text:
- Research article
- Open Access
Hematopoietic differentiation: a coordinated dynamical process towards attractor stable states
BMC Systems Biology volume 4, Article number: 85 (2010)
The differentiation process, proceeding from stem cells towards the different committed cell types, can be considered as a trajectory towards an attractor of a dynamical process. This view, taking into consideration the transcriptome and miRNome dynamics considered as a whole, instead of looking at few 'master genes' driving the system, offers a novel perspective on this phenomenon. We investigated the 'differentiation trajectories' of the hematopoietic system considering a genome-wide scenario.
We developed serum-free liquid suspension unilineage cultures of cord blood (CB) CD34+ hematopoietic progenitor cells through erythroid (E), megakaryocytic (MK), granulocytic (G) and monocytic (Mo) pathways. These cultures recapitulate physiological hematopoiesis, allowing the analysis of almost pure unilineage precursors starting from initial differentiation of HPCs until terminal maturation. By analyzing the expression profile of protein coding genes and microRNAs in unilineage CB E, MK, G and Mo cultures, at sequential stages of differentiation and maturation, we observed a coordinated, fully interconnected and scalable character of cell population behaviour in both transcriptome and miRNome spaces reminiscent of an attractor-like dynamics. MiRNome and transcriptome space differed for a still not terminally committed behaviour of microRNAs.
Consistent with their roles, the transcriptome system can be considered as the state space of a cell population, while the continuously evolving miRNA space corresponds to the tuning system necessary to reach the attractor. The behaviour of miRNA machinery could be of great relevance not only for the promise of reversing the differentiated state but even for tumor biology.
The hematopoietic system is a privileged observatory for the study of the highly coordinated process leading to differentiated cells. Hematopoietic proliferation and differentiation is sustained by a pool of multipotent self-renewing hematopoietic stem cells (HSCs), which give rise to a hierarchy of progenitor populations (HPCs) with more restricted lineage potential, ultimately leading to the production of all types of mature blood cells. HSCs/primitive HPCs give rise to a hierarchy of committed HPCs, functionally defined as burst-forming units (BFUs) or colony-forming units (CFUs). The earliest HPCs are multipotent and generate mixed colonies (CFU-GEMM: CFU-granulocytic, erythroid, macrophage, megakaryocyte). Multipotent HPCs differentiate and become gradually committed to specific lineages, i.e., HPCs of the erythroid series (early and late BFU-E and CFU-E) the megakaryocytic lineage (BFU-MK and CFU-MK) and the granulo-monocytic lineage (CFU-GM, CFU-G, CFU-M) . HSCs/HPCs differentiation is controlled by the combined effects of hematopoietic growth factors (HGFs), chromatin modifiers, transcription factors and microRNAs (miRNAs). MiRNAs are a new class of small non coding RNAs (~22 nucleotides), playing a key role in post-transcriptional regulation of gene expression [2, 3]. This occurs through degradation or translational repression of target mRNAs, by binding to their 3'-untranslated regions [4, 5]. The human genome encodes ~700 miRNAs http://microrna.sanger.ac.uk, located in introns, exons or intergenic regions [6, 7]. Several studies have demonstrated that miRNAs have unique developmental-specific expression or signature, since each tissue/cell type produces a specific set of miRNAs [8, 9].
Beside the enumeration of the different actors playing in the differentiation act, we have not to forget that the differentiation process can be considered as a trajectory towards an attractor (i.e. a specific cell type), interesting the cell as a whole with a coordinated change in metabolism, gene expression, shape, general responsiveness to environmental stimuli. In this view, the attention is no more focused on a single 'average' cell behavior with a sequential differential gene activation/repression program, but on a mutually interacting ensemble of cells in which the entire transcriptome pattern moves from the initial unstable attractor state (progenitor cells) towards another stable, end attractor, state (mature cells) . By analyzing both protein coding genes (PCGs) and miRNAs expression during hematopoiesis, we gave a proof-of-concept of this attractor-like dynamics and were able to characterize the coordinated and scalable character of cell population behaviour in both mRNAs and miRNAs spaces. These results allow to envisage a sort of biological statistical mechanics along the lines described by Bar-Yam and colleagues , in which the traditional "master genes" driven approach may be substituted by a global motion of the system as a whole.
We will also discuss the complementary role of transcriptome and miRNome and describe the miRNome as a sort of continuously adjusting control device of the cell population .
To investigate the cellular and molecular mechanisms underlying haematopoiesis, serum-free culture systems were developed for unilineage differentiation and maturation of cord blood CD34+HPCs through the erythroid (E), megakaryocytic (MK), granulocytic (G) or monocytic (Mo) pathways. The CD34+HPCs population is an heterogeneous population containing: i) very few HSCs (<0.1%), ii) minority of pluripotent HPCs (1-3%) and iii) majority (>90%) of committed progenitors (i.e. BFU-E, BFU-MK, CFU-GM). In these cultures, HPCs are stimulated by specific unilineage growth factors (HGFs) at a saturating level combined with appropriate dosages of multilineage or early acting HGFs (see material and methods). It has to be considered and is currently accepted that HGFs are permissive and not instructive molecule, thus addition of HGFs mainly induce proliferation, differentiation and maturation of unilineage committed progenitors. Therefore this type of culture allows to analyze 'pure' unilineage precursors at discrete sequential stages of differentiation/maturation process starting from committed progenitors, but do not allow to investigate early stages of hematopoiesis, such as cell fate decision and/or commitment of pluripotent HPCs.
Specifically, unilineage cultures of cord blood CD34+HPCs are derived by culturing purified HPCs, separated by positive selection immunomagnetic system for CD34+, in serum-free liquid suspension and induced to a) selective E growth by very low dosages of IL-3, GM-CSF, and a saturating erythropoietin (Epo) level, b) unilineage MK growth by saturating dosage of thrombopoietin (Tpo), c) unilineage G growth by low dose of IL-3 and GM-CSF combined with plateau level of G-CSF, and d) unilineage Mo by low dose of IL-6 and saturating dosage of Flt-3 and M-CSF.
The purity and the differentiation process of each type of these cultures was assessed by flow-cytometry: a gradual decrease of CD34+ marker during the first days of culture, followed by a progressively increasing expression of specific membrane markers (glycophorin A, CD41, CD15 and CD14, respectively) (Figure 1) was observed. Consistently, cell morphological analysis showed a gradual wave of maturation along the diverse differentiative pathways to terminal mature cells (Figure 1).
These culture systems, moving from committed progenitors to mature cells state, allow to analyze the existence, if any, of developmental trajectories during hematopoietic differentiation. To this end, we decided to selectively analyze the expression profiles of PCGs and miRNAs in unilineage culture of cord blood (CB) CD34+ cells (HPCs) through the erythroid, megakaryocytic, granulocytic and monocytic differentiation/maturation. Total RNA was extracted from CD34+ HPCs and at different days of culture, corresponding to discrete sequential stages of differentiation/maturation process, from each type of culture.
Microarray analysis was performed using the GeneChip Human Genome U133 Plus 2.0 oligonucleotide microarrays (Affymetrix) for gene expression profiling, and a custom in-house manufactured miRNA microarray platform (A-MEXP-620 version 3.0) to profile the expression of miRNAs (see additional files 1 and 2: Tables S1 and S2 and materials and methods for accession number).
The reliability of gene microarray results was inspected by comparing the variations in signal of well-known lineage-specific genes and the corresponding antigens/proteins as reported in current literature [13, 14]. In particular, the expression trend was evaluated for the above specific markers, plus other specific proteins such as erythropoietin receptor (EPOR), β-hemoglobin (HBB) and GATA1 for E unilineage , Spi1, MAFB and CEBPE for G and Mo cultures , GATA1 and FLI1 for MK lineage . Variation in the RNA expression of these markers as reported by microarray was always concordant with the expected protein modulation during differentiation (Figure 2A).
To validate miRNAs expression profile, six randomly chosen differentially expressed miRNAs were analyzed by real-time reverse-transcription polymerase chain reaction (RT-PCR) (Figure 2B). Moreover, several miRNAs showed comparable expression with previously reported trends (i.e. miR-221/222 , miR-451, miR-155 during erythropoiesis , miR-223 during granulopoiesis , and miR-17-5p, miR-20a, miR-106 during monocytopoiesis ). Regarding miRNAs expression profile during megakaryopoiesis, we noticed an incomplete overlap between our and previously published results [22, 23] possibly due to the different CD34+HPCs source (i.e. cord blood versus bone marrow).
In order to understand how PCGs and miRNAs expression patterns correlate with hematopoietic cell differentiation, both genome and miRNAs expression profiles were analyzed as global patterns by using the Gene Expression Dynamics Inspector (GEDI) software . GEDI displays a global picture of the state vector relative to both transcriptome and miRNome monitoring the cell population while it moves towards its differentiation fate.
Genome-wide expression profile of PCGs was representative of a redundant set of 54630 human sequences, with expression signal linear values ranging from 0.293 to 71337.
For GEDI analysis we used a subset of 17655 sequences (Expressed Sequences) having signal values >500 (an under-expression-threshold roughly corresponding to the 1% of maximum expression) in at least a sample (Figure 3A). Resulting representations revealed that after the first step of unilineage differentiation (from CD34+ to day 5-6 of culture) a distinctive lineage-specific gene expression profile was already recognizable. After this time minor changes occurred along the maturation processes to further define the differentiated expression profile (Figure 3B). Earliness of definition of recognizable lineage-specific patterns supports the idea that the mature differentiated cells can be considered as multidimensional attractors in the global expression space .
In dynamical terms, the stem/progenitor cells can be considered as an unstable state (unstable attractor) from where the system exits following perturbations (i.e. growth factors, cell-cell interaction), and migrates along the different trajectories in the multidimensional transcript profile space (state space) until reaching the stable ones (stable attractors), corresponding to the mature phenotypes. The transcript profile space is defined by the gene expression pattern; changes of the transcript profile results in changing the position of the cell population in the multidimesional space.
We then calculated the global similarity between samples on the entire expressed genome (17655 Expressed Sequences) in terms of Pearson correlation (r). As expected, given we are dealing with samples of the same general cell-type, genome-wide correlations are extremely high (Pearson r around 0.90), indicating a strong common order parameter influencing the expression level of the entire genome (Figure 4). Interestingly, we observed a monotonically decreasing correlation coefficient between CD34+HPCs and different lineages at increasing time, consistent with the increasing distance from the initial unstable stem/progenitor cell attractor. To further analyze the attractor like properties of the differentiation dynamics we calculated the first derivative of the trajectory change in terms of corr(t1) - corr(t0)/(t1 - t0), being t1 and t0 two different times of observations and corr (t) the relative correlation with initial state. As evident in Figure 3, the decrease in correlation from unit value is concentrated on the first time step, being the subsequent decrease in time much less pronounced consistently with a progressive decreasing of the derivative (data not shown). This flattening of the differentiation rate appears to be particularly pronounced in the case of erythroid differentiation, in which the major part of total displacement from initial state is accomplished in the first time step.
In order to demonstrate the scalability of gene expression dynamics (i.e. the possibility to get the same general picture of differentiation dynamics adopting different size views of the genome) different gene sets were selected. The 17655 Expressed Sequences were divided in 14636 Medium Expressed (maximum signal in the range 500-5000), 3019 High Expressed (signal >5000 in at least one sample) and 7642 Changed (signal variation >3× in respect to CD34+ in at least a sample) sequences subsets (Figure 3A).
The Expressed Sequences set gave exactly the same results in terms of monotonic linear decrease of correlation with CD34+HPCs pattern of all the other choices (Medium Expressed, High Expressed and Changed Sequences). The same progressive displacement from unstable CD34+HPCs attractor is highlighted with the Changed Sequences and with a random 250 genes extraction (Figure 4).
The same invariant character of displacement from progenitor cell line was observed in further subdivided sets (i.e. signal values >500 with 6-fold change between CD34+HPCs and mature cells) (data not shown).
Another proof-of-concept of the scalable character of differentiation process came from GEDI analysis performed on multiple subsets of 1000 or 100 sequences randomly picked up from the Expressed Sequence set (Figure 3B). The smallest subsets (100 genes) too showed stable, lineage-specific expression patterns, even if with a less monotonically decreasing correlation trend from CD34 with respect to bigger sets.
These results demonstrate the scalable character of the progressive displacement of the different lineages from their common stem/progenitor cells. Therefore, we can consider these analyzed cell populations as strongly connected dynamical systems in which the expression of different genes is highly correlated [26, 11].
Having stated that all the diverse differentiation trajectories monotonously move from their progenitors, we asked whether the mutual relations between different cell populations during the trajectory towards their fates are conserved. This corresponds to check for the maintaining of a stable differentiation rate across cell lines, so that their mutual distances (in terms of 1-r metrics with r the Pearson correlation coefficient), even if increasing during differentiation process, maintain their relative proportions. In order to answer this question, we used a multidimensional scaling (MDS) technique (Kruskal-Wish scaling), that allows for the construction of a bi-dimensional plot where the relative distances of the objects mirror the effective distances of the same objects computed over the multidimensional space, constituted by their expression levels over the entire genome (Figure 5A) . We computed such distances (Figure 5B) in three distinct temporal windows denominated step 1, step 2 and step 3 corresponding to 5-6, 9-11 and 14-15 differentiative days, respectively (Figure 5A). As shown, the relative position of the lineages remains strongly invariant during the entire time span analyzed so pointing to: i) the existence of distinct attractors towards which the gene expression dynamics of the cells are directed starting from day 5; ii) the progressive enlargement of the phase space spanned by the different cell kinds with the progression of the differentiation process, as marked by the increasing average radius correspondent to the increase of the between lineages distances as for expression patterns along an already decided and fixed direction. Thus, the distance between cell kinds increases as a consequence of the progression of differentiation process, but the relative positions of the different cell kinds are maintained. This indicates that the system continues along the same kind of trajectory, maintaining the overall shape that can be considered as the phase space of the hematopoietic differentiation process. The topological invariance of MDS space, even if not a conclusive proof, strongly points to the existence of a specific attractor for each cell kind in the transcriptome space. The distance space has a very important characteristic: the ability to detect the change of 'shape' of the studied object without the need of making any unjustified assumption. A linear trajectory maintaining its end-point (attractor) implies the increasing of size of the object (the data set made by the 4 lineages) without any modification in shape and thus maintaining the relation between different cell kinds positions linearly invariant. The distances between cell kinds monotonically increase in time but maintain invariant their relative proportions and thus the relative positions. As a consequence the distances between the trajectories are correlated in time (see figure 5, panels B and C).
Having stated the genome-wide expression is a suitable space for describing the differentiation trajectories of blood cell lineages, we turned our attention to the miRNA space.
The GEDI analysis of 627 miRNAs exhibits clear distinct expression patterns associated with different hematopoietic lineages, revealing distinctive signatures for each hematopoietic differentiative pathway (Figure 6A). Moreover defined, correlated and lineage-specific expression profiles were also obtained using subsets including 195 and 92 miRNA having in at least a sample signal >1 or >4% respectively of maximum signal.
In analogy with the transcriptome space, we observed a progressive linear displacement of miRNA patterns from the HPCs (Figure 6B), as shown by the trend of Pearson correlation coefficient of the four different lineages with CD34+HPCs population. These results were confirmed by analyzing a replicate microarray (additional file 3, Figure S1).
The scalability feature, observed in the transcriptome, is present in the miRNAs space as well: the analysis of a randomly extracted sample of 100 miRNA sequences (Figure 6B right) gave us exactly the same results of the full-rank space (627 miRNAs, Figure 6B left). The analysis performed with less than 50 randomly picked-up elements do not anymore constitute a reliable sampling giving rise to widely varying results from sample to sample. The scalability of microRNA regulation tells us that even this system 'acts as a whole' being a connected system with several mutual correlations between its elements.
The MDS reveals a profound difference with the transcriptome based picture (Figure 6C), showing that the topology of the miRNA space is much less invariant than the correspondent transcriptome space. The lack of invariance is mirrored by the absence of any linear relation between the mutual distances among lineages at different steps (r = -0.013 between step 1 and step 3 distances, Figure 6D) in the miRNA space. The difference between micro RNA and transcriptome spaces in terms of topological invariance of the mutual relations between cell kinds profiles is not due to the smaller number of variables considered for miRNome. As a matter of fact, the MDS based portraits computed over six different 627 genes random extractions gave completely coincident results with the ones obtained on the whole set (additional file 4, Figure S2). The mutual distances between the cell lineages computed at different steps along the development trajectory (steps 1, 2 and 3 as depicted in the additional file 4, Figure S2) where remarkably invariant between the whole set and random extractions with an average correlation between distance matrices r = 0.93. This gave rise to completely coincident MDS portraits. This result indicates that, while, in terms of transcriptome state, a cell population can be considered already committed to its fate at five days of differentiation and consequently following a differentiation trajectory with a fixed orientation as described by MDS, the miRNA pattern shows a still not finally committed shape until the end of the studied period.
