| 1. |
- Alam, Athar, et al.
(författare)
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The Role of ClpB in Bacterial Stress Responses and Virulence
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 8
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Forskningsöversikt (refereegranskat)abstract
- Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA+ proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.
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| 2. |
- Altincekic, Nadine, et al.
(författare)
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Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
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| 3. |
- Aroankins, Takwa S., et al.
(författare)
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The Hydrogen-Coupled Oligopeptide Membrane Cotransporter Pept2 is SUMOylated in Kidney Distal Convoluted Tubule Cells
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Protein post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) on lysine residues is a reversible process highly important for transcription and protein stability. In the kidney, SUMOylation appears to be important for the cellular response to aldosterone. Therefore, in this study, we generated a SUMOylation profile of the aldosterone-sensitive kidney distal convoluted tubule (DCT) as a basis for understanding SUMOylation events in this cell type. Using mass spectrometry-based proteomics, 1037 SUMO1 and 552 SUMO2 sites, corresponding to 546 SUMO1 and 356 SUMO2 proteins, were identified from a modified mouse kidney DCT cell line (mpkDCT). SUMOylation of the renal hydrogen-coupled oligopeptide and drug co-transporter (Pept2) at one site (K139) was found to be highly regulated by aldosterone. Using immunolabelling of mouse kidney sections Pept2 was localized to DCT cells in vivo. Aldosterone stimulation of mpkDCT cell lines expressing wild-type Pept2 or mutant K139R-Pept2, post-transcriptionally increased Pept2 expression up to four-fold. Aldosterone decreased wild-type Pept2 abundance in the apical membrane domain of mpkDCT cells, but this response was absent in K139R-Pept2 expressing cells. In summary, we have generated a SUMOylation landscape of the mouse DCT and determined that SUMOylation plays an important role in the physiological regulation of Pept2 trafficking by aldosterone.
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| 4. |
- Ballante, Flavio, et al.
(författare)
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Modified N-acyl-L-homoserine lactone compounds abrogate Las-dependent quorum-sensing response in human pathogen Pseudomonas aeruginosa
- 2023
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Ingår i: Frontiers in Molecular Biosciences. - : FRONTIERS MEDIA SA. - 2296-889X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Quorum sensing (QS) is a mode of cell-cell communication that bacteria use to sense population density and orchestrate collective behaviors. The common opportunistic human pathogen Pseudomonas aeruginosa employs QS to regulate a large set of genes involved in virulence and host-pathogen interactions. The Las circuit positioned on the top of the QS hierarchy in P. aeruginosa makes use of N-acyl-L-homoserine lactones (AHLs) as signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL). Disabling QS circuits by certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), has been proposed as a strategy to attenuate bacterial pathogenicity. In this study, four new AHL analogs were designed by incorporating a tert-butoxycarbonyl Boc group in amide and beta-keto (3-oxo) moiety. Compounds were evaluated on a molecular and phenotypic basis as a QSI using the screening strategy linked to the assignment of the Las QS system in P. aeruginosa. Using a LasR-based bioreporter, we found that the compounds decreased LasR-controlled light activity and competed efficiently with natural 3O-C12-HSL. The compounds reduced the production of the cognate 3O-C12-HSL and certain virulence traits, like total protease activity, elastase activity, pyocyanin production, and extracellular DNA release. Furthermore, a quantitative proteomic approach was used to study the effect of the compounds on QS-regulated extracellular proteins. Among the four compounds tested, one of them showed the most significant difference in the appearance of the 3O-C12-HSL-responsive reference proteins related to QS communication and virulence, i.e., a distinct activity as a QSI. Moreover, by combining experimental data with computational chemistry, we addressed the effect of LasR protein flexibility on docking precision and assessed the advantage of using a multi-conformational docking procedure for binding mode prediction of LasR modulators. Thus, the four new AHL compounds were tested for their interaction with the AHL-binding site in LasR to identify the key interferences with the activity of LasR. Our study provides further insight into molecular features that are required for small-molecule modulation of LasR-dependent QS communication in P. aeruginosa. This should facilitate rational design of the next generation of antivirulence tools to study and manipulate QS-controlled fitness in bacteria and, thereby, handle bacterial infections in a new way.
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| 5. |
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| 6. |
- Breimer, Michael, 1951, et al.
(författare)
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The Structural Complexity and Animal Tissue Distribution of N-Glycolylneuraminic Acid (Neu5Gc)-Terminated Glycans. Implications for Their Immunogenicity in Clinical Xenografting
- 2019
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- N-Glycolylneuraminic acid (Neu5Gc)-terminated glycans are present in all animal cells/tissues that are already used in the clinic such as bioprosthetic heart valves (BHV) as well as in those that potentially will be xenografted in the future to overcome end stage cell/organ failure. Humans, as a species lack this antigen determinant and can react with an immune response after exposure to Neu5Gc present in these products/cells/tissues. Genetically engineered source animals lacking Neu5Gc has been generated and so has animals that in addition lack the major alpha Gal xenoantigen. The use of cells/tissues/organs from such animals may improve the long-term performance of BHV and allow future xenografting. This review summarizes the present knowledge regarding structural complexity and tissue distribution of Neu5Gc on glycans of cells/tissue/organs already used in the clinic or intended for treatment of end stage organ failure by xenografting. In addition, we briefly discuss the role of anti-Neu5Gc antibodies in the xenorejection process and how knowledge about Neu5Gc structural complexity can be used to design novel diagnostics for anti-Neu5Gc antibody detection.
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| 7. |
- Brodin, L, et al.
(författare)
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α-Synuclein in the Synaptic Vesicle Liquid Phase: Active Player or Passive Bystander?
- 2022
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Ingår i: Frontiers in molecular biosciences. - : Frontiers Media SA. - 2296-889X. ; 9, s. 891508-
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Tidskriftsartikel (refereegranskat)abstract
- The protein α-synuclein, which is well-known for its links to Parkinson’s Disease, is associated with synaptic vesicles (SVs) in nerve terminals. Despite intensive studies, its precise physiological function remains elusive. Accumulating evidence indicates that liquid-liquid phase separation takes part in the assembly and/or maintenance of different synaptic compartments. The current review discusses recent data suggesting α-synuclein as a component of the SV liquid phase. We also consider possible implications of these data for disease.
