| 1. |
- Hibar, Derrek P., et al.
(författare)
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Novel genetic loci associated with hippocampal volume
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The hippocampal formation is a brain structure integrally involved in episodic memory, spatial navigation, cognition and stress responsiveness. Structural abnormalities in hippocampal volume and shape are found in several common neuropsychiatric disorders. To identify the genetic underpinnings of hippocampal structure here we perform a genome-wide association study (GWAS) of 33,536 individuals and discover six independent loci significantly associated with hippocampal volume, four of them novel. Of the novel loci, three lie within genes (ASTN2, DPP4 and MAST4) and one is found 200 kb upstream of SHH. A hippocampal subfield analysis shows that a locus within the MSRB3 gene shows evidence of a localized effect along the dentate gyrus, subiculum, CA1 and fissure. Further, we show that genetic variants associated with decreased hippocampal volume are also associated with increased risk for Alzheimer's disease (r(g) = -0.155). Our findings suggest novel biological pathways through which human genetic variation influences hippocampal volume and risk for neuropsychiatric illness.
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| 2. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 3. |
- Akter, Farhima, et al.
(författare)
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Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:11, s. 5040-5046
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Tidskriftsartikel (refereegranskat)abstract
- The chemical reactions used to make antibody DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of phi X174 gene A* protein and Z(mab2s) (A*-Zmab). The phi X174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab2s) protein moiety can bind to specific species of antibodies. The DNA in an A*-Zmab conjugate was attached to the A* protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A*-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin gamma 1 (IgG1) but not to rabbit IgG. This indicates the generality of the A*-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A*-Zmab-DNA conjugates up to 80% after a 15 min reaction. Finally, sensitive detection of human interferon-gamma (IFN-gamma) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.
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| 4. |
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| 5. |
- Allerbring, E. B., et al.
(författare)
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Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics
- 2012
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Ingår i: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 42:11, s. 2990-3000
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Tidskriftsartikel (refereegranskat)abstract
- The molecular basis underlying T-cell recognition of MHC molecules presenting altered peptide ligands is still not wellestablished. A hierarchy of T-cell activation by MHC class I-restricted altered peptide ligands has been defined using the T-cell receptor P14 specific for H-2Db in complex with the immunodominant lymphocytic choriomeningitis virus peptide gp33 (KAVYNFATM). While substitution of tyrosine to phenylalanine (Y4F) or serine (Y4S) abolished recognition by P14, the TCR unexpectedly recognized H-2Db in complex with the alanine-substituted semiagonist Y4A, which displayed the most significant structural modification. The observed functional hierarchy gp33 > Y4A > Y4S = Y4F was neither due to higher stabilization capacity nor to differences in structural conformation. However, thermodynamic analysis demonstrated that while recognition of the full agonist H-2Db/gp33 was strictly enthalpy driven, recognition of the weak agonist H-2Db/Y4A was instead entropy driven with a large reduction in the favorable enthalpy term. The fourfold larger negative heat capacity derived for the interaction of P14 with H-2Db/gp33 compared with H-2Db/Y4A can possibly be explained by higher water entrapment at the TCR/MHC interface, which is also consistent with the measured opposite entropy contributions for the interactions of P14 with both MHCs. In conclusion, this study demonstrates that P14 makes use of different strategies to adapt to structural modifications in the MHC/peptide complex.
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| 6. |
- Gallo, Selene, et al.