We can speculate on this lack of 'orientation invariance' of miRNA space, stressing the fact that while genome wide expression profile marks the 'average state' of the cell population at a given instant of time, the miRNA pattern can be equated to the 'fine controls' acting on the system to drive the trajectory towards its differentiation fate and actively maintaining the population on the attractor . In other words while the transcriptome is the 'location in space' of the system, the miRNA machinery, for its regulative role, can be considered as its 'instantaneous velocity vector'.
Discussion and Conclusions
The hematopoietic differentiation process permits to follow the dynamic of a system when moving from a common unstable progenitor state to different stable attractors correspondent to the distinct cell types, each characterized by a clearly distinguished profile in the multidimensional transcript profile space.
Our study showed that both transcriptome and miRNome gene expression profiles map cells into a multidimensional space, where the positions of differentiating cells progress along trajectories starting from undifferentiated CD34+ cells towards specific differentiation fates, in line to the existence of the attractor-like dynamics during hematopoiesis.
The transcriptome and miRNome spaces derive from the expression patterns of protein coding genes and miRNAs respectively, and coordinated changes in their expression patterns can be interpreted as trajectories of cell populations in a multidimensional space. The analysis of gene and miRNAs expression profiles gave a proof-of-concept of the scalable character of biological regulation circuits. While the increasing distance (i.e. the progressive decay of Pearson r) representing the increasing differences between the various differentiating cell types and the progenitor cells appears as an almost trivial result, it is important to highlight that the same behaviour is observed with widely different choices of gene subsets, including those genes that are routinely considered as 'noisy and unreliable information'. This implies the strong connectivity of the gene regulation network that can be considered as a unitary system so casting doubts about classical concepts like the existence of 'housekeeping genes' whose expression is largely invariant and decoupled from specific cell activity. As a matter of fact scalability (i.e. the possibility to observe the same general behaviour at different scales, here corresponding to different choices of genes) is a very important consequence of the statistical-mechanics like behaviour of cell populations that until now was only marginally taken into account [26, 28]. Under this paradigm the principal actor of the biologically relevant process is no more the single cell, but a cell ensemble which different sources of noise shape as a distribution of elements (cells) slightly differing between them in analogy with Maxwell-Boltzmann distribution of kinetic energy over an ensemble of particles. The energy landscapes typical of physical systems are mirrored, in the case of cell populations, by the so called epigenetic landscapes .
The proportionality of the mutual distances between different cell populations during the trajectory toward their fates was observed for embryonic stem cells by Aiba and colleagues , by using the epigenetic landscape model proposed by Conrad Waddington . During development, the epigenetic landscape is intended as a physical landscape made of hills and valleys in which a development trajectory is like a marble rolling according to the landscape shapes it encounters. In this model, the unstable stem cell population occupies the top of an hill, from which the system can escape following the different faces of the hill. In this case, two trajectories going along two different directions and remaining with the same bearing (driven by the same topology of the hill) while being progressively set apart between them, will maintain the same relative mutual orientation during all the process. We observed a similar dynamics by analysing unilineage HPCs differentiation using a multidimensional scaling technique: the distances between different cell lines, while increasing in time, maintain their mutual correlation/orientation in the transcriptome space. This correlation is no more present in the miRNA space. The dynamics of differentiation/maturation in the gene expression space has many features consistent with an attractor-like behaviour. Our experimental conditions do not allow for the ultimate proof of the existence of a proper attractor, i.e. the robustness of the differentiated cells to external perturbations, but the obtained results globally support the idea of attractor.
Both transcriptome and miRNome dynamics showed a scalable character and progressive displacement from stem/progenitor cells of the four different lineages during the differentiation process, but while the transcriptome trajectories conserved the relative relations between cell kinds distances this was not the case in the miRNA space.
This behaviour is consistent with the view of the transcriptome as a state space of a cell and miRNA space as a sort of "continuously adjusting" tuning system, affecting cell lineage evolution to reach the attractor.
The reconciliation of transcriptome and miRNome findings is not an easy task. We tried and look for a simple transcriptome-miRNome relation by computing the enrichment in hematopoietic related genes in the targets of miRNAs that vary significantly during the differentiation process. The enrichment level did not differ from a purely random choice of miRNAs (data not shown), thus not allowing for a single miRNome-transcriptome relation to be drawn. This lack of a simple correspondence between regulated genes and miRNA profile is consistent with the holistic character of attractor-like dynamics described. Moreover, these results are in line with the results of Liu and Kohane  and with the observation of Seitz H.  which propose that many computationally identified miRNA targets may actually be competitive inhibitors of miRNA function, preventing miRNAs from binding their authentic targets by sequestering them. Thus we must limit ourselves to the registration of a different dynamics of global miRNome with respect to transcriptome without going further into unjustified speculations.
Furthermore, our data do not allow to analyze whether or not cell population arrives at a more or less stable miRNA profile, anyway we believe that a relevant and continuously on-going tuning of the system by the miRNAs is needed to actively maintain the differentiated state. Moreover, considering that miRNAs represent a fundamental epigenetic regulatory mechanism, their behaviours suggest the possibility of their involvement in reprogramming or reversing differentiated cells. Reprogramming is the activation of a pre-existing gene expression program caused by stimulating a transition into the attractor that encodes such a program. MiRNAs modulation may be a reasonable way connecting the attractors according to Huang model .
The discovery of these dynamics in miRNA space should be of great relevance not only for the promise of reversing the differentiated state, but even for tumor biology. As a matter of fact while the differentiation process is the approaching to a stable attractor from an unstable one, the process of tumor formation can be considered as the escaping from the stable attractor correspondent to the normal tissue shifting to a less differentiated, embryonic-like mode correspondent to the tumor state. This consideration opens another important research avenue in cell system dynamics.
Our findings may have implications in the interpretative analysis of gene expression signature and possibly in the choice of future therapeutic options for cancer diseases. The consideration of gene expression as a whole was already applied for diagnosis purpose , but never on miRNA expression. Another important implication of this way to consider hematopoietic process is that the locations of cells in the multidimensional transcriptome and miRNome space can be used to identify cells of unknown origin, to determine the differentiation status of cells and to assess their multi-differentiative potency.
Cord blood human progenitor cells purification and cultures
Human cord blood was obtained after informed written consent and processed under approval of the Istituto Superiore di Sanità ethics committee.
Isolation of CD34+ cells from cord blood (CB), unilineage culture and morphological analysis were performed as described . Briefly, CD34+ cells were purified from CB by positive selection using the midi-MACS immunomagnetic separation system according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladabach, Germany). The purity of CD34+ cells was assessed by flow cytometry using a monoclonal PE-conjugated anti-CD34 antibody and was routinely over 95% (range comprised between 92 and 98%). CD34+ progenitors were cultured in serum-free medium in the presence of various recombinant human cytokine combinations. Serum-free medium was prepared as it follows: freshly prepared Iscove's modified Dulbecco's medium was supplemented with bovine serum albumin (10 mg/ml), pure human transferrin (700 mg/ml), human low-density lipoprotein (40 mg/ml), insulin (10 mg/ml), sodium pyruvate (10-4 mol/l), L-glutamine (2 × 10-3 mol/l), rare inorganic elements supplemented with iron sulfate (4 × 10-8 mol/l) and nucleosides (10 μg/ml each).