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| 8. |
- Brunel, Mathilde, et al.
(författare)
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Oleaginous yeast Rhodotorula toruloides biomass effect on the metabolism of Arctic char (Salvelinus alpinus)
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Sustainability issues arise when using fish oil and vegetable oils in fish feed production for aquaculture purposes. Microbial production of single cell oil is a potential alternative as a lipid ingredient in the production of fish feed. In this study, we replaced the vegetable oils with the oleaginous yeast R. toruloides biomass in the diet of Arctic char (S. alpinus) and investigated the effects on health and composition. Measurement of fish growth parameters showed a higher liver weight and hepatosomatic index in the experimental group of fish fed partly with yeast biomass compared to a control group fed a diet with vegetable oils. No significant differences in the lipid content of muscle and liver tissues were found. The fatty acid profiles in the muscle of both fish groups were similar while the experimental fish group had a higher amount of monounsaturated fatty acids in the liver. Histology of livers showed no significant difference in the number of lipid droplets. The size of hepatic lipid droplets seemed to be related to liver fat content. Quantification of metabolites in the liver revealed no differences between the fish groups while plasma metabolites involved in energy pathways such as alanine, 3-hydroxybutyrate, creatinine, serine, betaine, and choline were significantly higher in the experimental fish group.
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| 9. |
- Bungon, Theodore, et al.
(författare)
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Graphene FET Sensors for Alzheimer's Disease Protein Biomarker Clusterin Detection
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- We report on the fabrication and characterisation of graphene field-effect transistor (GFET) biosensors for the detection of Clusterin, a prominent protein biomarker of Alzheimer's disease (AD). The GFET sensors were fabricated on Si/SiO2 substrate using photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with linker molecules and anti-Clusterin antibodies. Concentration of linker molecules was also independently verified by absorption spectroscopy using the highly collimated micro-beam light of Diamond B23 beamline. The detection was achieved through the binding reaction between the antibody and varying concentrations of Clusterin antigen from 1 to 100 pg/mL, as well as specificity tests using human chorionic gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFETs were characterized using direct current (DC) 4-probe electrical resistance (4-PER) measurements, which demonstrated a limit of detection of the biosensors to be similar to 300 fg/mL (4 fM). Comparison with back-gated Dirac voltage shifts with varying concentration of Clusterin show 4-PER measurements to be more accurate, at present, and point to a requirement for further optimisation of the fabrication processes for our next generation of GFET sensors. Thus, we have successfully fabricated a promising set of GFET biosensors for the detection of Clusterin protein biomarker. The developed GFET biosensors are entirely generic and also have the potential to be applied to a variety of other disease detection applications such as Parkinson's, cancer, and cardiovascular.
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| 10. |
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| 11. |
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| 12. |
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| 13. |
- Chen, CZ, et al.
(författare)
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Identification of Tumor Mutation Burden, Microsatellite Instability, and Somatic Copy Number Alteration Derived Nine Gene Signatures to Predict Clinical Outcomes in STAD
- 2022
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Ingår i: Frontiers in molecular biosciences. - : Frontiers Media SA. - 2296-889X. ; 9, s. 793403-
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Tidskriftsartikel (refereegranskat)abstract
- Genomic features, including tumor mutation burden (TMB), microsatellite instability (MSI), and somatic copy number alteration (SCNA), had been demonstrated to be involved with the tumor microenvironment (TME) and outcome of gastric cancer (GC). We obtained profiles of TMB, MSI, and SCNA by processing 405 GC data from The Cancer Genome Atlas (TCGA) and then conducted a comprehensive analysis though “iClusterPlus.” A total of two subgroups were generated, with distinguished prognosis, somatic mutation burden, copy number changes, and immune landscape. We revealed that Cluster1 was marked by a better prognosis, accompanied by higher TMB, MSIsensor score, TMEscore, and lower SCNA burden. Based on these clusters, we screened 196 differentially expressed genes (DEGs), which were subsequently projected into univariate Cox survival analysis. We constructed a 9-gene immune risk score (IRS) model using LASSO-penalized logistic regression. Moreover, the prognostic prediction of IRS was verified by receiver operating characteristic (ROC) curve analysis and nomogram plot. Another independent Gene Expression Omnibus (GEO) contained specimens from 109 GC patients was designed as an external validation. Our works suggested that the 9‐gene‐signature prediction model, which was derived from TMB, MSI, and SCNA, was a promising predictive tool for clinical outcomes in GC patients. This novel methodology may help clinicians uncover the underlying mechanisms and guide future treatment strategies.
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| 14. |
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| 15. |
- Dam, Tommy, et al.
(författare)
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Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Forskningsöversikt (refereegranskat)abstract
- Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease.
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| 16. |
- Damiati, Safa A., et al.
(författare)
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Microfluidic Synthesis of Indomethacin-Loaded PLGA Microparticles Optimized by Machine Learning
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Several attempts have been made to encapsulate indomethacin (IND), to control its sustained release and reduce its side effects. To develop a successful formulation, drug release from a polymeric matrix and subsequent biodegradation need to be achieved. In this study, we focus on combining microfluidic and artificial intelligence (AI) technologies, alongside using biomaterials, to generate drug-loaded polymeric microparticles (MPs). Our strategy is based on using Poly (D,L-lactide-co-glycolide) (PLGA) as a biodegradable polymer for the generation of a controlled drug delivery vehicle, with IND as an example of a poorly soluble drug, a 3D flow focusing microfluidic chip as a simple device synthesis particle, and machine learning using artificial neural networks (ANNs) as an in silico tool to generate and predict size-tunable PLGA MPs. The influence of different polymer concentrations and the flow rates of dispersed and continuous phases on PLGA droplet size prediction in a microfluidic platform were assessed. Subsequently, the developed ANN model was utilized as a quick guide to generate PLGA MPs at a desired size. After conditions optimization, IND-loaded PLGA MPs were produced, and showed larger droplet sizes than blank MPs. Further, the proposed microfluidic system is capable of producing monodisperse particles with a well-controllable shape and size. IND-loaded-PLGA MPs exhibited acceptable drug loading and encapsulation efficiency (7.79 and 62.35%, respectively) and showed sustained release, reaching approximately 80% within 9 days. Hence, combining modern technologies of machine learning and microfluidics with biomaterials can be applied to many pharmaceutical applications, as a quick, low cost, and reproducible strategy.
