Lykke Pedersen

Modeling oscillatory control in NF-κB, p53 and Wnt signaling.

Oscillations are commonly observed in cellular behavior and span a wide range of timescales, from seconds in calcium signaling to 24 hours in circadian rhythms. In between lie oscillations with time periods of 1-5 hours seen in NF-κB, p53 and Wnt signaling, which play key roles in the immune system, cell growth/death and embryo development, respectively. In the first part of this article, we provide a brief overview of simple deterministic models of oscillations. In particular, we explain the mechanism of saturated degradation that has been used to model oscillations in the NF-κB, p53 and Wnt systems. The second part deals with the potential physiological role of oscillations. We use the simple models described earlier to explore whether oscillatory signals can encode more information than steady-state signals. We then discuss a few simple genetic circuits that could decode information stored in the average, amplitude or frequency of oscillations. The presence of frequency-detector circuit downstream of NF-κB or p53 would be a strong clue that oscillations are important for the physiological response of these signaling systems.

Independent component and pathway-based analysis of miRNA-regulated gene expression in a model of type 1 diabetes

BackgroundSeveral approaches have been developed for miRNA target prediction, including methods that incorporate expression profiling. However the methods are still in need of improvements due to a high false discovery rate. So far, none of the methods have used independent component analysis (ICA). Here, we developed a novel target prediction method based on ICA that incorporates both seed matching and expression profiling of miRNA and mRNA expressions. The method was applied on a cellular model of type 1 diabetes.ResultsMicrorray profiling identified eight miRNAs (miR-124/128/192/194/204/375/672/708) with differential expression. Applying ICA on the mRNA profiling data revealed five significant independent components (ICs) correlating to the experimental conditions. The five ICs also captured the miRNA expressions by explaining >97% of their variance. By using ICA, seven of the eight miRNAs showed significant enrichment of sequence predicted targets, compared to only four miRNAs when using simple negative correlation. The ICs were enriched for miRNA targets that function in diabetes-relevant pathways e.g. type 1 and type 2 diabetes and maturity onset diabetes of the young (MODY).ConclusionsIn this study, ICA was applied as an attempt to separate the various factors that influence the mRNA expression in order to identify miRNA targets. The results suggest that ICA is better at identifying miRNA targets than negative correlation. Additionally, combining ICA and pathway analysis constitutes a means for prioritizing between the predicted miRNA targets. Applying the method on a model of type 1 diabetes resulted in identification of eight miRNAs that appear to affect pathways of relevance to disease mechanisms in diabetes.

Plasma proteome analysis of patients with type 1 diabetes with diabetic nephropathy

BackgroundAs part of a clinical proteomics program focused on diabetes and its complications we are looking for new and better protein biomarkers for diabetic nephropathy. The search for new and better biomarkers for diabetic nephropathy has, with a few exceptions, previously focused on either hypothesis-driven studies or urinary based investigations. To date only two studies have investigated the proteome of blood in search for new biomarkers, and these studies were conducted in sera from patients with type 2 diabetes. This is the first reported in depth proteomic study where plasma from type 1 diabetic patients was investigated with the goal of finding improved candidate biomarkers to predict diabetic nephropathy. In order to reach lower concentration proteins in plasma a pre-fractionation step, either hexapeptide bead-based libraries or anion exchange chromatography, was performed prior to surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis.ResultsProteomic analysis of plasma from a cross-sectional cohort of 123 type 1 diabetic patients previously diagnosed as normoalbuminuric, microalbuminuric or macroalbuminuric, gave rise to 290 peaks clusters of which 16 were selected as the most promising biomarker candidates based on statistical performance, including independent component analysis. Four of the peaks that were discovered have been identified as transthyretin, apolipoprotein A1, apolipoprotein C1 and cystatin C. Several yet unidentified proteins discovered by this novel approach appear to have more potential as biomarkers for diabetic nephropathy.ConclusionThese results demonstrate the capacity of proteomic analysis of plasma, by confirming the presence of known biomarkers as well as revealing new biomarkers for diabetic nephropathy in plasma in type 1 diabetic patients.

A Wnt oscillator model for somitogenesis

We propose a model for the segmentation clock in vertebrate somitogenesis, based on the Wnt signaling pathway. The core of the model is a negative feedback loop centered around the Axin2 protein. Axin2 is activated by beta-catenin, which in turn is degraded by a complex of GSK3beta and Axin2. The model produces oscillatory states of the involved constituents with typical time periods of a few hours (ultradian oscillations). The oscillations are robust to changes in parameter values and are often spiky, where low concentration values of beta-catenin are interrupted by sharp peaks. Necessary for the oscillations is the saturated degradation of Axin2. Somite formation in chick and mouse embryos is controlled by a spatial Wnt gradient which we introduce in the model through a time-dependent decrease in Wnt3a ligand level. We find that the oscillations disappear as the ligand concentration decreases, in agreement with observations on embryos.