For erythroid unilineage culture, serum-free medium was supplemented with 0.01 U/ml IL-3, 0.001 ng/ml GM-CSF (PeproTech Inc. Rocky Hill, NJ, USA) and 3 U/ml erythropoietin (Amgen Thousand Oaks, CA, USA). In these culture conditions, progeny of cells 97 +/- 2% glycophorin-A cells were generated. For megakaryocytic unilineage culture, medium was supplemented with 100 ng/ml thrombopoietin (PeproTech). In these culture conditions, progeny of cells 94 +/- 3% CD41+ were generated. For granulocytic unilineage culture, medium was supplemented with 1 U/ml IL3, 0.1 ng/ml GM-CSF combined with plateau level of G-CSF (500 U/ml) (PeproTech). In these culture conditions, progeny of cells 80-85% CD15+ were generated. For monocytic unilineage culture, serum-free medium was supplemented with 1 ng/ml IL6, 100 ng/ml Flt3-ligand, combined with plateau level M-CSF (50 ng/ml) (PeproTech). In these cultures, progeny of cells 100 +/- 2% CD14+ were generated. For morphological analysis, cells were smeared on glass slides by cytospin centrifugation, stained with May-Grünwald-Giemsa and analyzed at 400× magnification under a microscope (Eclipse 1000, Nikon, Tokio, Japan) equipped with a digital camera.
Flow Cytometry analysis
Cells were washed and incubated 30 minutes on ice in the dark with 3-5 μg/ml PE conjugated monoclonal antibody to Glycophorin A (GPA), CD34, CD41, CD15 and CD14 or isotype control (Pharmingen, San Diego, CA, USA). Cells were then washed and analysed on a FACSCanto (Becton Dickinson, Franklin Lakes, NJ, USA). Acquisition and analysis were performed using Diva software (Becton Dickinson).
Microarrays and bioinformatic analysis
To analyze microRNAs expression in human hematopoietic cells, total RNA was obtained using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Microarray analysis was performed as described [35, 36]. Briefly, labeled targets from 5 μg of total RNA were used for hybridization on microarray chip containing 3766 probes in triplicate, including 627 human precursors miRNA genes. The probes (40-mer oligonucleotides) are spotted by contacting technologies and covalently attached to a polymeric matrix. The microarray were hybridized in 6 × SSPE/30% formamide at 25°C for 18 h, washed in 0.75 × TNT (Tris-HCl/sodium chloride/Tween) at 37°C for 40 min, and processed by using direct detection of the biotin-containing transcripts by Streptavidin - Alexa647 conjugate.
Processed slides were scanned by using Axon 4000 B Scanner (Molecular Device, Orleans Drive Sunnyvale, CA, USA). The expression level were analyzed by GenePix Pro 184.108.40.206 software.
Raw data were normalized and analyzed using the GeneSpring GX 7.3.1 software (Agilent, Foster City, CA, USA). Expression data were median centered. Statistical comparisons were performed by using the ANOVA (Analysis of Variance) tool, using the Benjamini and Hochberg correction for false positives reduction. Cluster analysis was performed using the Pearson correlation as measure of similarity. Microarray data are conform to the MIAME guidelines and have been deposited in ArrayExpress http://www.ebi.ac.uk/arrayexpress under the accession number E-MEXP-2148.
To analyze mRNAs expression in human hematopoietic cells, total RNA was purified from 1-12 × 106 cells using RNeasy Mini columns (Qiagen GmbH, Hilden, Germany). RNA was quantified spectroscopically (A260 nm) and quality was assessed by evaluating the A260/A280 ratio and analyzing electrophoretic migration of ribosomal RNAs in 1% agarose gel of a 65°C denaturated 1 μg aliquot. When the presence of genomic DNA was revealed, RNA samples were treated with DNaseI.
Gene expression profiling of 14 different samples, corresponding to 3-4 time points for each of the 4 differentiative pathways, was determined using the GeneChip Human Genome U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA) containing a redundant probe set of 54630 sequences. Sample labeling of a 5 μg RNA aliquot, hybridization, washing and scanning were performed according to standard manufacturer's procedures (Affymetrix). Raw data were normalized by first calculating the mean value of the signal of the housekeeping (HK) β-actin (ACTB), α-tubulin (TUBA1B), GAPDH and ubiquitin C (UBC) genes in each sample and then dividing signal values of each time point (ti) for the result of ratio HKmean(ti)/HKmean(t0). Microarray data are conform to the MIAME guidelines and were made publicly available by submission to ArrayExpress under the accession number E-MEXP-2146.
Genome-wide and miRNAs expression patterns was analyzed by using the Gene Expression Dynamics Inspector (GEDI, version 2.1) program [24, 25]. Briefly, this algorithm transforms high dimensional gene expression data into distinct two-dimensional color graphic representations deriving from the averaging over a suitable nearly-squared pixel grids, in which each pixel represents the mean value of a given group of genes (around 5-10 genes/pixel on average) having similar expression pattern among different samples. For GEDI analysis were used the log2-transformed linear normalized absolute values, with 20 (1st phase) and 80 (2nd phase) training iterations and a variable grid size (41 × 40, 11 × 10, 10 × 9, 8 × 7 or 6 × 5 according to sequence number) as settings. Sampling of PCGs for scalability analysis was performed on the 17655 Expressed Sequences set by ordering lines corresponding to different sequence ID on the basis of a new-randomly generated numerical column, and subsequently picking up multiple 100 or 1000-sized sequence data sets. For each analysis a negative control was included by applying the same procedure to a same-sized set of numbers randomly generated among a comparable range (1-50000 for PCGs and 1-100 for miRNAs). The global invariance of the aspect of the two dimensional color pattern relative to a given lineage is the image in light of the reaching of an attractor stable state encompassing the entire transcriptome, while the emergent character of the gene expression modification is implicit in the progressive averaging (analogous to the renormalization group of statistical mechanics) procedure. The scalable properties of the system were assessed by different choices of subsets of genes into which perform the analysis.
The analysis of both PCG and miRNA spaces in terms of system dynamics was performed using the Pearson correlation coefficient or product moment correlation. We applied this index in order to have a measure of the level of concordance of any two expression vectors correspondent to two different biological samples, x and y with n dimension (n = genes) and mean values of expressions and . Their mutual Pearson correlation, r = (x, y) is then defined as:
where , corresponding to the differences from the mean expression of each gene in the X and Y sample respectively and θ is the angle between two expression vectors. Geometrically, Eq. 1 shows that the correlation coefficient can be viewed as the cosine of the angle on n-dimensional space between the two vectors of data which have been shifted by the average to have mean zero. Angle q is a measure of the differences between the two vectors and consequently of the difference in expression pattern of the two sample, when θ = 0 (and consequently r = 1) the two expression patterns are completely coincident, and the two vectors are parallel. In the case of r = 0 (and consequently θ = 90 degrees) the two expression vectors are orthogonal, i.e. the expression patterns of the two samples are each other independent.
The measure Dij = 1-Rij with R = Pearson correlation coefficient between i and j samples can be considered as a between sample distance going from 0 (R = 1) for the total resemblance of the two samples to 1 as maximal possible distance (given the practical impossibility, in this particular case, of the scoring of a relevant negative correlation).
The Kruskal-Wish Multidimensional Scaling (MDS) technique was adopted to accommodate the studied samples into a bi-dimensional plane, maintaining as invariant as possible the original between samples distance. This technique, like any MDS procedure, projects the samples into an abstract space maintaining the initial between samples distances that in our case derive from the original n-dimensional (with n = number of genes) expression space through the agency of Pearson correlation.
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We thank CM Croce for providing supports for microarray analysis, A Zeuner for providing useful materials, A Sorrentino for reading the manuscript, M Biffoni for flow cytometry, A Cerio for technical assistance, G Loreto for graphics. This work was supported by grants from the Italy-USA Oncology Program, Istituto Superiore di Sanità, Rome, Italy.
NF carried out the molecular studies and drafted the manuscript, LC carried out the molecular studies, performed the GEDI analysis and drafted the manuscript, EP purified and prepared the unilineage hematopoietic cultures, AC participated in the design of the study, CGL performed microRNA arrays, GAC performed microRNA arrays and participated in the design of the study, SR performed the statistical analysis on the correlation between microRNAs and gene expression data, CP participated in its design and coordination, GM conceived the study, participated in its design and wrote the paper, AG performed the statistical analysis, participated in its design and wrote the paper. All the authors read and approved the final manuscript.
Nadia Felli, Luciano Cianetti contributed equally to this work.
Electronic supplementary material
Additional file 1: Table S1: Expression of selected genes during hematopoietic differentiation. (XLS 5 MB)
Additional file 2: Table S2: Expression of 627 human microRNA during hematopoietic differentiation. (XLS 354 KB)
Additional file 3: Figure S1: Pearson correlation (r) with CD34+ profile of the four different lineages. (DOC 205 KB)
Additional file 4: Figure S2: Correlation between whole set distance space and a representative random gene extraction (627 genes). (DOC 24 KB)
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Felli, N., Cianetti, L., Pelosi, E. et al. Hematopoietic differentiation: a coordinated dynamical process towards attractor stable states. BMC Syst Biol 4, 85 (2010). https://doi.org/10.1186/1752-0509-4-85
- Scalable Character
- Stable Attractor
- Epigenetic Landscape
- Cord Blood CD34
- miRNA Machinery

domain: biology


Mitchell, who voted for Trump in 2016 and in November, sent a letter to
‘It is unacceptable for political candidates to treat our election system as though we are a third- world nation and incite distrust of something so basic as the sanctity of our vote,’ Mitchell wrote in his scalding letter to House Republican Minority Leader Kevin McCarthy and Republican National Committee Chairwoman Ronna McDaniel.
Mitchell plans to retire from Congress at the end of his term next month.
Retiring Rep. Paul Mitchell of Michigan quit the Republican party on Monday and will become an independent in disgust over President Donald Trump’s bid to overturn the election results. He said on CNN Monday night: ‘I’ve had enough…This party has to stand for democracy first’
Trump and Mitchell pictured together above. Mitchell voted for Trump in 2016 and 2020 and says he agrees with 95 percent of his administration’s work, but says his effort to overturn election results is unconstitutional
The 64-year-old revealed he’s departing from the Republican party for its refusal to accept the election results.
‘I believe that any candidate has the ability to request a recount, to go through legal process if they think there’s some basis that the votes are not reasonable or concern about fraud. But we’ve gone through the process,’ Mitchell said in an interview with
‘It became clear to me that I could no longer be associated with the Republican Party, that leadership does not stand up and say the process, the election is over,’ he added.
He pointed out that he voted for Trump in both elections and agreed with much of his agenda.
‘As I said in my letter I voted for Donald Trump. I supported the administration policy 95, 96 percent of the time the last two terms,’ he said.
‘I’ve been active in the national and state party. But this party has to stand up for democracy first, for our Constitution first and not political considerations, not just a candidate, not simply for raw political power. And that’s what I feel is going on and I’ve had enough,’ he added.
Mitchell wrote a letter to House Republican Minority Leader Kevin McCarthy and Republican National Committee Chairwoman Ronna McDaniel on Monday announcing his disaffiliation
‘It is unacceptable for political candidates to treat our election system as though we are a third- world nation and incite distrust of something so basic as the sanctity of our vote,’ Mitchell wrote
Mitchell lashed out at his Republican peers calling their behavior in the election ‘disgusting’.
‘This election simply confirms for me it’s about power first and that, frankly, is disgusting and demoralizing,’ Mitchell said.
‘Losing is brutal, it’s personal and it hurts. But if you’re not willing to accept that, you should not be in political leadership,’ he added.
Mitchell called the slew of legal challenges to contest election results led by Trump allies as ‘ridiculous’ and expressed concern over the threats thrown at election officials who called the race for Biden.
‘When you can’t conduct an election without threats of violence, we become a third world nation. What are we, Venezuela?’ he said.
He’s now demanding accountability within his party.
‘It’s why I’m ending my tenure in Congress by speaking out, in this case, that our leadership owes us better than what we are seeing right now and until I see that, I’m going to be an independent because it’s simply unacceptable,’ he said.
Mitchell announced he’s breaking away from the GOP on Monday, the same day that the Electoral College’s results confirmed Joe Biden had indeed won the election.
Mitchell announced he’s breaking away from the GOP on Monday, the same day that the Electoral College’s results confirmed Joe Biden had indeed won the election
Mitchell (above) is now the second Republican member of Michigan’s congress to leave the party. Michigan Rep. Justin Amash first left the GOP last summer and became a Libertarian.
Mitchell is now the second Republican member of Michigan’s congress to leave the party. Michigan Rep. Justin Amash first left the GOP last summer and became a Libertarian after he called for Trump’s impeachment.
In Mitchell’s letter he slammed his Republican colleagues for supporting harmful conspiracy theories surrounding the election.
‘Republican leaders collectively sit back and tolerate unfounded conspiracy theories and “stop the steal” rallies without speaking out for our electoral process, which the Department of Homeland Security said was “the most secure in American history,” our nation will be damaged,’ Mitchell wrote.
‘I have spoken out clearly and forcefully in opposition to these messages. However, with the leadership of the Republican Party and our Republican Conference in the House actively participating in at least some of those efforts, I fear long-term harm to our democracy,’ he added.
Rep. Paul Mitchell’s letter in full
Dear Chairwoman McDaniel and Leader McCarthy:
The weeks since the 2020 General Election have been traumatic for many in our nation, both voters and those of us who took an oath to serve this nation.
As you well know, my voting record in Congress over the past two terms has been in line with President Trump and the administration’s policies more than 95 percent of the time. Further, I voted for President Trump in the 2020 General Election despite some reservations about four more years under his leadership. I felt that many policies achieved during the Trump administration had been positive for our nation, whereas the policies espoused by the Democratic Party were too radical and did not reflect my principles.
I have also worked hard to support each of you and your efforts. Ronna, you may recall that I spoke with you several times as you considered undertaking the role of RNC Chair. It was my strong belief that you could help lead our party and support its core principles, and I encouraged you to undertake the position. Kevin, I worked diligently with you as part of the Republican Leadership Team for both the 115th and 116th Congresses. I fervently whipped votes for our policies during both terms on some tough issues.
As an active supporter of the national GOP, the state GOP, the NRCC and individual candidates, I raised almost $800k in just 2 1/2 years for the NRCC to support Republican candidates supported by leadership. I have contributed personal funds, not simply PAC funds, to innumerable candidates at the Federal and state levels.
I agree that there have been some disconcerting aspects to this election. With more than 155 million people voting, both administrative errors and even some fraudulent voting likely occurred. Steps must be taken, by each state, to audit election results, validate ballots and process and report findings to ensure that every legal vote counts. Michigan clearly needs to do that, not just Wayne County. However, the president and his legal team have failed to provide substantive evidence of fraud or administrative failure on a scale large enough to impact the outcome of the election.
Ronna, you know Michigan politics well. President Trump did not lose Michigan because of Wayne County, but rather he lost because of dwindling support in areas including Kent and Oakland County, both previous Republican strongholds. In the 2020 election, President Trump lost Michigan by 154,000 votes, compared to 2016 when his margin of victory was slightly more than 10,700 votes.
I have stated publicly numerous times that when entering the political arena, a person must be willing to accept winning and losing with grace and maturity. Having personally experienced both winning and losing, the latter can be brutal.
Any candidate, including the president, is entitled to request recounts and pursue legal challenges they believe are appropriate if they possess evidence of wrongdoing. President Trump has undertaken or supported both of these options. Recounts have failed to significantly alter the vote outcome in any state and dozens of court cases have been summarily dismissed in both state and Federal courts across our nation.
It is unacceptable for political candidates to treat our election system as though we are a third- world nation and incite distrust of something so basic as the sanctity of our vote. Further, it is unacceptable for the president to attack the Supreme Court of the United States because its judges, both liberal and conservative, did not rule with his side or that “the Court failed him.” It was our Founding Fathers’ objective to insulate the Supreme Court from such blatant political motivations.
If Republican leaders collectively sit back and tolerate unfounded conspiracy theories and “stop the steal” rallies without speaking out for our electoral process, which the Department of Homeland Security said was “the most secure in American history,” our nation will be damaged. I have spoken out clearly and forcefully in opposition to these messages. However, with the leadership of the Republican Party and our Republican Conference in the House actively participating in at least some of those efforts, I fear long-term harm to our democracy.
The stability and strength of our democracy has been an ongoing concern for me. I expressed strong concerns about the president’s response to Charlottesville, the anti-immigrant “send them back” rhetoric, and even the racist comments of my own colleagues in the House.
I believe that raw political considerations, not constitutional or voting integrity concerns, motivate many in party leadership to support the “stop the steal” efforts, which is extremely disappointing to me. As elected members of Congress, we take an oath to “support and defend the Constitution of the United States,” not to preserve and protect the political interests of any individual, be it the president or anyone else, to the detriment of our cherished nation.
As a result, I am writing to advise you both that I am withdrawing from my engagement and association with the Republican Party at both the national and state level. I will support, contribute to, and fundraise for individual candidates who reflect the principles I hold dear. Further, by copy of this letter I am also advising Ms. Laura Cox, Chair of the Michigan GOP of this decision.
I am also requesting that the Clerk of the U.S. House of Representatives change my party affiliation to Independent for the remainder of my term in office. While admittedly symbolic, we all know that symbols matter.
Member of Congress


Of course, Giuliani's national profile expanded enormously because of 9/11. And while the press harps on that point, the more interesting part of the story lies elsewhere. The war on terror hasn't just changed Giuliani's profile as a crisis-leader, it's changed the attitudes of many Americans, particularly conservatives, about the central crisis facing the country. It's not that pro-lifers are less pro-life or that social conservatives are suddenly OK with homosexuality, gun control and other issues where Giuliani's dissent from mainstream conservative opinion would normally disqualify him. It's that they really, really believe the war on terror is for real. At conservative conferences, on blogs and on talk radio, pro-life issues have faded in their passion and intensity compared with the war on terror. Taken together, terrorism, Iraq and Islam have become the No. 1 social issue for conservative base of the party.
Note: I didn't say it's become the No. 1 foreign-policy or national-security issue for social conservatives. It's become the No. 1 social issue, at least for many of them.
Unambiguous polling data is hard to come by on this point, but the anecdotal data is enormous. From my e-mail alone, it's obvious. Books that frontally challenge Islam as a religion have become mainstays of conservative publishing. Meanwhile, Dinesh D'Souza's book, "The Enemy at Home," a passionate, socially conservative polemic calling for the American right to align itself with traditional Muslims against the domestic left and Islamic extremists, has found itself almost entirely undefended on the right for its perceived effort to "blame America first."
William Bennett, the famed "virtue czar," emphasizes the civilizational struggle more than any other and gets an enormous response from social conservatives. Even before the war on terror, evangelicals embraced Israel for myriad reasons, among them a theological affinity for the Jewish state and a faith that it is an imperiled sister democracy. Such convictions are only multiplied and personalized for these Americans by events since 9/11. At National Review's "conservative summit" last month, Romney talked at length about abortion but gave short shrift to the war, and the disappointment in the room was palpable.
That's not to say either Romney or Giuliani will win, but they're the ones to watch because they get to design their first impressions in a way other top-tier candidates like John McCain and Newt Gingrich can't. Romney and Giuliani, both immensely attractive, savvy and well-funded politicians, are in effect the canaries in the coal mine of conservative politics. If either emerges from the dark tunnels of primary season alive, it will tell us a great deal about the future of the GOP and American politics.


