidskriftsartikel (refereegranskat)abstract
- Background: Adverse drug events (ADEs) cause considerable costs in hospitals. However, little is known about costs caused by ADEs outside hospitals, effects on productivity, and how the costs are distributed among payers. Objective: To describe the direct and indirect costs caused by ADEs, and their distribution among payers. Furthermore, to describe the distribution of patient out-of-pocket costs and lost productivity caused by ADEs according to socio-economic characteristics. Method: In a random sample of 5025 adults in a Swedish county, prevalence-based costs for ADEs were calculated. Two different methods were used: 1) based on resource use judged to be caused by ADEs, and 2) as costs attributable to ADEs by comparing costs among individuals with ADEs to costs among matched controls. Payers of costs caused by ADEs were identified in medical records among those with ADEs (n = 596), and costs caused to individual patients were described by socio-economic characteristics. Results: Costs for resource use caused by ADEs were (sic)505 per patient with ADEs (95% confidence interval (sic)345-665), of which 38% were indirect costs. Compared to matched controls, the costs attributable to ADEs were (sic)1631, of which (sic)410 were indirect costs. The local health authorities paid 58% of the costs caused by ADEs. Women had higher productivity loss than men ((sic)426 vs. (sic)109, p = 0.018). Out-of-pocket costs displaced a larger proportion of the disposable income among low-income earners than higher income earners (0.7% vs. 0.2%-0.3%). Conclusion: We used two methods to identify costs for ADEs, both identifying indirect costs as an important component of the overall costs for ADEs. Although the largest payers of costs caused by ADEs were the local health authorities responsible for direct costs, employers and patients costs for lost productivity contributed substantially. Our results indicate inequalities in costs caused by ADEs, by sex and income. (C) 2016 Elsevier Inc. All rights reserved.
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| 21. |
- Niklasson, Markus, 1987-
(författare)
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Coding to cure : NMR and thermodynamic software applied to congenital heart disease research
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Regardless of scientific field computers have become pivotal tools for data analysis and the field of structural biology is not an exception. Here, computers are the main tools used for tasks including structural calculations of proteins, spectral analysis of nuclear magnetic resonance (NMR) spectroscopy data and fitting mathematical models to data. As results reported in papers heavily rely on software and scripts it is of key importance that the employed computational methods are robust and yield reliable results. However, as many scientific fields are niched and possess a small potential user base the task to develop necessary software often falls on researchers themselves. This can cause divergence when comparing data analyzed by different measures or by using subpar methods. Therein lies the importance of development of accurate computational methods that can be employed by the scientific community.The main theme of this thesis is software development applied to structural biology, with the purpose to aid research in this scientific field by speeding up the process of data analysis as well as to ensure that acquired data is properly analyzed. Among the original results of this thesis are three user-friendly software: COMPASS - a resonance assignment software for NMR spectroscopy data capable of analyzing chemical shifts and providing the user with suggestions to potential resonance assignments, based on a meticulous database comparison. CDpal - a curve fitting software used to fit thermal and chemical denaturation data of proteins acquired by circular dichroism (CD) spectroscopy or fluorescence spectroscopy. PINT - a line shape fitting and downstream analysis software forNMRspectroscopy data, designed with the important purpose to easily and accurately fit peaks in NMR spectra and extract parameters such as relaxation rates, intensities and volumes of peaks.This thesis also describes a study performed on variants of the life essential regulatory protein calmodulin that have been associated with the congenital life threatening heart disease long QT syndrome (LQTS). The study provided novel insights revealing that all variants are distinct from the wild type in regards to structure and dynamics on a detailed level; the presented results are useful for the interpretation of results from protein interaction studies. The underlying research of this paper makes use of all three developed software, which validates that all developed methods fulfil a scientific purpose and are capable of producing solid results.
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| 22. |
- Niklasson, Markus, 1987-, et al.
(författare)
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Comprehensive analysis of NMR data using advanced line shape fitting.
- 2017
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Ingår i: Journal of Biomolecular NMR. - : Springer. - 0925-2738 .- 1573-5001. ; 69:2, s. 93-99
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Tidskriftsartikel (refereegranskat)abstract
- NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application. The software is a completely redesigned version of PINT ( https://pint-nmr.github.io/PINT/ ). It features a graphical user interface and includes functionality for peak picking, editing of peak lists and line shape fitting. In addition, the obtained peak intensities can be used directly to extract, for instance, relaxation rates, heteronuclear NOE values and exchange parameters. In contrast to most available software the entire process from spectral visualization to preparation of publication-ready figures is done solely using PINT and often within minutes, thereby, increasing productivity for users of all experience levels. Unique to the software are also the outstanding tools for evaluating the quality of the fitting results and extensive, but easy-to-use, customization of the fitting protocol and graphical output. In this communication, we describe the features of the new version of PINT and benchmark its performance.
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| 23. |
- Olofsson, Annelie, et al.
(författare)
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Biochemical and functional characterization of Helicobacter pylori vesicles
- 2010
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:6, s. 1539-1555
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Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigor of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and NMR. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.
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| 24. |
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| 25. |
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| 26. |
- Petzold, Katja, 1981-, et al.
(författare)
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Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns.
- 2007
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 35:20, s. 6854-6861
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Tidskriftsartikel (refereegranskat)abstract
- The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function.
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| 27. |
- Petzold, Katja, et al.
(författare)
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Folding of the αΙΙ-spectrin SH3 domain under physiological salt conditions
- 2008
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier. - 0003-9861 .- 1096-0384. ; 474:1, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of II-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the II-spectrin SH3 domain.
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| 28. |
- Petzold, Katja, 1981-
(författare)
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NMR studies of host-pathogen interactions
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.
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| 29. |
- Petzold, Katja, et al.
(författare)
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RNA structure : adding a second dimension
- 2011
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Ingår i: Nature Chemistry. - : Springer Science and Business Media LLC. - 1755-4330 .- 1755-4349. ; 3:12, s. 913-915
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Tidskriftsartikel (refereegranskat)
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| 30. |
- Petzold, Katja, et al.
(författare)
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Semiconstant-Time P,H-COSY NMR : Analysis of Complex Mixtures of Phospholipids Originating from Helicobacter pylori
- 2009
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Ingår i: Journal of the American Chemical Society. - : ACS Publications ASAP. - 0002-7863 .- 1520-5126. ; 131:40, s. 14150-1
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Tidskriftsartikel (refereegranskat)abstract
- Lipids play a central role in numerous biological events, ranging from normal physiological processes to host−pathogen interactions. The proposed semiconstant-time 31P,1H−COSY NMR experiment provides identification of known and structural characterization of unknown phospholipids in complex membrane extracts with high sensitivity, based on the combination of their 1H and 31P chemical shifts and coupling patterns. Furthermore, the spectra allow quantification of phospholipid composition. Analysis of the phospholipid composition of Helicobacter pylori, the causative agent of peptic ulcer disease, showed the presence of uncommon phospholipids. This novel NMR approach allows the study of changes in membrane composition in response to biological stimuli and opens up the possibility of identifying soluble phosphorus species in a number of research fields.
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| 31. |
- Robertsson, Joacim, et al.
(författare)
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Folding of Spectrin's SH3 Domain in the Presence of Spectrin Repeats
- 2005
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Ingår i: Cellular & Molecular Biology Letters. ; 10, s. 595-612
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Tidskriftsartikel (refereegranskat)abstract
- The multifunctional protein spectrin contains several different structural motifs, such as spectrin repeats and a SH3 domain. Both triple-helix spectrin repeats and the SH3 domain are believed to form independent structural entities. In a-spectrins the SH3 domain is localized to repeat 9, where it is positioned between helix B and helix C in the repeat unit. The presence of SH3 in repeat 9 decreases the thermal stability considerably of this repeat unit while another insert in helix C does not seem to affect the stability. Addition of one or two adjacent repeat units increases the thermal stability from ca 25°C to ~41 and ~48°C, respectively. Despite the differences in thermal stability, the folding properties of peptides comprising the SH3 domain only or together with one or more repeats are more or less the same.
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| 32. |
- Sahin, Cagla, et al.
(författare)
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Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures
- 2023
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 145:19, s. 10659-10668
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Tidskriftsartikel (refereegranskat)abstract
- Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.
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| 33. |
- Schlagnitweit, Judith, et al.
(författare)
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Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy
- 2019
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 20, s. 2474-2478
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Tidskriftsartikel (refereegranskat)abstract
- Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy. Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution-state in-cell NMR spectroscopy (e.g., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.
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| 34. |
- Shaikh, M, et al.
(författare)
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Synthesis and NMR elucidation of homoisoflavanone analogues
- 2011
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Ingår i: Structural Chemistry. - : Springer Science and Business Media LLC. - 1040-0400 .- 1572-9001. ; 22:1, s. 161-166
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Tidskriftsartikel (refereegranskat)abstract
- A series of five homoisoflavanone analogues have been synthesized from the corresponding 3,5-methoxy phenols via chroman-4-one in three steps. The complete NMR elucidation of these homoisoflavanone analogues is reported. The use of 2D NMR techniques (COSY, NOESY, HSQC and HMBC) proved to be very useful tools in the elucidation of homoisoflavanone analogues. The homoisoflavanone analogues exhibit an AA'BB' spin pattern in the ring B of the homoisoflavanone. These homoisoflavanone analogues are potential antifungal and anti-inflammatory agents.
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| 35. |
- Steiner, Emilie, et al.
(författare)
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Capturing Excited States in the Fast-Intermediate Exchange Limit in Biological Systems Using (HNMR)-H-1 Spectroscopy
- 2016
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Ingår i: ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. - : WILEY-V C H VERLAG GMBH. - 1433-7851 .- 1521-3773. ; 55:51, s. 15869-15872
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Tidskriftsartikel (refereegranskat)abstract
- Changes in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high-energy states. In this study, we present a versatile H-1 R-1 RD experiment that not only extends the exchange timescales at least three times beyond the rate limits of C-13/N-15 R-1 and ten times for CPMG experiments, but also makes use of easily accessible probes, thus allowing a general description of biologically important excited states. This technique can be used to extract chemical shifts for the structural characterization of excited states and to elucidate complex excited states.
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| 36. |
- Thiele, Christina Marie, et al.
(författare)
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EASY ROESY : reliable cross-peak integration in adiabatic symmetrized ROESY
- 2009
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 15:3, s. 585-588
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Tidskriftsartikel (refereegranskat)abstract
- Estimates of intramolecular distances are essential for structure determination. For medium-sized molecules, ROESY NMR is the method of choice for obtaining distances. However, the integration of ROESY cross-peaks is problematic due to the offset dependence of theintegrals and/or TOCSY artefacts. We here present EASY ROESY (rEliable Adiabatic SYmmetrized ROESY), which yields reliable intramolecular distances without sample-specific setup.
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