A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Expression and Localization of microRNAs in Perinatal Rat Pancreas: Role of miR-21 in Regulation of Cholesterol Metabolism

Objective To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas. Research Design and Methods RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells. Results Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21. Conclusions The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.

Finding diabetic nephropathy biomarkers in the plasma peptidome by high-throughput magnetic bead processing and MALDI-TOF-MS analysis

Purpose and experimental design: Diabetic nephropathy (DN) is the most common cause of end‐stage renal disease and improved biomarkers would help identify high‐risk individuals. The aim of this study was to discover candidate biomarkers for DN in the plasma peptidome in an in‐house cross‐sectional cohort (n=122) of type 1 diabetic patients diagnosed with normo‐, micro‐, and macroalbuminuria.

miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer

Ovarian cancer presents as an aggressive, advanced stage cancer with widespread metastases that depend primarily on multicellular spheroids in the peritoneal fluid. To identify new druggable pathways related to metastatic progression and spheroid formation, we integrated microRNA and mRNA sequencing data from 293 tumors from The Cancer Genome Atlas (TCGA) ovarian cancer cohort. We identified miR-509-3p as a clinically significant microRNA that is more abundant in patients with favorable survival in both the TCGA cohort (P = 2.3E–3), and, by in situ hybridization (ISH), in an independent cohort of 157 tumors (P < 1.0E–3). We found that miR-509-3p attenuated migration and disrupted multi-cellular spheroids in HEYA8, OVCAR8, SKOV3, OVCAR3, OVCAR4 and OVCAR5 cell lines. Consistent with disrupted spheroid formation, in TCGA data miR-509-3ps most strongly anti-correlated predicted targets were enriched in components of the extracellular matrix (ECM). We validated the Hippo pathway effector YAP1 as a direct miR-509-3p target. We showed that siRNA to YAP1 replicated 90% of miR-509-3p-mediated migration attenuation in OVCAR8, which contained high levels of YAP1 protein, but not in the other cell lines, in which levels of this protein were moderate to low. Our data suggest that the miR-509-3p/YAP1 axis may be a new druggable target in cancers with high YAP1, and we propose that therapeutically targeting the miR-509-3p/YAP1/ECM axis may disrupt early steps in multi-cellular spheroid formation, and so inhibit metastasis in epithelial ovarian cancer and potentially in other cancers.

Actogram analysis of free-flying migratory birds : new perspectives based on acceleration logging

The use of accelerometers has become an important part of biologging techniques for large-sized birds with accelerometer data providing information about flight mode, wing-beat pattern, behaviour and energy expenditure. Such data show that birds using much energy-saving soaring/gliding flight like frigatebirds and swifts can stay airborne without landing for several months. Successful accelerometer studies have recently been conducted also for free-flying small songbirds during their entire annual cycle. Here we review the principles and possibilities for accelerometer studies in bird migration. We use the first annual actograms (for red-backed shrike Lanius collurio) to explore new analyses and insights that become possible with accelerometer data. Actogram data allow precise estimates of numbers of flights, flight durations as well as departure/landing times during the annual cycle. Annual and diurnal rhythms of migratory flights, as well as prolonged nocturnal flights across desert barriers are illustrated. The shifting balance between flight, rest and different intensities of activity throughout the year as revealed by actogram data can be used to analyse exertion levels during different phases of the life cycle. Accelerometer recording of the annual activity patterns of individual birds will open up a new dimension in bird migration research.

Dickkopf1 - A New Player in Modelling the Wnt Pathway

The Wnt signaling pathway transducing the stabilization of β-catenin is essential for metazoan embryo development and is misregulated in many diseases such as cancers. In recent years models have been proposed for the Wnt signaling pathway during the segmentation process in developing embryos. Many of these include negative feedback loops where Axin2 plays a key role. However, Axin2 null mice show no segmentation phenotype. We therefore propose a new model where the negative feedback involves Dkk1 rather than Axin2. We show that this model can exhibit the same type of oscillations as the previous models with Axin2 and as observed in experiments. We show that a spatial Wnt gradient can consistently convert this temporal periodicity into the spatial periodicity of somites, provided the oscillations in new cells arising in the presomitic mesoderm are synchronized with the oscillations of older cells. We further investigate the hypothesis that a change in the Wnt level in the tail bud during the later stages of somitogenesis can lengthen the time period of the oscillations and hence the size and separation of the later somites.