(författare)
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Functional connectivity signatures of major depressive disorder: machine learning analysis of two multicenter neuroimaging studies
- 2023
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Ingår i: Molecular Psychiatry. - : SPRINGERNATURE. - 1359-4184 .- 1476-5578. ; 28:7, s. 3013-3022
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Tidskriftsartikel (refereegranskat)abstract
- The promise of machine learning has fueled the hope for developing diagnostic tools for psychiatry. Initial studies showed high accuracy for the identification of major depressive disorder (MDD) with resting-state connectivity, but progress has been hampered by the absence of large datasets. Here we used regular machine learning and advanced deep learning algorithms to differentiate patients with MDD from healthy controls and identify neurophysiological signatures of depression in two of the largest resting-state datasets for MDD. We obtained resting-state functional magnetic resonance imaging data from the REST-meta-MDD (N = 2338) and PsyMRI (N = 1039) consortia. Classification of functional connectivity matrices was done using support vector machines (SVM) and graph convolutional neural networks (GCN), and performance was evaluated using 5-fold cross-validation. Features were visualized using GCN-Explainer, an ablation study and univariate t-testing. The results showed a mean classification accuracy of 61% for MDD versus controls. Mean accuracy for classifying (non-)medicated subgroups was 62%. Sex classification accuracy was substantially better across datasets (73-81%). Visualization of the results showed that classifications were driven by stronger thalamic connections in both datasets, while nearly all other connections were weaker with small univariate effect sizes. These results suggest that whole brain resting-state connectivity is a reliable though poor biomarker for MDD, presumably due to disease heterogeneity as further supported by the higher accuracy for sex classification using the same methods. Deep learning revealed thalamic hyperconnectivity as a prominent neurophysiological signature of depression in both multicenter studies, which may guide the development of biomarkers in future studies.
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| 7. |
- Grimm, Sebastian, et al.
(författare)
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Affinity maturation of an affibody molecule binding to human Raf-1 via non-targeted in vitro evolution
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The use of in vitro protein library technologies for the generation of high affinity binding proteins often includes an affinity maturation step, involving the construction of secondary libraries from which second generation variants with improved affinities are selected. We describe a novel approach for the affinity maturation of affibody molecules based on stepwise in vitro molecular evolution, involving cycles of whole gene error-prone PCR amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display for selection. Starting with a human Raf-1 binding affibody molecule of an initial 2 μM dissociation constant (KD), the in vitro evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor based monitoring of the collective target binding ability of expressed pools obtained after each selection cycle. After a first cycle of diversification and selection, no increase in the hRaf-1 target binding of the pool was observed, whereas after two cycles, a significant increase in the binding response was seen. DNA sequencing showed that an alanine to valine substitution in an earlier variegated position had been effectively enriched, and was present in 11% and 83% of all clones after cycle one and two, respectively, either alone or in combination with other substitutions. Further studies on a subset of individual variants isolated after cycle two showed that the observed A27V substitution alone accounted for a 13-fold increase in affinity, predominantly through increased on-rate kinetics. Additional substitutions in framework or non-framework positions typically resulted in a further two-fold increase in affinity. Interestingly, thermal melting point (Tm) analyses showed that an increased affinity could be associated with either slightly higher or lower Tm values, compared to the parental variant. All investigated variants showed excellent refolding properties, and the selectivity of the affinity matured hRaf-1 binders had not been compromised by the substitutions, as analyzed using a multiplexed bead-based binding assay involving 77 recombinant human control protein fragments.
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| 8. |
- Grimm, Sebastian, et al.
(författare)
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Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 29:5, s. 534-542
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Tidskriftsartikel (refereegranskat)abstract
- The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.
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| 9. |
- Grimm, Sebastian, 1980-
(författare)
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Ribosome display for selection and evolution of affibody molecules
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway. In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.
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| 10. |
- Grimm, Sebastian, et al.
(författare)
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Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
- 2011
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Ingår i: Molecular Biotechnology. - : Springer Science and Business Media LLC. - 1073-6085 .- 1559-0305. ; 48:3, s. 263-276
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Tidskriftsartikel (refereegranskat)abstract
- Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) recognition and dissociation constants (K (D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.
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| 11. |
- Grimm, Sebastian, et al.
(författare)
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Selection and characterisation of affibody molecules inhibiting the interaction between Ras and Raf in vitro
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784. ; 27:6, s. 766-773
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Tidskriftsartikel (refereegranskat)abstract
- Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed
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| 12. |
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| 13. |
- Grimm, Sebastian, et al.
