A5021082839- CRISPR and Genetic Engineering
- interferon and immune responses
- CAR-T cell therapy research
- Immune Cell Function and Interaction
- RNA and protein synthesis mechanisms
- Advanced biosensing and bioanalysis techniques
- Single-cell and spatial transcriptomics
- Hepatitis C virus research
- Virus-based gene therapy research
- Immune Response and Inflammation
- Cancer Immunotherapy and Biomarkers
- RNA Research and Splicing
- Receptor Mechanisms and Signaling
- RNA Interference and Gene Delivery
- Mast cells and histamine
- Inflammasome and immune disorders
- Systemic Lupus Erythematosus Research
- Neuropeptides and Animal Physiology
- vaccines and immunoinformatics approaches
- Immunotherapy and Immune Responses
- Pluripotent Stem Cells Research
- Epigenetics and DNA Methylation
- Advanced Proteomics Techniques and Applications
- Cancer Genomics and Diagnostics
- Melanoma and MAPK Pathways
Vienna Biocenter
2015-2016
Ludwig Cancer Research
2016
Austrian Academy of Sciences
2007-2013
CeMM Research Center for Molecular Medicine
2006-2013
Robert Koch Institute
2005-2009
University of Kansas
2001-2002
University of Regensburg
2001
Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second copy. However, no completely haploid line has been described, hampering accessibility subset genes. The near-haploid HAP1 contains single copy all chromosomes except heterozygous 30-megabase fragment Chromosome 15. This large encompasses 330 genes integrated on long arm 19. Here, we employ CRISPR/Cas9-based strategy to excise this...
Abstract Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, introduction foreign DNA sequences tag an endogenous gene is still cumbersome as it requires synthesis or cloning homology templates. Here we present a strategy enables tagging loci one generic donor plasmid. It contains interest flanked two gRNA recognition sites allow excision from Co-transfection cells...
Dasatinib is a small-molecule kinase inhibitor used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). We have analyzed kinases targeted by dasatinib using an unbiased chemical proteomics approach to detect binding proteins directly from lysates CML cells. Besides Abl and Src kinases, we identified Tec Btk Tec, but not Itk, as major binders dasatinib. The activity was inhibited nanomolar concentrations in vitro cultured gatekeeper residue critical determinant...
Aicardi–Goutières syndrome (AGS) is a rare inherited autoimmune disease caused by mutations in genes encoding the RNase H2 subunits A, B, and C; DNase three prime repair exonuclease 1 (TREX1); sterile alpha motif (SAM) domain HD domain-containing protein (SAMHD1). Using unbiased affinity purification coupled to mass spectrometry, we identify SAMHD1 as nucleic-acid-binding displaying preference for RNA over DNA. In contrast TREX1 complex, has no obvious nuclease activity. addition,...
TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) NAP1 TBKBP2, AZI2) whose functional relationship to TBK1 IKK-i is poorly understood. We performed a systematic affinity purification–mass spectrometry approach derive comprehensive TBK1/IKK-i molecular network. The most salient feature the network mutual exclusive interaction adaptors kinases,...
Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator functional avidity in mice. Here, we investigate mechanistic role CISH regulating human solid tumors demonstrate CRISPR/Cas9 disruption enhances response checkpoint...
Understanding how cells respond differently to perturbation is crucial in cell biology, but existing methods often fail accurately quantify and interpret heterogeneous single-cell responses. Here we introduce the perturbation-response score (PS), a method diverse responses at level. Applied datasets such as Perturb-seq, PS outperforms quantifying partial gene perturbations. further enables dosage analysis without needing titrate perturbations, identifies 'buffered' 'sensitive' response...
Abstract Background On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation central proteome, which is set proteins ubiquitously expressed in all cells. Composition and properties proteome are investigated through bioinformatics analyses. Results We experimentally identify a comprising 1,124 that abundantly cells using state art mass spectrometry protein identification bioinformatics. The main represented functions...
