Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific ...events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.
Full text
Available for:
IJS, NUK, SBMB, UL, UM, UPUK
Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been ...described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.
miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. ...Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes - endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In the last years miRNAs have increasingly been recognised as potent posttranscriptional regulators of gene expression. Possibly, miRNAs exert their action on virtually any biological process by ...simultaneous regulation of numerous genes. The importance of miRNA-based regulation in health and disease has inspired research to investigate diverse aspects of miRNA origin, biogenesis, and function. Despite the recent rapid accumulation of experimental data, and the emergence of functional models, the complexity of miRNA-based regulation is still far from being well understood. In particular, we lack comprehensive knowledge as to which cellular processes are regulated by which miRNAs, and, furthermore, how temporal and spatial interactions of miRNAs to their targets occur. Results from large-scale functional analyses have immense potential to address these questions. In this review, we discuss the latest progress in application of high-content and high-throughput functional analysis for the systematic elucidation of the biological roles of miRNAs.
Full text
Available for:
FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Determining composition, shape, and size of nanoparticles dispersed in a complex matrix is necessary in the assessment of toxicity, for regulatory actions, and environmental monitoring. Many types of ...nanoparticles that are currently used in consumer products contain more than one metal which are often not uniformly distributed ( e.g. , core–shell nanoparticles). This compositional and structural complexity makes their characterization difficult. In this study, we investigate the capability of single particle inductively coupled plasma mass spectrometry (spICP-MS) using time-of-flight (TOF) and quadrupole (Q) mass analyzers to determine the composition, size distribution, and concentration of a series of nanoparticles that are used in a variety of industrial applications: BiVO 4 , (Bi 0.5 Na 0.5 )TiO 3 and steel (which contains Fe, Cr, Ni, Mo) nanoparticles. In addition, we tested both types of mass analyzers with Au-core/Ag-shell nanoparticles, which are well-characterized and have already been used for assessment of multi-element capabilities of spICP-MS. The results confirm that both types of mass analyzers produce accurate estimations of the size of Au-core/Ag-shell particles. For other multi-element nanoparticles, spICP-MS provided the size of aggregates and/or agglomerates in the prepared suspensions. In general, particle size detection limits ( d LOD ) of spICP-TOFMS instruments with values of 29 nm for Ti, 14 nm for Mo, and 7 nm for Au, are smaller than those obtained for the quadrupole instruments. This study finds that only spICP-TOFMS can accurately assess the elemental composition of nano-steel particles. By contrast, spICP-QMS is limited to the detection of 2 elements in an individual particle and the elemental composition of nano-steel particles is less accurate. In general, spICP-TOFMS was able to quantify multiple elements with high precision and that currently makes it the first choice for multi-element detection of unknown nanoparticles.
The understanding of miRNA target interactions is still limited due to conflicting data and the fact that high-quality validation of targets is a time-consuming process. Faster methods like ...high-throughput screens and bioinformatics predictions are employed but suffer from several problems. One of these, namely the potential occurrence of downstream (i.e. secondary) effects in high-throughput screens has been only little discussed so far. However, such effects limit usage for both the identification of interactions and for the training of bioinformatics tools. In order to analyse this problem more closely, we performed time-dependent microarray screening experiments overexpressing human miR-517a-3p, and, together with published time-dependent datasets of human miR-17-5p, miR-135b and miR-124 overexpression, we analysed the dynamics of deregulated genes. We show that the number of deregulated targets increases over time, whereas seed sequence content and performance of several miRNA target prediction algorithms actually decrease over time. Bioinformatics recognition success of validated miR-17 targets was comparable to that of data gained only 12 h post-transfection. We therefore argue that the timing of microarray experiments is of critical importance for detecting direct targets with high confidence and for the usability of these data for the training of bioinformatics prediction tools.
Full text
Available for:
BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering ...(si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
We applied 14-mer 2'-OMe RNAs as inhibitors of selected micro RNAs. To improve their properties, we introduced a trimethoxystilbene residue at the 5'-terminus and three 2'-fluoro-2'-deoxynucleotides ...at the 3'-terminus to obtain potent inhibitors, whose mismatch discrimination is substantially better than that of typically applied >18-mers.
Determining composition, shape, and size of nanoparticles dispersed in a complex matrix is necessary in the assessment of toxicity, for regulatory actions, and environmental monitoring. Many types of ...nanoparticles that are currently used in consumer products contain more than one metal which are often not uniformly distributed (
e.g.
, core-shell nanoparticles). This compositional and structural complexity makes their characterization difficult. In this study, we investigate the capability of single particle inductively coupled plasma mass spectrometry (spICP-MS) using time-of-flight (TOF) and quadrupole (Q) mass analyzers to determine the composition, size distribution, and concentration of a series of nanoparticles that are used in a variety of industrial applications: BiVO
4
, (Bi
0.5
Na
0.5
)TiO
3
and steel (which contains Fe, Cr, Ni, Mo) nanoparticles. In addition, we tested both types of mass analyzers with Au-core/Ag-shell nanoparticles, which are well-characterized and have already been used for assessment of multi-element capabilities of spICP-MS. The results confirm that both types of mass analyzers produce accurate estimations of the size of Au-core/Ag-shell particles. For other multi-element nanoparticles, spICP-MS provided the size of aggregates and/or agglomerates in the prepared suspensions. In general, particle size detection limits (
d
LOD
) of spICP-TOFMS instruments with values of 29 nm for Ti, 14 nm for Mo, and 7 nm for Au, are smaller than those obtained for the quadrupole instruments. This study finds that only spICP-TOFMS can accurately assess the elemental composition of nano-steel particles. By contrast, spICP-QMS is limited to the detection of 2 elements in an individual particle and the elemental composition of nano-steel particles is less accurate. In general, spICP-TOFMS was able to quantify multiple elements with high precision and that currently makes it the first choice for multi-element detection of unknown nanoparticles.
In this study, we investigate the capability of spICP-MS using time-of-flight and quadrupole mass analyzers to determine composition size distribution and concentration.
PDF