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| 17. |
- Davies, James S., et al.
(författare)
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Selective Nutrient Transport in Bacteria : Multicomponent Transporter Systems Reign Supreme
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Forskningsöversikt (refereegranskat)abstract
- Multicomponent transporters are used by bacteria to transport a wide range of nutrients. These systems use a substrate-binding protein to bind the nutrient with high affinity and then deliver it to a membrane-bound transporter for uptake. Nutrient uptake pathways are linked to the colonisation potential and pathogenicity of bacteria in humans and may be candidates for antimicrobial targeting. Here we review current research into bacterial multicomponent transport systems, with an emphasis on the interaction at the membrane, as well as new perspectives on the role of lipids and higher oligomers in these complex systems.
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| 18. |
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| 19. |
- Dubackic, Marija, et al.
(författare)
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On the Cluster Formation of α-Synuclein Fibrils
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated with Parkinson’s disease. These intracellular inclusions, called Lewy bodies, also contain significant amounts of lipids. To better understand such accumulations, it should be important to study α-Synuclein fibril formation under conditions where the fibrils lump together, mimicking what is observed in Lewy bodies. In the present study, we have therefore investigated the overall structural arrangements of α-synuclein fibrils, formed under mildly acidic conditions, pH = 5.5, in pure buffer or in the presence of various model membrane systems, by means of small-angle neutron scattering (SANS). At this pH, α-synuclein fibrils are colloidally unstable and aggregate further into dense clusters. SANS intensities show a power law dependence on the scattering vector, q, indicating that the clusters can be described as mass fractal aggregates. The experimentally observed fractal dimension was d = 2.6 ± 0.3. We further show that this fractal dimension can be reproduced using a simple model of rigid-rod clusters. The effect of dominatingly attractive fibril-fibril interactions is discussed within the context of fibril clustering in Lewy body formation.
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| 20. |
- Edwards, Steven J., et al.
(författare)
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High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy
- 2020
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional cell cultures are able to better mimic the physiology and cellular environments found in tissuesin vivocompared to cells grown in two dimensions. In order to study the structure and function of cells in 3-D cultures, light microscopy is frequently used. The preparation of 3-D cell cultures for light microscopy is often destructive, including physical sectioning of the samples, which can result in the loss of 3-D information. In order to probe the structure of 3-D cell cultures at high resolution, we have explored the use of expansion microscopy and compared it to a simple immersion clearing protocol. We provide a practical method for the study of spheroids, organoids and tumor-infiltrating immune cells at high resolution without the loss of spatial organization. Expanded samples are highly transparent, enabling high-resolution imaging over extended volumes by significantly reducing light scatter and absorption. In addition, the hydrogel-like nature of expanded samples enables homogenous antibody labeling of dense epitopes throughout the sample volume. The improved labeling and image quality achieved in expanded samples revealed details in the center of the organoid which were previously only observable following serial sectioning. In comparison to chemically cleared spheroids, the improved signal-to-background ratio of expanded samples greatly improved subsequent methods for image segmentation and analysis.
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| 21. |
- Elsholz, Alexander K. W., et al.
(författare)
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Functional Diversity of AAA plus Protease Complexes in Bacillus subtilis
- 2017
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 4
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Forskningsöversikt (refereegranskat)abstract
- Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.
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| 22. |
- Finka, Andrija, et al.
(författare)
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Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones
- 2015
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 2:JUN
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Forskningsöversikt (refereegranskat)abstract
- Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases.
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| 23. |
- Gao, Menghan, et al.
(författare)
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A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Oncolytic adenovirus is one of the most promising treatments against cancer and is widely evaluated clinically. During high titer production, “Wild-type-” like replication-competent adenovirus (RCA) contaminants can be generated through recombination events due to the DNA sequence similarity between oncolytic virus and host cells. These RCA contaminants raise various safety concerns in clinics. Cell culture-based methods have been developed to detect RCA contaminants in replication-deficient adenovirus vectors. These methods were based on that only RCA contaminants, but not the vectors, are able to grow in and lyse the test cell line. However, these methods are not suitable for distinguishing RCA contaminants from the oncolytic adenovirus products because both can replicate in test cell lines. Herein, we reported a qPCR-based method to quantify RCA contaminants quickly and reliably in E1B-deleted oncolytic adenovirus products. This method is based on specific detection of the E1B gene, which can be acquired during production via recombination events between viral and host cell DNA. The assay is sensitive with the limit of detection at 10 VP of the RCA contaminants and the limit of quantification at 75 VP of the RCA contaminants in each 40 µL qPCR reaction. We have also validated the method on virus batches produced in the non-GMP and GMP conditions. Our results showed that this qPCR-based method was reliable and robust for detecting and quantifying RCA contaminants in oncolytic adenovirus products. The method may also be adapted for other oncolytic adenoviruses products by switching primer sets.
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| 24. |
- Grinkevich, Vera V., et al.
(författare)
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Novel Allosteric Mechanism of Dual p53/MDM2 and p53/MDM4 Inhibition by a Small Molecule
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.
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| 25. |
- Hasan, Mahmudul, et al.