The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- class-specific HDAC inhibitors available, these drugs do not allow a comprehensive understanding individual function, or the therapeutic potential isoform-specific targeting. To systematically compare impact catalytic functions HDAC1, HDAC2 HDAC3, we generated human HAP1 lines expressing catalytically inactive enzymes. Using this genetic toolbox effect inhibition with effects...
Hepatitis C virus (HCV) is a positive‐strand RNA that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) essential for viral replication. Here, the kinase Raf‐1 was identified as novel cellular binding partner of NS5A, to C‐terminal domain NS5A. colocalizes NS5A in replication complex. The interaction results increased phosphorylation at serine 338. Integrity crucial replication: inhibition by small‐molecule inhibitor BAY43‐9006...
Species isoforms of histamine H<sub>2</sub>-, H<sub>3</sub>-, and H<sub>4</sub>-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H<sub>1</sub>R (hH<sub>1</sub>R) guinea pig (ghH<sub>1</sub>R). We coexpressed hH<sub>1</sub>R gpH<sub>1</sub>R with regulators G-protein signaling Sf9 insect cells analyzed GTPase activity G<sub>q</sub>-proteins. Small agonists showed similar effects at gpH<sub>1</sub>R, whereas bulkier...
It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H<sub>2</sub>-receptor (H<sub>2</sub>R) agonists are lower at H<sub>2</sub>R human neutrophils than guinea pig atrium. To elucidate these differences, we analyzed fusion proteins (hH<sub>2</sub>R) (gpH<sub>2</sub>R), respectively, short splice variant G<sub>sα</sub>(G<sub>sαS</sub>) expressed in Sf9 cells. The small GTPase assay antagonists inhibiting histamine-stimulated GTP hydrolysis by...
Abstract The human histamine H 2 ‐receptor (hH R) couples to G s ‐proteins activate adenylyl cyclase and q phospholipase C, but C activation has not consistently been observed. aim of this study was compare coupling hH R insect mammalian ‐ in Spodoptera frugiperda (Sf9) cells. Interaction with proteins assessed coexpressed or receptor‐G α fusion that enhance efficiency. efficiently coupled cyclase. However, poorly as by the lack enhancement histamine‐stimulated steady‐state GTP hydrolysis...
The role of hepatitis C virus (HCV) protein non-structural (NS) 5A in HCV-associated pathogenesis is still enigmatic. To investigate the vivo NS5A for viral persistence and virus-associated a transgenic (Tg) mouse model was established. Mice with liver-targeted transgene expression were generated using albumin promoter. Alterations hepatic immune response determined by Western blot, infection lymphocytic choriomeningitis (LCMV), transient NS3/4A Tg mice hydrodynamic injection. Cytotoxic T...
Chromosomal translocations are a hallmark of cancer cells and give rise to fusion oncogenes. To gain insight into the mechanisms governing tumorigenesis, adequate model cell lines required. We employ versatile CRISPR/Cas system engineer in which chromosomal either generated de novo (CD74-ROS1) or existing reverted back original configuration (BCR-ABL1). this end, we co-apply two guide RNAs artificially generate breakpoints screen for spontaneous events by PCR. The approach use is efficient...
Abstract Recognition of foreign DNA by cytosolic innate immune receptors triggers the production IFN‐β. However, it is unclear whether different types ligands are recognized similar and resulting response distinct from endosomal TLR response. To address these questions, we compared two most commonly used (IFN‐stimulatory (ISD) poly(dAdT)) assessed minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures competition experiments suggest that ISD...
The availability of CRISPR/Cas9 technology has enabled the rapid establishment gene knockouts in many cell types and even whole organisms. However, conditional inactivation essential genes remains a challenge. We devised an approach named DECAI (DEgradation based on Cre-regulated- Artificial Intron). It utilizes small cassette just 201 nucleotides that is inserted into coding exon target using homology-directed repair. As its sequence derived from artificial intron, removed by splicing...
Abstract Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes phenotypes cannot be studied lower It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics human cells. Here, we establish a approach based on highly robust sensitive multiplexed RNA sequencing of mutant We conduct 10 parallel using collection engineered haploid isogenic cell lines knockouts covering...