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text:
Credit Facility – Make Purchases Without Worrying About Lack Of Funds & Repay The Amount At Their Convenience. Pay & Get Paid By ACH, Wire
Credit Facilities Definition
Zil Money is a financial technology company, not a bank or an FDIC member. Zil Money offers banking services through partnership with FDIC member banks Silicon Valley Bank, Texas National Bank, and East-West Bank.
Unlocking Business Potential: Embracing The Power Of Credit Facilities. Fast And Easy Funding Solution For Your Business. Easy Repayment
Use Credit Facilities Provided By Zil Money And Improve Your Business Fast And Easy Funding Solution For Your Business
Credit Facilities Fast And Easy Funding Solution For Your Business Set Up One-Time/Recurring Faster Same-Day ACH, Printable Check, eChecks

domain: finance


text:
Should You Invest in Exchange Traded Funds?
There are thousands of ETFs available. Should you invest in them?
Have A Question About This Topic?
Retiring earlier than expected can be disheartening. Learn steps that can help you smoothe the road ahead.
Without your knowing, your investment portfolio could be off-kilter.
Every so often, you’ll hear about Social Security benefits running out. But is there truth to the fears, or is it all hype?

domain: economics


Who: The Advertising Standards Authority (ASA)
Where: United Kingdom
When: 14 June 2023
Law stated as at: 4 August 2023
The ASA has taken significant strides towards more effective regulation through the integration of technology, specifically artificial intelligence (AI), to monitor and regulate online advertising. Since 2021, the ASA has been dedicating resources to bolster its data science capabilities and construct a specialised team to address the unique challenges posed by regulating online advertising in the digital age.
The focus of its technological advancement is the development of the Active Ad Monitoring system, which uses AI to identify proactively online ads that violate rules and regulations. While the ASA has historically demonstrated a proactive approach to regulation, the scale of online advertising has posed considerable difficulties in efficiently monitoring compliance. A creation of the ASA’s data science team, the system is designed to sift through the huge volume of online ads and provide intelligence to ASA experts, streamlining their efforts.
The system is comprised of three components:
- Ad capture at scale. The system captures ads from social media, search and display using a mix of public sources, our own internal monitoring tools and proprietary datasets.
- AI-based filtering. Machine learning models are configured to spot the ads that are most likely to be relevant to a given issue, or to have specific compliance problems.
- Expert review. ASA experts can browse and search content related to their work via a web interface that allows them to quickly assess issues, and identify problematic examples for action.
The ASA has said that developing the capabilities of the system will be an ongoing project. At the moment, it is only focused on the subset of issues the ASA is focusing on. However, it has already had a massive impact and currently processes more than 100,000 ads each month. Notably, the system played a pivotal role in the ASA’s efforts to regulate advertising related to cryptocurrency services during 2021/2022. This involved not only the development of rules in collaboration with partners like the Financial Conduct Authority and industry stakeholders but also the monitoring of compliance and prompt actions against non-compliant ads.
Previously the ASA has been reliant on labour-intensive manual searches and public complaints to identify non-compliant ads. Now, the Active Ad Monitoring system automatically captures relevant ads and employs machine learning algorithms to flag potentially non-compliant advertisement which are then subsequently reviewed and acted upon by ASA experts. Each week, the compliance team receives a list of flagged ads, along with explanations of rule violations, enabling swift and effective actions to secure compliance and remove non-compliant ads from platforms.
This integration of AI technology and data-driven processes has yielded significant results. The Active Ad Monitoring system has already led to the amendment or withdrawal of hundreds of non-compliant ads. The ASA’s commitment extends beyond investment in AI and it is a focused on creating real-world applications that translate into a reduction of non-compliant ads.
Why this matters:
The ASA’s adoption of AI-powered Active Ad Monitoring system is pivotal as it empowers efficient, proactive, and scalable enforcement of online advertising regulations. In the ever-expanding digital landscape, AI swiftly identifies non-compliant ads, safeguarding consumers from deceptive content and ensuring fair competition. This innovative approach sets a standard for technology-driven regulatory practices, optimises resource allocation and fosters industry accountability, shaping a more transparent and responsible advertising ecosystem.


Bitcoin has risen above $10,000 for the first time in almost a month as protests in U.S. cities continue to intensify.
At around 23:05 UTC (7:05 p.m. Eastern Time), bitcoin (BTC) rose from $9,895 to around $10,429 over the course of 30 minutes as a new wave of volatility greeted the end of the daily closing period for June 1.
The large spike in BTC's price comes as violence erupts on the sixth day of protests over the death of George Floyd at the hands of Minneapolis police. BTC has since cooled slightly and is currently changing hands for around $10,191, according to CoinDesk's Bitcoin Price Index.
Bitcoin has been gyrating between $8,600 and $10,000 for a period of around 32 days and finally broke above the $10,000 psychological resistance in convincing fashion as traders sought to capitalize on a bullish technical setup.
"ETH broke out already, BTC now having its turn," said Joshua Green, chief investment officer at Orthogonal Trading.
"The macro and fundamental scenario is very bullish, and the market has been shorting into $10,000, under-allocated above $10,000," Green added.
Other notable cryptos are also having their time in the sun with the likes of eos (EOS), cardano (ADA), bitcoin cash (BCH) and litecoin (LTC) having climbed around 5% over a 24-hour period.
The leader in news and information on cryptocurrency, digital assets and the future of money, CoinDesk is a media outlet that strives for the highest journalistic standards and abides by a strict set of editorial policies. CoinDesk is an independent operating subsidiary of Digital Currency Group, which invests in cryptocurrencies and blockchain startups. As part of their compensation, certain CoinDesk employees, including editorial employees, may receive exposure to DCG equity in the form of stock appreciation rights, which vest over a multi-year period. CoinDesk journalists are not allowed to purchase stock outright in DCG.
Learn more about Consensus 2023, CoinDesk’s longest-running and most influential event that brings together all sides of crypto, blockchain and Web3. Head to consensus.coindesk.com to register and buy your pass now.


text:
Skyfall review: A Bond movie about Bond
James R | On 28, Sep 2021
Director: Sam Mendes
Cast: Daniel Craig, Javier Bardem, Judi Dench, Naomie Harris, Ralph Fiennes
Skyfall has already frequently been described as the best Bond movie of all time. It’s not. But that’s partly because it’s not a Bond movie – it’s a movie about Bond. And that’s something very special indeed.
But Bond isn’t the only one under the microscope: M (Dench) is too. Dogged by a decision she makes during the blistering opening set piece, she finds herself under attack from an old enemy with Ralph Fiennes’ government official (Mallory) snapping at her heels and no 007 to defend her.
An espionage thriller that explores the shady past of the head of MI6? Ever since Skyfall’s synopsis was revealed, it sounded like a different direction for 007. But while the first thought might be Christopher Nolan’s dark reboot of Batman, Sam Mendes’ take on Bond owes a greater debt to Spooks.
John Logan’s script, co-written with series stalwarts Neal Purvis and Robert Wade, feels like a smart spy flick that just happens to feature Daniel Craig. There’s even a hint of that fan favourite theory that “007” is a moniker handed down between agents over the years. As for M, who has been a character in her own right since GoldenEye, Judi Dench’s sweary and stubborn boss is every bit the female Harry Pearce, even finding the time to deploy some Tennyson among the treachery. A surprising amount of the action unfolds on UK soil, too, including one spectacular scene on the London Underground.
But Sam Mendes makes sure this is bigger and better than any BBC production. He brings the hushed intelligence of Spooks to the fray but regularly takes time out to blow up buildings and derail trains with exhilarating precision. He’s helped no end by cinematographer Roger Deakins, who adds his own trademark attention to detail, along with the most striking addition to the Bond formula: the silhouette. For the first time in the franchise’s history, 007’s world literally becomes a place of shadows and reflections, a ghostly style echoed in Daniel Kleinman’s haunting titles sequence. The best Bond film of all time? Skyfall is certainly the most beautiful.
Despite all these changes, though, the film is clearly Bond 23. Unlike Die Another Day’s 20th birthday bash, 007’s 50th anniversary is treated with just the right amount of wry humour: a final tableau cheekily sets up the future, while familiar faces such as Q return with a wink. “What were you expecting?” snarks Ben Whishaw’s boffin, after handing Craig a gun. “An exploding pen? We don’t do that anymore.” Another gag involving the Aston Martin DB5 sees Craig hitting Connery levels of charisma. His style and sense of humour, though, is unique among Bonds: the chuckles come from disbelief at what he’s doing rather than laughs from smug one-liners, with everyone including him aware that he’s not invincible – not that it stops him chasing down villains with a dogged recklessness. He’s the most psychologically complex 007 to date, even without the stubble.
And what to say of Craig’s nemesis? Sporting a bleach-blond wig and a uniquely camp menace, Javier Bardem’s Silva is a classic Bond villain in the Sean Bean mould, never letting his physical deformity distract from a highly personal vendetta – one that dials M for Matriarch as much as it does Murder.
There are minor quibbles. Dialogue occasionally slips into clumsy mode (“I have new information!” chirps Naomie Harris’ scene-stealing yet slightly wasted field agent, Eve, to justify her appearance in Shanghai halfway through) and Adele’s theme tune is harmlessly derivative. More disappointing is Thomas Newman’s score, which underscores the GoldenEye comparison by pulling an Eric Serra; the composer goes for an electronic-tinged soundtrack, shoehorning in the Bond theme at random moments to remind us what we’re watching with barely a trace of Adele’s title number.
Music aside, though, Skyfall is a perfect cinematic balancing act, one that squares Silva and James as two offsprings of the same system and ties Bond’s back-story tightly to its barn-storming, booby-trapped finale. Skyfall is a Bond movie and not a Bond movie. It’s a blockbuster with a cast of 12 characters. It’s a secret agent movie that copies Home Alone. It’s an action film directed by Sam Mendes. More than anything, though, it’s absolutely brilliant.
Where to watch online in the UK:
This review was originally published in 2014.