(författare)
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Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The essential enzyme ribonucleotide reductase (RNR) is highly regulated both at the level of overall activity and substrate specificity. Studies of class I, aerobic RNRs have shown that overall activity is downregulated by the binding of dATP to a small domain known as the ATP-cone often found at the N-terminus of RNR subunits, causing oligomerization that prevents formation of a necessary alpha(2)beta(2) complex between the catalytic (alpha(2)) and radical generating (beta(2)) subunits. In some relatively rare organisms with RNRs of the subclass NrdAi, the ATP-cone is found at the N-terminus of the beta subunit rather than more commonly the alpha subunit. Binding of dATP to the ATP-cone in beta results in formation of an unusual beta(4) tetramer. However, the structural basis for how the formation of the active complex is hindered by such oligomerization has not been studied. Here we analyse the low-resolution three-dimensional structures of the separate subunits of an RNR from subclass NrdAi, as well as the alpha(4)beta(4) octamer that forms in the presence of dATP. The results reveal a type of oligomer not previously seen for any class of RNR and suggest a mechanism for how binding of dATP to the ATP-cone switches off catalysis by sterically preventing formation of the asymmetrical alpha(2)beta(2) complex.
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| 26. |
- Hellsten, Sofie V, et al.
(författare)
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Nutritional Stress Induced by Amino Acid Starvation Results in Changes for Slc38 Transporters in Immortalized Hypothalamic Neuronal Cells and Primary Cortex Cells
- 2018
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Ingår i: Frontiers in Molecular Biosciences. - : FRONTIERS MEDIA SA. - 2296-889X. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Amino acid sensing and signaling is vital for cells, and both gene expression and protein levels of amino acid transporters are regulated in response to amino acid availability. Here, the aim was to study the regulation of all members of the SLC38 amino acid transporter family, Slc38a1-11 , in mouse brain cells following amino acid starvation. We reanalyzed microarray data for the immortalized hypothalamic cell line N25/2 subjected to complete amino acid starvation for 1, 2, 3, 5, or 16 h, focusing specifically on the SLC38 family. All 11 Slc38 genes were expressed in the cell line, and Slc38a1, Slc38a2, and Slc38a 7 were significantly upregulated at 5 h and most strongly at 16 h. Here, protein level changes were measured for SLC38A7 and the orphan family member SLC38A11 which has not been studied under different amino acid starvation condition at protein level. At 5 h, no significant alteration on protein level for either SLC38A7 or SLC38A11 could be detected. In addition, primary embryonic cortex cells were deprived of nine amino acids, the most common amino acids transported by the SLC38 family members, for 3 h, 7 h or 12 h, and the gene expression was measured using qPCR. Slc38a1, Slc38a2, Slc38a5, Slc38a6, Slc38a9, and Slc38a10 were upregulated, while Slc38a3 and Slc38a7 were downregulated. Slc38a8 was upregulated at 5 h and downregulated at 12 h. In conclusion, several members from the SLC38 family are regulated depending on amino acid levels and are likely to be involved in amino acid sensing and signaling in brain.
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| 27. |
- Huang, Shan, et al.
(författare)
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Truncated lubricin glycans in osteoarthritis stimulate the synoviocyte secretion of VEGFA, IL-8, and MIP-1α : Interplay between O-linked glycosylation and inflammatory cytokines
- 2022
-
Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- The primary aim of the study was to identify inflammatory markers relevant for osteoarthritis (OA)-related systemic (plasma) and local (synovial fluid, SF) inflammation. From this, we looked for inflammatory markers that coincided with the increased amount of O-linked Tn antigen (GalNAcα1-Ser/Thr) glycan on SF lubricin. Inflammatory markers in plasma and SF in OA patients and controls were measured using a 44-multiplex immunoassay. We found consistently 29 markers detected in both plasma and SF. The difference in their concentration and the low correlation when comparing SF and plasma suggests an independent inflammatory environment in the two biofluids. Only plasma MCP-4 and TARC increased in our patient cohort compared to control plasma. To address the second task, we concluded that plasma markers were irrelevant for a direct connection with SF glycosylation. Hence, we correlated the SF-inflammatory marker concentrations with the level of altered glycosylation of SF-lubricin. We found that the level of SF-IL-8 and SF-MIP-1α and SF-VEGFA in OA patients displayed a positive correlation with the altered lubricin glycosylation. Furthermore, when exposing fibroblast-like synoviocytes from both controls and OA patients to glycovariants of recombinant lubricin, the secretion of IL-8 and MIP-1α and VEGFA were elevated using lubricin with Tn antigens, while lubricin with sialylated and nonsialylated T antigens had less or no measurable effect. These data suggest that truncated glycans of lubricin, as found in OA, promote synovial proinflammatory cytokine production and exacerbate local synovial inflammation.
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| 28. |
- Jansson, Malin, et al.
(författare)
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Prognostic Value of Stromal Type IV Collagen Expression in Small Invasive Breast Cancers
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Breast cancer is the most common cause of cancer death among women worldwide. Localized breast cancer can be cured by surgery and adjuvant therapy, but mortality remains high for tumors that metastasize early. Type IV collagen is a basement membrane protein, and breach of this extracellular matrix structure is the first step of cancer invasion. Type IV collagen is found in the stroma of many cancers, but its role in tumor biology is unclear. Here, expression of type IV collagen in the stroma of small breast cancers was analyzed, correlated to clinically used prognostic biomarkers and patient survival. The findings were further validated in an independent gene expression data cohort. Tissue samples from 1,379 women with in situ and small invasive breast cancers (<= 15 mm) diagnosed in 1986-2004 were included. Primary tumor tissue was collected into tissue microarrays. Type IV collagen expression in tissues was visualized using immunohistochemistry. Gene expression data was extracted from the Cancer Genome Atlas database. Out of 1,379 women, 856 had an invasive breast cancer and type IV collagen staining was available for 714 patients. In Kaplan-Meier analysis high type IV collagen expression was significantly associated (p = 0.026) with poorer breast cancer specific survival. There was no correlation of type IV collagen expression to clinically used prognostic biomarkers. High type IV collagen expression was clearly associated to distant metastasis (p = 0.002). In an external validation cohort (n = 1,104), high type IV collagen mRNA expression was significantly (p = 0.041) associated with poorer overall survival, with overexpression of type IV collagen mRNA in metastatic tissue. Stromal type IV collagen expression in the primary tumor correlates to poor breast cancer specific survival most likely due to a higher risk of developing distant metastasis. This ECM protein may function as biomarker to predict the risk of future metastatic disease in patients with breast cancers.
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| 29. |
- Joshi, Bishnu, et al.
(författare)
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Transcriptome Profiling of Staphylococcus aureus Associated Extracellular Vesicles Reveals Presence of Small RNA-Cargo
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial extracellular vesicles (EVs) have a vital role in bacterial pathogenesis. However, to date, the small RNA-cargo of EVs released by the opportunistic pathogen Staphylococcus aureus has not been characterized. Here, we shed light on the association of small RNAs with EVs secreted by S. aureus MSSA476 cultured in iron-depleted bacteriologic media supplemented with a subinhibitory dosage of vancomycin to mimic infection condition. Confocal microscopy analysis on intact RNase-treated EVs indicated that RNA is associated with EV particles. Transcriptomic followed by bioinformatics analysis of EV-associated RNA revealed the presence of potential gene regulatory small RNAs and high levels of tRNAs. Among the EV-associated enriched small RNAs were SsrA, RsaC and RNAIII. Our finding invites new insights into the potential role of EV-associated RNA as a modulator of host-pathogen interaction.
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| 30. |
- Kettisen, Karin, et al.
(författare)
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Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Due to its compatible oxygen-transporting abilities, hemoglobin (Hb) is a protein of interest in the development of artificial oxygen therapeutics. Despite continuous formulation attempts, extracellular Hb solution often exhibits undesirable reactions when applied in vivo. Therefore, protein engineering is frequently used to examine alternative ways of controlling the unwanted reactions linked to cell-free Hb solutions. In this study, three mutants of human fetal hemoglobin (HbF) are evaluated; single mutants αA12D and αA19D, and a double mutant αA12D/A19D. These variants were obtained by site-directed mutagenesis and recombinant production in E. coli, and carry negative charges on the surface of the α-subunit at the designated mutation sites. Through characterization of the mutant proteins, we found that the substitutions affected the protein in several ways. As expected, the isoelectric points (pIs) were lowered, from 7.1 (wild-type) down to 6.6 (double mutant), which influenced the anion exchange chromatographic procedures by shifting conditions toward higher conductivity for protein elution. The biological and physiological properties of HbF could be improved by these small modifications on the protein surface. The DNA cleavage rate associated with native HbF could be reduced by 55%. In addition, the negatively charged HbF mutant had an extended circulation time when examined in a mouse model using top load Hb additions. At the same time, the mutations did not affect the overall structural integrity of the HbF molecule, as determined by small-angle X-ray scattering. In combination with circular dichroism and thermal stability, modest structural shifts imposed by the mutations could possibly be related to changes in secondary structure or reorganization. Such local deformations were too minor to be determined within the resolution of the structural data; and overall, unchanged oxidation and heme loss kinetics support the conclusion that the mutations did not adversely affect the basic structural properties of Hb. We confirm the value of adding negatively charged residues onto the surface of the protein to improve the global functions of recombinant Hb.
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| 31. |
- Kettisen, Karin, et al.
(författare)
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Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant
- 2023
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.
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| 32. |
- Koder Hamid, Mona, et al.
(författare)
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Molecular dynamics simulations of the adsorption of an intrinsically disordered protein: Force field and water model evaluation in comparison with experiments
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- This study investigates possible structural changes of an intrinsically disordered protein (IDP) when it adsorbs to a solid surface. Experiments on IDPs primarily result in ensemble averages due to their high dynamics. Therefore, molecular dynamics (MD) simulations are crucial for obtaining more detailed information on the atomistic and molecular levels. An evaluation of seven different force field and water model combinations have been applied: (A) CHARMM36IDPSFF + CHARMM-modified TIP3P, (B) CHARMM36IDPSFF + TIP4P-D, (C) CHARMM36m + CHARMM-modified TIP3P, (D) AMBER99SB-ILDN + TIP3P, (E) AMBER99SB-ILDN + TIP4P-D, (F) AMBERff03ws + TIP4P/2005, and (G) AMBER99SB-disp + disp-water. The results have been qualitatively compared with those of small-angle X-ray scattering, synchrotron radiation circular dichroism spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. The model IDP corresponds to the first 33 amino acids of the N-terminal of the magnesium transporter A (MgtA) and is denoted as KEIF. With a net charge of +3, KEIF is found to adsorb to the anionic synthetic clay mineral Laponite® due to the increase in entropy from the concomitant release of counterions from the surface. The experimental results show that the peptide is largely disordered with a random coil conformation, whereas the helical content (α- and/or 310-helices) increased upon adsorption. MD simulations corroborate these findings and further reveal an increase in polyproline II helices and an extension of the peptide conformation in the adsorbed state. In addition, the simulations provided atomistic resolution of the adsorbed ensemble of structures, where the arginine residues had a high propensity to form hydrogen bonds with the surface. Simulations B, E, and G showed significantly better agreement with experiments than the other simulations. Particularly noteworthy is the discovery that B and E with TIP4P-D water had superior performance to their corresponding simulations A and D with TIP3P-type water. Thus, this study shows the importance of the water model when simulating IDPs and has also provided an insight into the structural changes of surface-active IDPs induced by adsorption, which may play an important role in their function.
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| 33. |
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| 34. |
- Kohler, Verena, 1992-, et al.
(författare)
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Reversible protein assemblies in the proteostasis network in health and disease
- 2023
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- While proteins populating their native conformations constitute the functional entities of cells, protein aggregates are traditionally associated with cellular dysfunction, stress and disease. During recent years, it has become clear that large aggregate-like protein condensates formed via liquid-liquid phase separation age into more solid aggregate-like particles that harbor misfolded proteins and are decorated by protein quality control factors. The constituent proteins of the condensates/aggregates are disentangled by protein disaggregation systems mainly based on Hsp70 and AAA ATPase Hsp100 chaperones prior to their handover to refolding and degradation systems. Here, we discuss the functional roles that condensate formation/aggregation and disaggregation play in protein quality control to maintain proteostasis and why it matters for understanding health and disease.
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| 35. |
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| 36. |
- Lau, Elizabeth C. H. T., et al.
