Background: Acute Myeloid leukemia is a heterogeneous disease that requires novel targeted treatment options tailored to the patients' specific microenvironment and blast phenotype.
Methods: We characterized bone marrow and/or blood samples of 37 AML patients and healthy donors by high dimensional flow cytometry and RNA sequencing using computational analysis. In addition, we performed ex vivo ADCC assays using allogeneic NK cells isolated from healthy donors and AML patient material to test the cytotoxic potential of CD25 Mab (also referred to as RG6292 and RO7296682) or isotype control antibody on regulatory T cells and CD25+ AML cells.
Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology.
View Article and Find Full Text PDFAngelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal , a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein.
View Article and Find Full Text PDFThe natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs.
View Article and Find Full Text PDFProteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action.
View Article and Find Full Text PDFHepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a high mortality rate due to limited treatment options. Hence, the response of HCC to different cancer immunotherapies is being intensively investigated in clinical trials. Immune checkpoint blockers (ICB) show promising results, albeit for a minority of HCC patients.
View Article and Find Full Text PDFAntisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets.
View Article and Find Full Text PDFThe successful development of high-affinity gapmer antisense oligonucleotide (ASO) therapeutics containing locked nucleic acid (LNA) or constrained ethyl (cEt) substitutions has been hampered by the risk of hepatotoxicity. Here, we present an in vitro approach using transfected mouse fibroblasts to predict the potential hepatic liabilities of LNA-modified ASOs (LNA-ASOs), validated by assessing 236 different LNA-ASOs with known hepatotoxic potential. This in vitro assay accurately reflects in vivo findings and relates hepatotoxicity to RNase H1 activity, off-target RNA downregulation, and LNA-ASO-binding affinity.
View Article and Find Full Text PDFCompartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals.
View Article and Find Full Text PDFNon-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds.
View Article and Find Full Text PDFBackground: In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.
Results: Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research.
Purpose: Cell surface proteins are the primary means for a cell to sense and interact with its environment and their dysregulation has been linked to numerous diseases. In particular, the identification of proteins specific to a single tissue type or to a given disease phenotype may enable the characterization of novel therapeutic targets. We tested here the feasibility of a cell surface proteomics approach to identify pertinent markers directly in a clinically relevant tissue.
View Article and Find Full Text PDFRecent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates.
View Article and Find Full Text PDFCopper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20-fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow-up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift.
View Article and Find Full Text PDFThe propagation of phosphorylation downstream of receptor tyrosine kinases is a key dynamic cellular event involved in signal transduction, which is often deregulated in disease states such as cancer. Probing phosphorylation dynamics is therefore crucial for understanding receptor tyrosine kinases' function and finding ways to inhibit their effects. MS methods combined with metabolic labeling such as stable isotope labeling with amino acids in cell culture (SILAC) have already proven successful in deciphering temporal phosphotyrosine perturbations.
View Article and Find Full Text PDFBackground: Whole transcriptome analyses are an essential tool for understanding disease mechanisms. Approaches based on next-generation sequencing provide fast and affordable data but rely on the availability of annotated genomes. However, there are many areas in biomedical research that require non-standard animal models for which genome information is not available.
View Article and Find Full Text PDFThe stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12 and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction.
View Article and Find Full Text PDFWe have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3'-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand.
View Article and Find Full Text PDFThe long-tailed macaque, also referred to as cynomolgus monkey (Macaca fascicularis), is one of the most important nonhuman primate animal models in basic and applied biomedical research. To improve the predictive power of primate experiments for humans, we determined the genome sequence of a Macaca fascicularis female of Mauritian origin using a whole-genome shotgun sequencing approach. We applied a template switch strategy that uses either the rhesus or the human genome to assemble sequence reads.
View Article and Find Full Text PDFRenal tubulo-interstitial fibrosis is a non-specific process, representing the final common pathway for all kidney diseases, irrespective of their initial cause, histological injury, or etiology, leading to gradual expansion of the fibrotic mass which destroys the normal structure of the tissue and results in organ dysfunction and, ultimately, in end-stage organ failure. Proteomic studies of the fibrotic pathophysiological mechanisms have been performed in cell cultures, animal models and human tissues, addressing some of the key issues. This article will review proteomic contribution to the raising current knowledge on renal fibrosis biology and also mention seminal open questions to which proteomic techniques and proteomists could fruitfully contribute.
View Article and Find Full Text PDFRationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs).
Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments.
cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e.
View Article and Find Full Text PDFBackground: A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Ialpha, PKA-IIalpha, PKA-IIbeta) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging.
Results: The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%.
J Phys Chem B
February 2007
The homodimeric catabolite activator protein (CAP) is a bacterial DNA binding transcription regulator whose activity is controlled by the binding of the intracellular mediator cyclic adenosine monophosphate (cAMP). Each CAP subunit consists of a cyclic nucleotide and a DNA binding domain. Here, we investigate the structural features of the ligand-bound CAP in aqueous solution by molecular dynamics simulations based on the available X-ray structures (Passner et al.
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