domain: entertainment


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text:
From The Spun:
Bucs head coach Bruce Arians announced on Monday that Brown is still donning a walking boot, meaning it’s probably a longshot he plays against Washington this Sunday.
Brown is dealing with an ankle injury. He had been using crutches to get around, but eventually moved to a walking boot.
It was previously believed the Bucs’ recent bye would allow Brown enough time to get healthy. But it sounds like he may end up missing another week or two.
“Bruce Arians said Antonio Brown is still in a walking boot,” wrote ESPN’s Jenna Laine via Twitter. “He’s less confident about him and Rob Gronkowski returning this week as he is about some other players. He believes Richard Sherman will be good to go.”
The last time the Buccaneers took the field – without Antonio Brown, that is – they were beat by a Trevor Siemian-led New Orleans Saints team. It was an ugly outing by the 2020-21 champs.
Buccaneers fans are starting to get worried about Brown’s status.
Others think this opens the door for the Buccaneers to make a run after Odell Beckham Jr.
OBJ is expected to become a free agent on Tuesday after he clears waivers. Tampa Bay could be a landing spot.
OBJ to Tampa Bay makes too much sense, especially if Antonio Browns ends up missing the next few weeks.
For now, Tom Brady will have to rely on Mike Evans and Chris Godwin this Sunday versus Washington.

domain: news


text:
Brantley Gilbert - Just As I Am: Platinum Edition 180g Vinyl 2LP
Brantley Gilbert Just As I Am: Platinum Edition 180g Vinyl 2LP
Brantley Gilbert is tearing up the road to Country music with his upcoming album Just As I Am (The Valory Music Co.), which is already burning up the airwaves and ushering in summer with his first new work in four years. Like the album's title, the songs are an expression of who he is at this time in his life. The rings, the chains, the faith, the no apologies - "If You Want A Bad Boy," you'll find one in Brantley's first song on his new album. Love him or not, his latest offering is Just As I Am and it is an album of unforgettable memories - for him and everyone who hears it.
Brantley doesn't just write songs, he shares from the heart the chapters of his life through music. He is adamant about the fact that the devoted "BG Nation" is made up of his friends, not fans. He's created a high-energy, in-your-face collection with plenty of adrenaline and testosterone. He also evokes a softer connection, proving a tough guy can be tender, too. No matter the song, he plays the guitar like an extension of his voice – an ebb and flow of emotion that resonates with millions.
Well-worn denim, metal in many forms and a pulled-down ball cap may frame Brantley, but the true art comes from what you don't see. "I sing for the people who have helped me do my thing," he says. His inspiration for writing comes from all aspects of his life, but he knows when he gets an "ear worm" it is a song that he needs to write now; he just has to give it legs and wings. That's just what happened with Brantley's #1 single "Bottoms Up."
Brantley is excited about the renewed direction of his music as the more he lives, the more real life stories he has to share with others. He's recruiting a new generation of fans who are less interested in genre and all about expressing themselves through the songs they sing and blurring the lines a bit. Effortlessly Just As I Am becomes the universal soundtrack from your best first date, the last dance of a good-bye and memories in between. This bad boy will make you want to crawl out of the bedroom window, give you the courage to start a new relationship and inspire you to close your eyes and see the candlelight, starlight, spotlights and twilight of his life...or grab your crew for a good time.
When putting the chapters of his life to music, Brantley believes if you write a song the right way, you're still in love with someone every time you sing it, you're still pissed when you were an idiot, and you're still ready to party with your friends. Brantley gives the world a taste of who he's looking for in "My Baby's Guns N' Roses." It's this passion for life that fuels his music. You feel the emotions running through each song: the longing of a guitar, the pounding heart of the drums, the changing gears on a dime as the music drives you 100 mph down one road, only to jump the curb and go another direction entirely.
This album contains new anthems and clearly shows who Brantley Gilbert is now and what he's come through to get where he is today. His music is deep, like the man of substance singing it, and you'll find yourself wanting to fall in love one minute and get into some trouble the next. Life may be where Brantley's songs start, but you can expect to have a lot of fun with his music. His songs are as serious as you make them.
Brantley Gilbert Just As I Am: Track Listing:
1. If You Want a Bad Boy
2. 17 Again
3. Bottoms Up
4. That Was Us
5. I'm Gone
6. My Baby's Guns N' Roses
7. Lights of My Hometown
8. One Hell of an Amen
9. Small Town Throwdown (feat. Justin Moore and Thomas Rhett)
10. Let It Ride
11. My Faith in You
13. Read Me My Rights
14. Grown Ass Man
15. Bottoms Up (Remix) (feat. T.I.)
16. Do What The Night Wants
17. Stone Cold Sober
18. Same Old Song
19. Just As I Am
We Also Recommend

domain: artistic


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|"Nice breakfast with fresh waffle maker."||(in 2 reviews)|
|""Friendly staff , great location.""||(in 55 reviews)|
|"Nice staff and clean rooms"||(in 45 reviews)|
|"The overwhelming smell of Detol?"||(in 4 reviews)|
|"room was filthy"||(in 35 reviews)|
|"Pool and hot tub was closed."||(in 16 reviews)|
|room was clean||in 167 reviews|
|staff was friendly||in 125 reviews|
|pool and hot tub||in 99 reviews|
|breakfast was good||in 86 reviews|
|good location||in 53 reviews|
|front desk||in 39 reviews|
|bed was very comfortable||in 26 reviews|
|fridge and microwave||in 25 reviews|
|next door||in 11 reviews|
|air conditioning unit||in 10 reviews|
|smell in the room||in 8 reviews|
|waffle maker||in 7 reviews|
|cigarette smoke||in 6 reviews|
|pet friendly||in 5 reviews|
|ground floor||in 4 reviews|
|sink in bathroom||in 3 reviews|
|killing flies||in 2 reviews|
Research before paying
Pool was not working which is the reason why I chose the hotel. Overpriced for no pool.
For the money it's perfect
$58/night on kayak.com and you get a clean, comfortable room with TV, Wi-Fi, fridge, microwave and coffee maker. The staff are attentive, accommodating and curteous. Then you get a pretty decent little continental breakfast in the morning and access to a pool and hot tub. Sign me up any day! Anyone complaining on here has a steak taste on a hamburger budget.
Located in Okanagan Valley, Travelodge by Wyndham Salmon Arm BC is the perfect base for those wanting to visit the area's well-known wineries. It also provides an indoor pool, a Jacuzzi and free Wi-Fi.
The hotel has 36 rooms and has been recently refurbished. Staff are available 24/7 and can assist with booking tours and tickets.
The hotel's rooms are comfortable and offer a kitchenette that is equipped with a microwave and a refrigerator. Hot drinks can be made with the provided tea and coffee supplies. They are also fitted with cable/satellite channels, a hair dryer and a spa bath.
Travelodge by Wyndham Salmon Arm BC has easy access to Downtown Salmon Arm. There is also the added convenience of many nearby restaurants and cafés.
based on 341 reviews
Policies vary by room type and provider.
Guests travelling with new or existing direct bookings for stays at Travelodge by Wyndham Salmon Arm through 30 June 2020 will have their cancellation or change penalties waived up to 24 hours prior to arrival. For more information, visit the Wyndham Group Response to COVID-19 page.
Travelodge by Wyndham Salmon Arm is located at 2401 Trans Canada Highway, 1.1 km from the centre of Salmon Arm. Salmon Arm Golf Club is the closest landmark to Travelodge by Wyndham Salmon Arm.
Check-in time is 3:00 PM and check-out time is 11:00 AM at Travelodge by Wyndham Salmon Arm.
Yes, Travelodge by Wyndham Salmon Arm offers free Wi-Fi.
Yes, Travelodge by Wyndham Salmon Arm offers free parking.
No, Travelodge by Wyndham Salmon Arm does not offer free airport shuttle service.
Travelodge by Wyndham Salmon Arm is 79.7 km from Kamloops. Travelodge by Wyndham Salmon Arm is 81.9 km from Kelowna.
Yes, Travelodge by Wyndham Salmon Arm is a pet-friendly hotel.
Yes, Travelodge by Wyndham Salmon Arm has a pool on-site.
Travelodge by Wyndham Salmon Arm has implemented the following measures:
Daily cleaning, increased availability of sanitation products.
Lobbies with safe social distancing.
Minimal contact with hotel personnel and other guests.