(författare)
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Single domain affinity proteins for the detection of the genome organizer protein SATB1
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules and VHH antibody fragments are two classes of affinity proteins, both characterized by a small size and a single subunit domain structure. Here, we report the selection and characterization of affinity proteins from both classes against the special AT rich sequence binding protein SATB1, a suggested marker protein for aggressive and metastasizing breast cancer. The selected VHH antibody fragments originate from a nonimmune phage display library, while affibody molecules were selected from a library constructed in vitro and displayed on ribosomes. It was observed that selected molecules recognizing one of three conserved DNA-binding domains of SATB1, also recognized its close homologue SATB2 while several of the selected molecules from both classes binding to other regions selectively recognized SATB1. Two of these SATB1 selective molecules, VHH clone 2D2 and affibody molecule clone ZSATB1:2, performed well in differentimmunotechnology applications including ELISA, WB, IF and pull-out experiments and gave a selective staining of endogenous SATB1 in Jurkat T cells. These molecules may thus become useful tools, either alone or in combination, for the selective detection of SATB1 in breast tumor specimens. Due to their small size in comparison to immunoglobulins, such single domain binding proteins may be suitable for high resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy, where the resolution may get constrained by the size of the affinity reagent.
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| 14. |
- Javaheripour, Nooshin, et al.
(författare)
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Altered resting-state functional connectome in major depressive disorder : a mega-analysis from the PsyMRI consortium
- 2021
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Ingår i: Translational Psychiatry. - : Springer Nature. - 2158-3188. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Major depressive disorder (MDD) is associated with abnormal neural circuitry. It can be measured by assessing functional connectivity (FC) at resting-state functional MRI, that may help identifying neural markers of MDD and provide further efficient diagnosis and monitor treatment outcomes. The main aim of the present study is to investigate, in an unbiased way, functional alterations in patients with MDD using a large multi-center dataset from the PsyMRI consortium including 1546 participants from 19 centers (). After applying strict exclusion criteria, the final sample consisted of 606 MDD patients (age: 35.8 +/- 11.9 y.o.; females: 60.7%) and 476 healthy participants (age: 33.3 +/- 11.0 y.o.; females: 56.7%). We found significant relative hypoconnectivity within somatosensory motor (SMN), salience (SN) networks and between SMN, SN, dorsal attention (DAN), and visual (VN) networks in MDD patients. No significant differences were detected within the default mode (DMN) and frontoparietal networks (FPN). In addition, alterations in network organization were observed in terms of significantly lower network segregation of SMN in MDD patients. Although medicated patients showed significantly lower FC within DMN, FPN, and SN than unmedicated patients, there were no differences between medicated and unmedicated groups in terms of network organization in SMN. We conclude that the network organization of cortical networks, involved in processing of sensory information, might be a more stable neuroimaging marker for MDD than previously assumed alterations in higher-order neural networks like DMN and FPN.
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| 15. |
- Mohnke, Sebastian, et al.
(författare)
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Further evidence for the impact of a genome-wide-supported psychosis risk variant in ZNF804A on the Theory of Mind network
- 2014
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Ingår i: Neuropsychopharmacology. - : Nature Publishing Group. - 0893-133X .- 1740-634X. ; 39:5, s. 1196-1205
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Tidskriftsartikel (refereegranskat)abstract
- The single-nucleotide polymorphism (SNP) rs1344706 in ZNF804A is one of the best-supported risk variants for psychosis. We hypothesized that this SNP contributes to the development of schizophrenia by affecting the ability to understand other people's mental states. This skill, commonly referred to as Theory of Mind (ToM), has consistently been found to be impaired in schizophrenia. Using functional magnetic resonance imaging, we previously showed that in healthy individuals rs1344706 impacted on activity and connectivity of key areas of the ToM network, including the dorsomedial prefrontal cortex, temporo-parietal junction, and the posterior cingulate cortex, which show aberrant activity in schizophrenia patients, too. We aimed to replicate these results in an independent sample of 188 healthy German volunteers. In order to assess the reliability of brain activity elicited by the ToM task, 25 participants performed the task twice with an interval of 14 days showing excellent accordance in recruitment of key ToM areas. Confirming our previous results, we observed decreasing activity of the left temporo-parietal junction, dorsomedial prefrontal cortex, and the posterior cingulate cortex with increasing number of risk alleles during ToM. Complementing our replication sample with the discovery sample, analyzed in a previous report (total N=297), further revealed negative genotype effects in the left dorsomedial prefrontal cortex as well as in the temporal and parietal regions. In addition, as shown previously, rs1344706 risk allele dose positively predicted increased frontal-temporo-parietal connectivity. These findings confirm the effects of the psychosis risk variant in ZNF804A on the dysfunction of the ToM network.
