RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological ...interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.
ABSTRACT
We have obtained new solutions and methods for the process of thermal Comptonization. We modify the solution to the kinetic equation of Sunyaev and Titarchuk to allow its application up to ...mildly relativistic electron temperatures and optical depths $\gtrsim {1}$. The solution can be used for spectral fitting of X-ray spectra from astrophysical sources. We also have developed an accurate Monte Carlo method for calculating spectra and timing properties of thermal Comptonization sources. The accuracy of our kinetic equation solution is verified by comparison with the Monte Carlo results. We also compare our results with those of other publicly available methods. Furthermore, based on our Monte Carlo code, we present distributions of the photon emission times and the evolution of the average photon energy for both up and down scattering.
We review how the recent increase in X-ray and radio data from black hole and neutron star binaries can be merged together with theoretical advances to give a coherent picture of the physics of the ...accretion flow in strong gravity. Both long term X-ray light curves, X-ray spectra, the rapid X-ray variability and the radio jet behaviour are consistent with a model where a standard outer accretion disc is truncated at low luminosities, being replaced by a hot, inner flow which also acts as the launching site of the jet. Decreasing the disc truncation radius leads to softer spectra, as well as higher frequencies (including quasi periodic oscillations, QPOs) in the power spectra, and a faster jet. The collapse of the hot flow when the disc reaches the last stable orbit triggers the dramatic decrease in radio flux, as well as giving a qualitative (and often quantitative) explanation for the major hard-soft spectral transition seen in black holes. The neutron stars are also consistent with the same models, but with an additional component due to their surface, giving implicit evidence for the event horizon in black holes. We review claims of observational data which conflict with this picture, but show that these can also be consistent with the truncated disc model. We also review suggested alternative models for the accretion flow which do not involve a truncated disc. The most successful of these converge on a similar geometry, where there is a transition at some radius larger than the last stable orbit between a standard disc and an inner, jet dominated region, with the X-ray source associated with a mildly relativistic outflow, beamed away from the disc. However, the observed uniformity of properties between black holes at different inclinations suggests that even weak beaming of the X-ray emission may be constrained by the data. After collapse of the hot inner flow, the spectrum in black hole systems can be dominated by the disc emission. Its behaviour is consistent with the existence of a last stable orbit, and such data can be used to estimate the black hole spin. By contrast, these systems can also show very different spectra at these high luminosities, in which the disc spectrum (and probably structure) is strongly distorted by Comptonization. The structure of the accretion flow becomes increasingly uncertain as the luminosity approaches (and exceeds) the Eddington luminosity, though there is growing evidence that winds may play an important role. We stress that these high Eddington fraction flows are key to understanding many disparate and currently very active fields such as ULX, Narrow Line Seyfert 1's, and the growth of the first black holes in the Early Universe. PUBLICATION ABSTRACT
Abstract
We systematically analyse all publicly available XMM-Newton spectra of radio-quiet PG quasars. The soft X-ray excess in these objects is well modelled by an additional, cool, Compton ...scattering region. However, the remarkably constant temperature derived for this component over the whole sample requires a puzzling fine tuning of the parameters. Instead, we propose that the soft excess is an artifact of strong, relativistically smeared, partially ionized absorption. The strong jump in opacity at ∼0.7 keV from O vii, O viii and iron can lead to an apparent soft excess below this energy, which is trivially constant since it depends on atomic processes. This can have a dramatic effect on the derived spectrum, which has implications for fitting the relativistic smearing of the reflected iron line emission from the disc.
Active galactic nuclei and quasars are thought to be scaled-up versions of Galactic black hole binaries, powered by accretion onto supermassive black holes with masses of 106-109 , as opposed to the ...∼10 in binaries (here is the solar mass). One example of the similarities between these two types of systems is the characteristic rapid X-ray variability seen from the accretion flow. The power spectrum of this variability in black hole binaries consists of a broad noise with multiple quasi-periodic oscillations superimposed on it. Although the broad noise component has been observed in many active galactic nuclei, there have hitherto been no significant detections of quasi-periodic oscillations. Here we report the discovery of an ∼1-hour X-ray periodicity in a bright active galaxy, RE J1034+396. The signal is highly statistically significant (at the 5.6 level) and very coherent, with quality factor Q > 16. The X-ray modulation arises from the direct vicinity of the black hole.
We investigate the observed correlation between the 2–10 keV X-ray luminosity (in unit of the Eddington luminosity; l
X ≡ L
X/L
Edd) and the photon index (Γ) of the X-ray spectrum for both black hole ...X-ray binaries (BHBs) and active galactic nuclei (AGNs). We construct a large sample, with 10− 9 ≲ l
X ≲ 10− 1. We find that Γ is positively and negatively correlated with l
X when l
X ≳ 10− 3 and 10− 6.5 ≲ l
X ≲ 10− 3, respectively, while Γ is nearly a constant when l
X ≲ 10− 6.5. We explain the above correlation in the framework of a coupled hot accretion flow–jet model. The radio emission always comes from the jet while the X-ray emission comes from the accretion flow and jet when l
X is above and below 10−6.5, respectively. More specifically, we assume that with the increase of mass accretion rate, the hot accretion flow develops into a clumpy and further a disc–corona two-phase structure because of thermal instability. We argue that such kind of two-phase accretion flow can explain the observed positive correlation.
Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in ...vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for
differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene
we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome ...era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.
We build a simple model of the optical/ultraviolet (UV) emission from irradiation of the outer disc by the inner disc and coronal emission in black hole binaries. We apply this to the broad-band ...Swift data from the outburst of the black hole binary XTE J1817−330 to confirm previous results that the optical/UV emission in the soft state is consistent with a reprocessing a constant fraction of the bolometric X-ray luminosity. However, this is very surprising as the disc temperature drops by more than a factor of 3 in the soft state, which should produce a marked change in the reprocessing efficiency. The easiest way to match the observed constant reprocessed fraction is for the disc skin to be highly ionized (as suggested 30 yr ago by van Paradijs), so that the bulk of the disc flux is reflected and only the hardest X-rays heat the disc. The constant reprocessed fraction also favours direct illumination of the disc over a scattering origin as the optical depth/solid angle of any scattering material (wind/corona) over the disc should decrease as the source luminosity declines. By contrast, the reprocessed fraction increases very significantly (by a factor of ∼6) as the source enters the hard state. This dramatic change is not evident from X-ray/UV flux correlations as it is masked by bandpass effects. However, it does not necessarily signal a change in emission, for example, the emergence of the jet dominating the optical/UV flux as the reflection albedo must change with the dramatic change in spectral shape.
Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype ...that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.
Synopsis
The breast cancer drug palbociclib arrests cells in G1 phase by CDK4/6 inhibition, but also causes cellular senescence. Thermal proteome profiling shows that the latter is mediated by increased proteasome activation through reduced ECM29 binding.
Mass spectrometry‐based cellular thermal shift assay (MS‐CeTSA) analysis of CDK4/6 inhibitor palbociclib targets in MCF7 human breast cancer cells identifies protein complexes including the 20S proteasome.
Palbociclib treatment increases proteasome activity independently of the ubiquitin pathway.
Palbociclib activates the proteasome by disassociating the proteolysis‐inhibiting scaffold protein ECM29.
Proteasome activation is required for palbociclib‐induced cellular senescence, which is sensitive to the proteasome inhibitor bortezomib.
The proteasome is a novel downstream target for the anti‐cancer drug palbociclib inhibiting proliferation by reduced proteasomal association with its inhibitor EMC29.
PDF