domain: travel


text:
Hi! Simply click below and type your query.
Our experts will reply you very soon.
Tour Code : SBJ.T.0015
Duration : 3 Nights / 4 Days
Destination Covered : Puri, Bhubaneswar, Chilika Lake, Nandankanan, Jagannath Temple, Rajarani Temple, Lingaraj Temple
Tour Activities : Bird Watching, Beaches Sightseeing, Monuments & Historical Places, Sightseeing
Tour Themes : Religious & Pilgrimage, Culture & Heritage, Affordable & Budget Tours, Monuments & Historical Places, Women Friendly Tours
In this travel plan you are exploring Odisha (formerly Orissa), an eastern Indian state on the Bay of Bengal, is known for its tribal cultures and its many ancient Hindu temples. The capital, Bhubaneswar, is home to hundreds of temples, notably the intricately-carved Mukteshvara. The Lingaraj Temple complex, dating to the 11th century, is set around sacred Bindusagar Lake. So, above all the places with local sightseeing are waiting for your visit. SUNBIRD team will provide you all the services with hassle free process.
Price & Rates
|No of pax||Age Limit||Price per pax (Rs)|
|No of pax Adult||Age Limit Above 09 years||Price per pax (Rs) INR 14000 / Adult|
|City||Hotel Name||Star Rating|
|City Bhubaneswar||Hotel Name King's Court||Star Rating 3|
|City Puri||Hotel Name JBS Chariot||Star Rating 2|
Cancellation & Refund Policy
Packages by Theme

domain: geography


text:
Amazing place to eat! Eugenio is an amazing waitor, the food is out of this world amazing! So so good and highly suggest you come here for a real good Italian meal :)
Delicious Very nice restaurant with variety of foods and drinks. Very reasonably priced and very well maintained so dining there was a pleasure. Food excellent and service even better. Very crowdy so reservation is advisable.
Great food with local pasta Great food with local pasta recipes! Our waiters, Bogdan and Iaia, made our dinner an enjoyable experience. We appreciated the customer service.
Address Via della Paglia, 1/2/3, 00153 Roma RM, Italy
Phone +39 06 580 6404
Maps VFQ9+WP Rome, Metropolitan City of Rome, Italy Open Maps

domain: food


text:
If you are ready to step out of the normal crafting zone and enter the cool crafting zone, then you need to make this Seal Pop Up Card Template.
Kids love making these cards! They resemble a fun little seal and you can see their tongue. How fun it is to make this seal talk as well.
Seals are so much fun to make because they are so interesting. You can teach your kids that seals have a layer of blubber to help them stay warm in the very cold winters.
How to Make a Seal Pop Up Card Template
What you need:
- Seal pop up template
- Red construction paper
The first step you are going to take is to print out the seal pop up template!
As you can see, the template also has directions to complete the steps.
You can assist your child in cutting this template out if they need it.
Once it’s cut out, go ahead and fold it hamburger style.
Ever so carefully, go ahead and cut along the dotted lines where the mouth is.
Once the mouth is cut out, you are going to start folding it as instructed. Take the top part of the mouth and fold it away from the face.
Take the bottom part of the mouth and fold it out and away from the face.
As you can see, this will make the mouth part pop out.
Ever so carefully, take the template and fold it back in half, facing forward.
This is how you will glue the template to the red paper.
Glue everything but the mouth to the red paper.
The gluing is always the fun part!
Now you can fold the whole card in half, with the red paper included.
And there you have a fun Seal Pop Up Card Template. This is the perfect card to hand out to friends and family or just enjoy at home!


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Edo State Governor, Comrade Adams Oshiomhole, yesterday ordered the seizure of wares displayed along walkways on the newly constructed Oba Market roads.
Oshiomhole also ordered the traders to immediately evacuate refuse that littered the market surroundings.
It was the governor’s first official work of the New Year and he was peeved at the refuse heaps around the Oba Ovoranmwen Square and the littering of impounded vehicles along the road especially near the Oba Market Police Station.
Governor Oshiomhole ordered the vehicles to be towed away and warned the Divisional Police Officer of the station to desist from parking impounded vehicles along the road.
At the Benin Museum ground, Oshiomhole ordered the arrest of the manager for illegal operation and non-maintenance of the place.
Heaps of refuse littered the museum ground and some parts were leased out by the manager to business operators.
A woman caught cooking on the machine connected to the water fountain at the Museum was also arrested.
The manager said the place was given to the woman and others for use because of demands by fun seekers.
Oshiomhole later ordered that the museum ground be sealed up until further notice.
In an interview with newsmen, Oshiomhole said he was to be back to work in the New Year and assured that the contractors will return to site next week.


text:
Worries that downtown Portland’s new hotels would simply draw customers from nearby competitors appear to have been unfounded.
According to Matthew Arrants of Pinnacle Advisory Group, a brokerage that focuses on the hospitality industry, hotels on the city’s peninsula reported a 70 percent occupancy rate in 2015 – well above the state’s average of 56.7 percent. The U.S. average is 66 percent.
Arrants, who spoke Thursday before a crowd of more than 800 at the annual Maine Real Estate & Development Association conference, attributed the performance of Portland’s hotels to the city’s rising popularity as a tourist destination, identifying leisure travelers as the primary source for the increase in business.
“All the operators are happy because they haven’t had a rate war – yet,” he said.
In Maine and across the country, hotel operators for the past seven years have seen a steady rise in occupancy rates, room rates and revenue earned per available room, Arrant said. However, he said those gains have begun to taper off.
“It’s not bad, but just not as great as it’s been,” he said, adding that there are no discernible signs that the hospitality sector’s ongoing growth will reverse course anytime soon.
Although it doesn’t look like any new hotels are planned for downtown Portland, other parts of the state will add hundreds of new rooms in 2016, Arrant said. Among them are 135 rooms at the Cliff House, the first part of a two-phase project that will ultimately add 235 rooms to the landmark Cape Neddick hotel by 2017; a 73-room Hampton Inn in Kennebunk; an 83-room Hampton Inn and Suites in Kittery; and a 90-room Hampton Inn across from the Oxford Casino.


text:
Last week Hugh Savill of the Association of British Insurers (ABI) said an extension to Article 50 seemed inevitable… now the EU Council has agreed to delay the UK’s departure from the European Union, but that means the ball is right back in Britain’s court.
“After a lengthy discussion, the council today also agreed, subject to a successful vote next week, that in order to provide time for the UK Parliament to agree and ratify a Brexit deal, the date of our departure will now be extended to May 22,” said Prime Minister Theresa May in her March 21 press statement at the EU Council.
“If Parliament does not agree a deal next week, the EU Council will extend Article 50 until April 12. At this point we would either leave with no deal, or put forward an alternative plan.”
May has not had success in getting support for her now twice-rejected proposed withdrawal agreement. The silver lining for her though is that more Members of Parliament aren’t in favour of leaving without an exit plan in place.
Meanwhile the UK Prime Minister stressed how crucial it is for the House of Commons to pass a Brexit deal in order to put a stop to the uncertainty and facilitate a smooth and orderly divorce with the EU.
“I hope we can all agree, we are now at the moment of decision,” stated May. “I will make every effort to ensure that we are able to leave with a deal and move our country forward.”
Last month ABI director general Huw Evans called choosing the no-deal scenario an act of economic recklessness the UK would live to regret, asserting that exiting the world’s single biggest trading block with only the World Trade Organisation rules to fall back on would be wholly inadequate.
This despite the insurance industry having done “everything possible to prepare for no deal, including transferring an estimated 29 million insurance contracts and the establishment of nearly 40 EU subsidiaries and branches to minimise disruption to customers.”
Meanwhile a ‘Revoke Article 50’ petition, which is pushing for Britain to remain in the EU, has now gathered over two million signatures.


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Human tissues possess cells to regenerate themselves when damaged. These cells, including stem cells and other types of progenitors, are able, upon a certain type of stimuli, to regenerate the tissue by:
Generating new cells
Secrete a matrix that will form the new tissue
These abilities are not specific to a tissue meaning that stem cells from bone marrow or adipose tissue are able to give rise to new tissue.
STEMCIS specializes in harvesting and purifying adipose tissue's cells from a patient to cure his/her own pathology, especially for the musculoskeletal and urogenital systems pathologies. Therefore, STEMCIS focuses its research on the regenerative potential of cells contained in adipose tissue.
Adipose tissue cells are widely investigated in clinical trials to assess their potential in many applications.

domain: medical


text: Hand Drawn SMOKE Elements motion graphics pack features a 24 frames per second, 4K resolution animation package of hand-drawn cartoon smoke effects you can use as an overlay to your video presentation. You can use elements in projects like youtube blogs, explainer videos, children shows, sports, infographic, commercials and much more! Use it to create titles, transitions, logo reveals and spice up your videos! Alpha channel included, just drop it into your project. Enjoy! Check out our portfolio for more elements.

domain: design