(författare)
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Gold-iron oxide (Au/Fe3O4) magnetic nanoparticles as the nanoplatform for binding of bioactive molecules through self-assembly
- 2023
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Nanomedicine plays a crucial role in the development of next-generation therapies. The use of nanoparticles as drug delivery platforms has become a major area of research in nanotechnology. To be effective, these nanoparticles must interact with desired drug molecules and release them at targeted sites. The design of these "nanoplatforms" typically includes a functional core, an organic coating with functional groups for drug binding, and the drugs or bioactive molecules themselves. However, by exploiting the coordination chemistry between organic molecules and transition metal centers, the self-assembly of drugs onto the nanoplatform surfaces can bypass the need for an organic coating, simplifying the materials synthesis process. In this perspective, we use gold-iron oxide nanoplatforms as examples and outline the prospects and challenges of using self-assembly to prepare drug-nanoparticle constructs. Through a case study on the binding of insulin on Au-dotted Fe3O4 nanoparticles, we demonstrate how a self-assembly system can be developed. This method can also be adapted to other combinations of transition metals, with the potential for scaling up. Furthermore, the self-assembly method can also be considered as a greener alternative to traditional methods, reducing the use of chemicals and solvents. In light of the current climate of environmental awareness, this shift towards sustainability in the pharmaceutical industry would be welcomed.
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| 37. |
- León-González, José Alberto, et al.
(författare)
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Folding and Evolution of a Repeat Protein on the Ribosome
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Life on earth is the result of the work of proteins, the cellular nanomachines that fold into elaborated 3D structures to perform their functions. The ribosome synthesizes all the proteins of the biosphere, and many of them begin to fold during translation in a process known as cotranslational folding. In this work we discuss current advances of this field and provide computational and experimental data that highlight the role of ribosome in the evolution of protein structures. First, we used the sequence of the Ankyrin domain from the Drosophila Notch receptor to launch a deep sequence-based search. With this strategy, we found a conserved 33-residue motif shared by different protein folds. Then, to see how the vectorial addition of the motif would generate a full structure we measured the folding on the ribosome of the Ankyrin repeat protein. Not only the on-ribosome folding data is in full agreement with classical in vitro biophysical measurements but also it provides experimental evidence on how folded proteins could have evolved by duplication and fusion of smaller fragments in the RNA world. Overall, we discuss how the ribosomal exit tunnel could be conceptualized as an active site that is under evolutionary pressure to influence protein folding.
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| 38. |
- Liu, Yuanfeng, et al.
(författare)
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Exploring Gut Microbiota in Patients with Colorectal Disease Based on 16S rRNA Gene Amplicon and Shallow Metagenomic Sequencing
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- The gastrointestinal tract, the largest human microbial reservoir, is highly dynamic. The gut microbes play essential roles in causing colorectal diseases. In the present study, we explored potential keystone taxa during the development of colorectal diseases in central China. Fecal samples of some patients were collected and were allocated to the adenoma (Group A), colorectal cancer (Group C), and hemorrhoid (Group H) groups. The 16S rRNA amplicon and shallow metagenomic sequencing (SMS) strategies were used to recover the gut microbiota. Microbial diversities obtained from 16S rRNA amplicon and SMS data were similar. Group C had the highest diversity, although no significant difference in diversity was observed among the groups. The most dominant phyla in the gut microbiota of patients with colorectal diseases were Bacteroidetes, Firmicutes, and Proteobacteria, accounting for >95% of microbes in the samples. The most abundant genera in the samples were Bacteroides, Prevotella, and Escherichia/Shigella, and further species-level and network analyses identified certain potential keystone taxa in each group. Some of the dominant species, such as Prevotella copri, Bacteroides dorei, and Bacteroides vulgatus, could be responsible for causing colorectal diseases. The SMS data recovered diverse antibiotic resistance genes of tetracycline, macrolide, and beta-lactam, which could be a result of antibiotic overuse. This study explored the gut microbiota of patients with three different types of colorectal diseases, and the microbial diversity results obtained from 16S rRNA amplicon sequencing and SMS data were found to be similar. However, the findings of this study are based on a limited sample size, which warrants further large-scale studies. The recovery of gut microbiota profiles in patients with colorectal diseases could be beneficial for future diagnosis and treatment with modulation of the gut microbiota. Moreover, SMS data can provide accurate species- and gene-level information, and it is economical. It can therefore be widely applied in future clinical metagenomic studies.
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| 39. |
- López-Guajardo, A., et al.
(författare)
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Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6
- 2023
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis. Copyright © 2023 López-Guajardo, Zafar, Al Hennawi, Rossi, Alrwaili, Medcalf, Dunning, Nordgren, Pettersson, Estabrook, Hawkins and Gad.
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| 40. |
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| 41. |
- Magnusson, Rasmus, et al.
(författare)
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RNA-sequencing and mass-spectrometry proteomic time-series analysis of T-cell differentiation identified multiple splice variants models that predicted validated protein biomarkers in inflammatory diseases
- 2022
-
Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Profiling of mRNA expression is an important method to identify biomarkers but complicated by limited correlations between mRNA expression and protein abundance. We hypothesised that these correlations could be improved by mathematical models based on measuring splice variants and time delay in protein translation. We characterised time-series of primary human naive CD4(+) T cells during early T helper type 1 differentiation with RNA-sequencing and mass-spectrometry proteomics. We performed computational time-series analysis in this system and in two other key human and murine immune cell types. Linear mathematical mixed time delayed splice variant models were used to predict protein abundances, and the models were validated using out-of-sample predictions. Lastly, we re-analysed RNA-seq datasets to evaluate biomarker discovery in five T-cell associated diseases, further validating the findings for multiple sclerosis (MS) and asthma. The new models significantly out-performing models not including the usage of multiple splice variants and time delays, as shown in cross-validation tests. Our mathematical models provided more differentially expressed proteins between patients and controls in all five diseases. Moreover, analysis of these proteins in asthma and MS supported their relevance. One marker, sCD27, was validated in MS using two independent cohorts for evaluating response to treatment and disease prognosis. In summary, our splice variant and time delay models substantially improved the prediction of protein abundance from mRNA expression in three different immune cell types. The models provided valuable biomarker candidates, which were further validated in MS and asthma.
