Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines.

A challenge facing the human pluripotent stem cell (hPSC) field is the variability observed in differentiation potential of hPSCs. Variability can lead to time consuming and costly optimisation to yield the cell type of interest. This is especially relevant for the differentiation of hPSCs towards the endodermal lineages. Endodermal cells have the potential to yield promising new knowledge and therapies for diseases affecting multiple organ systems, including lung, thymus, intestine, pancreas and liver, as well as applications in regenerative medicine and toxicology. Providing a means to rapidly, cheaply and efficiently assess the differentiation potential of multiple hPSCs is of great interest. To this end, we have developed a rapid small molecule based screen to assess the endodermal potential (EP) of hPSCs, based solely on definitive endoderm (DE) morphology. This drastically reduces the cost and time to identify lines suitable for use in deriving endodermal lineages. We demonstrate the efficacy of this screen using 10 different hPSCs, including 4 human embryonic stem cell lines (hESCs) and 6 human induced pluripotent stem cell lines (hiPSCs). The screen clearly revealed lines amenable to endodermal differentiation, and only lines that passed our morphological assessment were capable of further differentiation to hepatocyte like cells (HLCs)

Characterising anomalous transport in accretion disks from X-ray observations

Whilst direct observations of internal transport in accretion disks are not yet possible, measurement of the energy emitted from accreting astrophysical systems can provide useful information on the physical mechanisms at work. Here we examine the unbroken multi-year time variation of the total X-ray flux from three sources: Cygnus X-1 , the microquasar GRS 1915+105 , and for comparison the nonaccreting Crab nebula. To complement previous analyses, we demonstrate that the application of advanced statistical methods to these observational time-series reveals important contrasts in the nature and scaling properties of the transport processes operating within these sources. We find the Crab signal resembles Gaussian noise; the Cygnus X-1 signal is a leptokurtic random walk whose self-similar properties persist on timescales up to three years; and the GRS 1915+105 signal is similar to that from Cygnus X-1, but with self-similarity extending possibly to only a few days. This evidence of self-similarity provides a robust quantitative characterisation of anomalous transport occuring within the systems

Statistical characterisation of full-disk EUV/XUV solar irradiance and correlation with solar activity

We investigate the distribution of fluctuations in solar irradiance when integrated over the full disk, obtained using extreme ultraviolet/soft X-ray observations from the SOHO CELIAS/SEM instrument. This time series sums over both the contributions of single distinguishable flares, and of many other processes. By detrending we select events with timescales of less than a few hours such as waves, slow flows, and CMEs. The statistics generated by this range of phenomena can be characterised by power-law-tailed distributions. We show that (i) during the high-activity period 2000 Jan-June the tail exponent a(T)=1.5 +/- 0.1; (ii) during the low-activity period 1996 Jan-June a(T)=3.0 +/- 0.2; and (iii) in general a(T) decreases with increasing activity.Comment: 4 pages, 5 figures; v.2 R-squared goodness of fits adde

Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

3D tissue culture provides a physiologically relevant and genetically tractable system for studying normal and malignant human tissues. Despite this, gene-silencing studies using siRNA has proved difficult. In this study, we have identified a cause for why traditional siRNA transfection techniques are ineffective in eliciting gene silencing in situ within 3D cultures and proposed a simple method for significantly enhancing siRNA entry into spheroids/organoids. In 2D cell culture, the efficiency of gene silencing is significantly reduced when siRNA complexes are prepared in the presence of serum. Surprisingly, in both 3D tumour spheroids and primary murine organoids, the presence of serum during siRNA preparation rapidly promotes entry and internalization of Cy3-labelled siRNA in under 2 hours. Conversely, siRNA prepared in traditional low-serum transfection media fails to gain matrigel or spheroid/organoid entry. Direct measurement of CTNNB1 mRNA (encoding β-catenin) from transfected tumour spheroids confirmed a transient but significant knockdown of β-catenin when siRNA:liposome complexes were formed with serum, but not when prepared in the presence of reduced-serum media (Opti-MEM). Our studies suggest a simple modification to standard lipid-based transfection protocols facilitates rapid siRNA entry and transient gene repression, providing a platform for researchers to improve siRNA efficiency in established 3D cultures

Astronomy & Astrophysics Characterising anomalous transport in accretion disks from X-ray observations

Abstract. Whilst direct observations of internal transport in accretion disks are not yet possible, measurement of the energy emitted from accreting astrophysical systems can provide useful information on the physical mechanisms at work. Here we examine the unbroken multi-year time variation of the total X-ray flux from three sources: Cygnus X-1, the microquasar GRS 1915+105, and for comparison the nonaccreting Crab nebula. To complement previous analyses, we demonstrate that the application of advanced statistical methods to these observational time-series reveals important contrasts in the nature and scaling properties of the transport processes operating within these sources. We find the Crab signal resembles Gaussian noise; the Cygnus X-1 signal is a leptokurtic random walk whose self-similar properties persist on timescales up to three years; and the GRS 1915+105 signal is similar to that from Cygnus X-1, but with self-similarity extending possibly to only a few days. This evidence of self-similarity provides a robust quantitative characterisation of anomalous transport occuring within the systems

Dynamics of tournaments: the soccer case

A random walk-like model is considered to discuss statistical aspects of tournaments. The model is applied to soccer leagues with emphasis on the scores. This competitive system was computationally simulated and the results are compared with empirical data from the English, the German and the Spanish leagues and showed a good agreement with them. The present approach enabled us to characterize a diffusion where the scores are not normally distributed, having a short and asymmetric tail extending towards more positive values. We argue that this non-Gaussian behavior is related with the difference between the teams and with the asymmetry of the scores system. In addition, we compared two tournament systems: the all-play-all and the elimination tournaments.Comment: To appear in EPJ

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5

Correction: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma.

Present: The originally supplied Figure 5 contains duplicate total-ERK panels. Correct: The proper Figure 5 appears below. The authors sincerely apologize for this error

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene