| 1. |
- Johansson, Patrik, et al.
(författare)
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A Patient-Derived Cell Atlas Informs Precision Targeting of Glioblastoma
- 2020
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 32:2
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Tidskriftsartikel (refereegranskat)abstract
- Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells, We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.
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| 2. |
- Genshaft, Alex S., et al.
(författare)
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Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction
- 2016
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Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1 (TM) system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.
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| 3. |
- Tarbier, M, et al.
(författare)
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Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells
- 2020
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1, s. 5445-
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.
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| 4. |
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| 5. |
- Batool, Tahira, et al.
(författare)
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Upregulated BMP-Smad signaling activity in the glucuronyl C5-epimerase knock out MEF cells
- 2019
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Ingår i: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 54, s. 122-129
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Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of glucuronic acid to iduronic acid in the process of heparan sulfate biosynthesis. Targeted interruption of the gene, Glce,in mice resulted in neonatal lethality with varied defects in organ development. To understand the molecular mechanisms of the phenotypes, we used mouse embryonic fibroblasts (MEF) as a model to examine selected signaling pathways. Our earlier studies found reduced activities of FGF-2, GDNF, but increased activity of sonic hedgehog in the mutant cells. In this study, we focused on the bone morphogenetic protein (BMP) signaling pathway. Western blotting detected substantially elevated endogenous Smad1/5/8 phosphorylation in the Hsepi mutant (KO) MEF cells, which is reverted by re-expression of the enzyme in the KO cells. The mutant cells displayed an enhanced proliferation and elevated alkaline phosphatase activity, marking higher differentiation, when cultured in osteogenic medium. The high level of Smad1/5/8 phosphorylation was also found in primary calvarial cells isolated from the KO mice. Analysis of the genes involved in the BMP signaling pathway revealed upregulation of a number of BMP ligands, but reduced expression of several Smads and BMP antagonist (Grem1) in the KO MEF cells. The results suggest that Hsepi expression modulates BMP signaling activity, which, at least partially, is associated with defected molecular structure of heparan sulfate expressed in the cells.
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| 6. |
- Mattisson, Jonas, et al.
(författare)
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Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Mosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.
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| 7. |
- Schuster, Jens, Assistant Professor, 1972-, et al.
(författare)
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ZEB2 haploinsufficient Mowat-Wilson syndrome induced pluripotent stem cells show disrupted GABAergic transcriptional regulation and function
- 2022
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Ingår i: Frontiers in Molecular Neuroscience. - : Frontiers Media SA. - 1662-5099. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Mowat-Wilson syndrome (MWS) is a severe neurodevelopmental disorder caused by heterozygous variants in the gene encoding transcription factor ZEB2. Affected individuals present with structural brain abnormalities, speech delay and epilepsy. In mice, conditional loss of Zeb2 causes hippocampal degeneration, altered migration and differentiation of GABAergic interneurons, a heterogeneous population of mainly inhibitory neurons of importance for maintaining normal excitability. To get insights into GABAergic development and function in MWS we investigated ZEB2 haploinsufficient induced pluripotent stem cells (iPSC) of MWS subjects together with iPSC of healthy donors. Analysis of RNA-sequencing data at two time points of GABAergic development revealed an attenuated interneuronal identity in MWS subject derived iPSC with enrichment of differentially expressed genes required for transcriptional regulation, cell fate transition and forebrain patterning. The ZEB2 haploinsufficient neural stem cells (NSCs) showed downregulation of genes required for ventral telencephalon specification, such as FOXG1, accompanied by an impaired migratory capacity. Further differentiation into GABAergic interneuronal cells uncovered upregulation of transcription factors promoting pallial and excitatory neurons whereas cortical markers were downregulated. The differentially expressed genes formed a neural protein-protein network with extensive connections to well-established epilepsy genes. Analysis of electrophysiological properties in ZEB2 haploinsufficient GABAergic cells revealed overt perturbations manifested as impaired firing of repeated action potentials. Our iPSC model of ZEB2 haploinsufficient GABAergic development thus uncovers a dysregulated gene network leading to immature interneurons with mixed identity and altered electrophysiological properties, suggesting mechanisms contributing to the neuropathogenesis and seizures in MWS.
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| 8. |
- Al-Amin, Rasel A., PhD student, 1983-, et al.
(författare)
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Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:31, s. 10999-11009
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The ability to monitor target engagement in cellular contexts is a key for successful drug discovery and also valuable in clinical routine. A cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. The CETSA combined with mass spectrometry (MS) detection can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis is slow, requires substantial amounts of the sample material, and often misses proteins of specific interest. Here, we combined the CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of a set of 67 proteins from small amounts of the sample material treated with kinase inhibitors. The results were concordant with the corresponding analyses read out via MS. Our approach allows analyses of large numbers of specific target proteins at high sensitivity in limited sample aliquots. Highly sensitive multiplex CETSA-PEA assays are therefore promising for monitoring drug-target engagement in small sample aliquots in the course of drug development and potentially in clinical settings.
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| 9. |
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| 10. |
- Löfgren, Sara E., et al.
(författare)
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A 3 '-Untranslated Region Variant Is Associated With Impaired Expression of CD226 in T and Natural Killer T Cells and Is Associated With Susceptibility to Systemic Lupus Erythematosus
- 2010
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Ingår i: Arthritis and Rheumatism. - : Wiley. - 0004-3591 .- 1529-0131. ; 62:11, s. 3404-3414
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Tidskriftsartikel (refereegranskat)abstract
- Objective. Costimulatory receptor CD226 plays an important role in T cell activation, differentiation, and cytotoxicity. This study was undertaken to investigate the genetic association of CD226 with susceptibility to systemic lupus erythematosus (SLE) and to assess the functional implications of this association. Methods. Twelve tag single-nucleotide polymorphisms (SNPs) in CD226 were typed in 1,163 SLE patients and 1,482 healthy control subjects from Europe or of European ancestry. Analyses of association were performed by single-marker Cochran-Mantel-Haenszel meta-analysis, followed by haplotype analysis. Gene expression was analyzed by quantitative real-time polymerase chain reaction analyses of RNA from peripheral blood mononuclear cells, and by fluorescence-activated cell sorter analysis. To study the functional impact of the associated variants, luciferase reporter constructs containing different portions of the 3'-untranslated region (3'-UTR) of the gene were prepared and used in transfection experiments. Results. A 3-variant haplotype, rs763361; rs34794968; rs727088 (ATC), in the last exon of CD226 was associated with SLE (P = 1.3 x 10(-4), odds ratio 1.24, 95% confidence interval 1.11-1.38). This risk haplotype correlated with low CD226 transcript expression and low CD226 protein levels on the surface of CD4+ and CD8+ T cells and natural killer T (NKT) cells. NK cells expressed high levels of CD226, but this expression was independent of the haplotype. Reporter assays with deletion constructs indicated that only the presence of rs727088 could account for the differences in the levels of luciferase transcripts. Conclusion. This study identified an association of CD226 with SLE in individuals of European ancestry. These data support the importance of the 3'-UTR SNP rs727088 in the regulation of CD226 transcription both in T cells and in NKT cells.
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