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| 42. |
- Majid, Abbasi, et al.
(författare)
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miR-124-3p Suppresses the Invasiveness and Metastasis of Hepatocarcinoma Cells via Targeting CRKL
- 2020
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X .- 2296-889X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Abnormal expressions of microRNAs are involved in growth and progression of human cancers including hepatocellular carcinoma (HCC). An adaptor protein CRKL plays a pivotal role in HCC growth, whereas miR-124-3p downregulation is associated with clinical stage and the poor survival of patients. However, the relationship between miR-124-3p and CRKL and the molecular mechanisms through which they regulate HCC metastasis remains unclear. In the current work, we explored miR-124-3p and its correlation with CRKL expression in HCC patient tissues. We found that miR-124-3p deficiency is inversely co-related with CRKL overexpression in tumorous tissues of HCC patients, which was also consistent in HCCLM3 and Huh7 HCC cell lines. Target validation data shows that miR-124-3p directly targets CRKL. The overexpression of miR-124-3p reverses the CRKL expression at both mRNA and protein levels and inhibits the cell development, migration, and invasion. Mechanistic investigations showed that CRKL downregulation suppresses the ERK pathway and EMT process, and concomitant decrease in invasion and metastasis of HCC cells. The expressions of key molecules in the ERK pathway such as RAF, MEK, ERK1/2, and pERK1/2 and key promoters of EMT such as N-cadherin and vimentin were downregulated, whereas E-cadherin, a key suppression indicator of EMT, was upregulated. MiR-124-3p-mediated CRKL suppression led to BAX/BCL-2 increase and C-JUN downregulation, which inhibited the cell proliferation and promoted the apoptosis in HCC cells. Collectively, our data illustrates that miR-124-3p acts as an important tumor-suppressive miRNA to suppress HCC carcinogenesis through targeting CRKL. The miR-124-3p-CRKL axial regulated pathway may offer valuable indications for cancer research, diagnosis, and treatment.
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| 43. |
- Malik, Yashpal S., et al.
(författare)
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SARS-CoV-2 Spike Protein Extrapolation for COVID Diagnosis and Vaccine Development
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 8
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Forskningsöversikt (refereegranskat)abstract
- Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to coronavirus disease 2019 (COVID-19) pandemic affecting nearly 71.2 million humans in more than 191 countries, with more than 1.6 million mortalities as of 12 December, 2020. The spike glycoprotein (S-protein), anchored onto the virus envelope, is the trimer of S-protein comprised of S1 and S2 domains which interacts with host cell receptors and facilitates virus-cell membrane fusion. The S1 domain comprises of a receptor binding domain (RBD) possessing an N-terminal domain and two subdomains (SD1 and SD2). Certain regions of S-protein of SARS-CoV-2 such as S2 domain and fragment of the RBD remain conserved despite the high selection pressure. These conserved regions of the S-protein are extrapolated as the potential target for developing molecular diagnostic techniques. Further, the S-protein acts as an antigenic target for different serological assay platforms for the diagnosis of COVID-19. Virus-specific IgM and IgG antibodies can be used to detect viral proteins in ELISA and lateral flow immunoassays. The S-protein of SARS-CoV-2 has very high sequence similarity to SARS-CoV-1, and the monoclonal antibodies (mAbs) against SARS-CoV-1 cross-react with S-protein of SARS-CoV-2 and neutralize its activity. Furthermore, in vitro studies have demonstrated that polyclonal antibodies targeted against the RBD of S-protein of SARS-CoV-1 can neutralize SARS-CoV-2 thus inhibiting its infectivity in permissive cell lines. Research on coronaviral S-proteins paves the way for the development of vaccines that may prevent SARS-CoV-2 infection and alleviate the current global coronavirus pandemic. However, specific neutralizing mAbs against SARS-CoV-2 are in clinical development. Therefore, neutralizing antibodies targeting SARS-CoV-2 S-protein are promising specific antiviral therapeutics for pre-and post-exposure prophylaxis and treatment of SARS-CoV-2 infection. We hereby review the approaches taken by researchers across the world to use spike gene and S-glycoprotein for the development of effective diagnostics, vaccines and therapeutics against SARA-CoV-2 infection the COVID-19 pandemic.
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| 44. |
- Mannervik, Bengt, et al.
(författare)
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Glutathione Transferases as Efficient Ketosteroid Isomerases
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 8
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Forskningsöversikt (refereegranskat)abstract
- In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M−1s−1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3β-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s−1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.
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| 45. |
- Marinaro, G., et al.
(författare)
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Wall-free droplet microfluidics for probing biological processes by high-brilliance X-ray scattering techniques
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Forskningsöversikt (refereegranskat)abstract
- Here we review probing biological processes initiated by the deposition of droplets on surfaces by micro- and nanobeam X-ray scattering techniques using synchrotron radiation and X-ray free-electron laser sources. We review probing droplet evaporation on superhydrophobic surfaces and reactions with substrates, basics of droplets deposition and flow simulations, droplet deposition techniques and practical experience at a synchrotron beamline. Selected applications with biological relevance will be reviewed and perspectives for the latest generation of high-brilliance X-ray sources discussed.
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| 46. |
- Mhashal, AR, et al.
(författare)
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Exploring the Conformational Impact of Glycine Receptor TM1-2 Mutations Through Coarse-Grained Analysis and Atomistic Simulations
- 2022
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Ingår i: Frontiers in molecular biosciences. - : Frontiers Media SA. - 2296-889X. ; 9, s. 890851-
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Tidskriftsartikel (refereegranskat)abstract
- Pentameric ligand-gated ion channels (PLGICs) are a family of proteins that convert chemical signals into ion fluxes through cellular membranes. Their structures are highly conserved across all kingdoms from bacteria to eukaryotes. Beyond their classical roles in neurotransmission and neurological disorders, PLGICs have been recently related to cell proliferation and cancer. Here, we focus on the best characterized eukaryotic channel, the glycine receptor (GlyR), to investigate its mutational patterns in genomic-wide tumor screens and compare them with mutations linked to hyperekplexia (HPX), a Mendelian neuromotor disease that disrupts glycinergic currents. Our analysis highlights that cancer mutations significantly accumulate across TM1 and TM2, partially overlapping with HPX changes. Based on 3D-clustering, conservation, and phenotypic data, we select three mutations near the pore, expected to impact GlyR conformation, for further study by molecular dynamics (MD). Using principal components from experimental GlyR ensembles as framework, we explore the motions involved in transitions from the human closed and desensitized structures and how they are perturbed by mutations. Our MD simulations show that WT GlyR spontaneously explores opening and re-sensitization transitions that are significantly impaired by mutations, resulting in receptors with altered permeability and desensitization properties in agreement with HPX functional data.