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| 16. |
- Satizabal, Claudia L., et al.
(författare)
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Genetic architecture of subcortical brain structures in 38,851 individuals
- 2019
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Ingår i: Nature Genetics. - : Nature Publishing Group. - 1061-4036 .- 1546-1718. ; 51:11, s. 1624-
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Tidskriftsartikel (refereegranskat)abstract
- Subcortical brain structures are integral to motion, consciousness, emotions and learning. We identified common genetic variation related to the volumes of the nucleus accumbens, amygdala, brainstem, caudate nucleus, globus pallidus, putamen and thalamus, using genome-wide association analyses in almost 40,000 individuals from CHARGE, ENIGMA and UK Biobank. We show that variability in subcortical volumes is heritable, and identify 48 significantly associated loci (40 novel at the time of analysis). Annotation of these loci by utilizing gene expression, methylation and neuropathological data identified 199 genes putatively implicated in neurodevelopment, synaptic signaling, axonal transport, apoptosis, inflammation/infection and susceptibility to neurological disorders. This set of genes is significantly enriched for Drosophila orthologs associated with neurodevelopmental phenotypes, suggesting evolutionarily conserved mechanisms. Our findings uncover novel biology and potential drug targets underlying brain development and disease.
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| 17. |
- Thompson, Paul M., et al.
(författare)
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The ENIGMA Consortium : large-scale collaborative analyses of neuroimaging and genetic data
- 2014
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Ingår i: BRAIN IMAGING BEHAV. - : Springer Science and Business Media LLC. - 1931-7557 .- 1931-7565. ; 8:2, s. 153-182
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Tidskriftsartikel (refereegranskat)abstract
- The Enhancing NeuroImaging Genetics through Meta-Analysis (ENIGMA) Consortium is a collaborative network of researchers working together on a range of large-scale studies that integrate data from 70 institutions worldwide. Organized into Working Groups that tackle questions in neuroscience, genetics, and medicine, ENIGMA studies have analyzed neuroimaging data from over 12,826 subjects. In addition, data from 12,171 individuals were provided by the CHARGE consortium for replication of findings, in a total of 24,997 subjects. By meta-analyzing results from many sites, ENIGMA has detected factors that affect the brain that no individual site could detect on its own, and that require larger numbers of subjects than any individual neuroimaging study has currently collected. ENIGMA's first project was a genome-wide association study identifying common variants in the genome associated with hippocampal volume or intracranial volume. Continuing work is exploring genetic associations with subcortical volumes (ENIGMA2) and white matter microstructure (ENIGMA-DTI). Working groups also focus on understanding how schizophrenia, bipolar illness, major depression and attention deficit/hyperactivity disorder (ADHD) affect the brain. We review the current progress of the ENIGMA Consortium, along with challenges and unexpected discoveries made on the way.
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| 18. |
- Tolmachev, Vladimir, et al.
(författare)
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Molecular design of radiocopper-labelled Affibody molecules
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Cu-CB-TE2A-GEEE-ZHER2:342 was 16 ± 6%ID/g and tumor-to-blood ratio was 181 ± 52. In conclusion, a combination of the cross-bridged CB-TE2A chelator and Gly-Glu-Glu-Glu spacer is preferable for radiocopper labelling of Affibody molecules and, possibly, other scaffold proteins having high renal re-absorption.
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| 19. |
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