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| 47. |
- Nawaz, Muhammad, et al.
(författare)
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Extracellular Vesicles, Tunneling Nanotubes, and Cellular Interplay: Synergies and Missing Links.
- 2017
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Ingår i: Frontiers in molecular biosciences. - : Frontiers Media SA. - 2296-889X. ; 4
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Forskningsöversikt (refereegranskat)abstract
- The process of intercellular communication seems to have been a highly conserved evolutionary process. Higher eukaryotes use several means of intercellular communication to address both the changing physiological demands of the body and to fight against diseases. In recent years, there has been an increasing interest in understanding how cell-derived nanovesicles, known as extracellular vesicles (EVs), can function as normal paracrine mediators of intercellular communication, but can also elicit disease progression and may be used for innovative therapies. Over the last decade, a large body of evidence has accumulated to show that cells use cytoplasmic extensions comprising open-ended channels called tunneling nanotubes (TNTs) to connect cells at a long distance and facilitate the exchange of cytoplasmic material. TNTs are a different means of communication to classical gap junctions or cell fusions; since they are characterized by long distance bridging that transfers cytoplasmic organelles and intracellular vesicles between cells and represent the process of heteroplasmy. The role of EVs in cell communication is relatively well-understood, but how TNTs fit into this process is just emerging. The aim of this review is to describe the relationship between TNTs and EVs, and to discuss the synergies between these two crucial processes in the context of normal cellular cross-talk, physiological roles, modulation of immune responses, development of diseases, and their combinatory effects in tissue repair. At the present time this review appears to be the first summary of the implications of the overlapping roles of TNTs and EVs. We believe that a better appreciation of these parallel processes will improve our understanding on how these nanoscale conduits can be utilized as novel tools for targeted therapies.
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| 48. |
- Nilsson, Patrik, et al.
(författare)
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Design of experiments to investigate multi-additive cellulose nanocrystal films
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose nanocrystal (CNC) suspensions can self-assemble into chiral nematic films upon the slow evaporation of water. These films are brittle, as indicated by their fracturing instead of plastically deforming once they are fully elastically deformed. This aspect can be mediated to some extent by plasticizing additives, such as glucose and glycerol, however, few reports consider more than one additive at a time or address the influence of additive content on the homogeneity of the self-assembled structure. In this work, design of experiments (DoE) was used to empirically model complex film compositions, attempting to relate additive concentrations in dilute suspension to film properties, and to understand whether outcome specific predictions are possible using this approach. We demonstrate that DoE can be used to predict film properties in multi-additive systems, without consideration given to the different phenomena that occur along the drying process or to the nature of the additives. Additionally, a homogeneity metric is introduced in relation to chiral nematic organization in CNC films, with most of the additive-containing compositions in this work found to reduce the homogeneity of the self-assembly relative to pure CNC films. Copyright © 2022 Nilsson, Engström, Kaschuk, Vapaavuori, Larsson and Abitbol.
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| 49. |
- Oldoni, Emanuela, et al.
(författare)
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Tackling the translational challenges of multi-omics research in the realm of European personalised medicine : A workshop report
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Personalised medicine (PM) presents a great opportunity to improve the future of individualised healthcare. Recent advances in -omics technologies have led to unprecedented efforts characterising the biology and molecular mechanisms that underlie the development and progression of a wide array of complex human diseases, supporting further development of PM. This article reflects the outcome of the 2021 EATRIS-Plus Multi-omics Stakeholder Group workshop organised to 1) outline a global overview of common promises and challenges that key European stakeholders are facing in the field of multi-omics research, 2) assess the potential of new technologies, such as artificial intelligence (AI), and 3) establish an initial dialogue between key initiatives in this space. Our focus is on the alignment of agendas of European initiatives in multi-omics research and the centrality of patients in designing solutions that have the potential to advance PM in long-term healthcare strategies.
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| 50. |
- Oliveira, Daniel V., et al.
(författare)
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Molecular Chaperone BRICHOS Inhibits CADASIL-Mutated NOTCH3 Aggregation In Vitro
- 2022
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media SA. - 2296-889X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common familial form of stroke, which is caused by mutations located in the epidermal growth factor (EGF)-like repeats of the NOTCH3 gene. Mutations cause the NOTCH3 (N3) protein to misfold and aggregate. These aggregates will be a component of granular osmiophilic material, which when accumulated around the arteries and arterioles is believed to cause the degradation of vascular smooth muscle cells (VSMC). VSMC degradation affects blood flow regulation and leads to white matter and neuronal death. Currently, there is no treatment for CADASIL. The dementia-relevant BRICHOS domain is a small multitalented protein with functions that include ATP-independent chaperone-like properties. BRICHOS has been shown to prevent the aggregation of both fibrillar and non-fibrillar structures. Therefore, the objective of this study is to investigate whether BRICHOS exhibits anti-aggregating properties on a recombinant CADASIL-mutated N3 protein consisting of the first five repeats of EGF (EGF(1-5)), harboring a cysteine instead of an arginine in the position 133, (R133C). We found that the N3 EGF(1-5) R133C mutant is more prone to aggregate, while the wildtype is more stable. Recombinant human Bri2 BRICHOS is able to interact and stabilize the R133C-mutated N3 protein in a dose-dependent manner. These results suggest an anti-aggregating impact of BRICHOS on the N3 EGF(1-5) R133C protein, which could be a potential treatment for CADASIL.
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