Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition.

RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.

The influence of heat and cold waves on mortality in Russian subarctic cities with varying climates.

Publications on ambient temperature-related mortality among Arctic or subarctic populations are extremely rare. While circumpolar areas cover large portions of several European countries, Canada, and the USA, the population of these territories is relatively small, and the data needed for statistical analysis of the health impacts of extreme temperature events are frequently insufficient. This study utilizes standard time series regression techniques to estimate relative increases in cause- and age-specific daily mortality rates during heat waves and cold spells in four Russian cities with a subarctic climate. The statistical significance of the obtained effect estimates tends to be greater in the continental climate than in the marine climate. A small meta-analysis was built around the obtained site-specific health effects. The effects were homogeneous and calculated for the selected weather-dependent health outcomes. The relative risks of mortality due to ischemic heart disease, all diseases of the circulatory system, and all non-accidental causes during cold spells in the age group ≥ 65 years were 1.20 (95% CI: 1.11-1.29), 1.14 (1.08-1.20), and 1.12 (1.07-1.17), respectively. Cold spells were more harmful to the health of the residents of Murmansk, Archangelsk, and Magadan than heat waves, and only in Yakutsk, heat waves were more dangerous. The results of this study can help the public health authorities develop specific measures for the prevention of excess deaths during cold spells and heat waves in the exposed subarctic populations.

GATA2/3-TFAP2A/C transcription factor network couples human pluripotent stem cell differentiation to trophectoderm with repression of pluripotency.

To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4 Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis.

Сardiovascular mortality during heat waves in temperate climate: an association with bioclimatic indices.

The authors studied the relative predictive powers of several bioclimatic indices as predictors of population mortality during heat waves. Daily mean and maximum values of air temperature, Humidex, apparent, and physiological equivalent temperatures (PETs) were examined. The numbers of daily deaths and daily meteorological data in Rostov-on-Don (southern Russia) were used. The study period spanned April-September between 1999 and 2011. The eight selected bioclimatic indices were used to identify heat waves and calculate the expected increases in mortality during such events from Poisson generalized linear model of daily death counts. All of the bioclimatic indices considered were positively and significantly associated with mortality during heat waves. The best predictor was chosen from a set of similar models by maximization of relative mortality risk estimates. Having compared the relative increases and their significance levels in several cause- and age-specific mortality rates, the authors concluded that PET was the most powerful predictor.

Negative regulation of the EGFR-MAPK cascade by actin-MAL-mediated Mig6/Errfi-1 induction.

We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF.

Mortality related to air pollution with the moscow heat wave and wildfire of 2010.

BACKGROUND: Prolonged high temperatures and air pollution from wildfires often occur together, and the two may interact in their effects on mortality. However, there are few data on such possible interactions. METHODS: We analyzed day-to-day variations in the number of deaths in Moscow, Russia, in relation to air pollution levels and temperature during the disastrous heat wave and wildfire of 2010. Corresponding data for the period 2006-2009 were used for comparison. Daily average levels of PM10 and ozone were obtained from several continuous measurement stations. The daily number of nonaccidental deaths from specific causes was extracted from official records. Analyses of interactions considered the main effect of temperature as well as the added effect of prolonged high temperatures and the interaction with PM10. RESULTS: The major heat wave lasted for 44 days, with 24-hour average temperatures ranging from 24°C to 31°C and PM10 levels exceeding 300 µg/m on several days. There were close to 11,000 excess deaths from nonaccidental causes during this period, mainly among those older than 65 years. Increased risks also occurred in younger age groups. The most pronounced effects were for deaths from cardiovascular, respiratory, genitourinary, and nervous system diseases. Continuously increasing risks following prolonged high temperatures were apparent during the first 2 weeks of the heat wave. Interactions between high temperatures and air pollution from wildfires in excess of an additive effect contributed to more than 2000 deaths. CONCLUSIONS: Interactions between high temperatures and wildfire air pollution should be considered in risk assessments regarding health consequences of climate change.

The influence of meteorological and geomagnetic factors on acute myocardial infarction and brain stroke in Moscow, Russia.

Evidence of the impact of air temperature and pressure on cardiovascular morbidity is still quite limited and controversial, and even less is known about the potential influence of geomagnetic activity. The objective of this study was to assess impacts of air temperature, barometric pressure and geomagnetic activity on hospitalizations with myocardial infarctions and brain strokes. We studied 2,833 myocardial infarctions and 1,096 brain strokes registered in two Moscow hospitals between 1992 and 2005. Daily event rates were linked with meteorological and geomagnetic conditions, using generalized linear model with controls for day of the week, seasonal and long-term trends. The number of myocardial infarctions decreased with temperature, displayed a U-shaped relationship with pressure and variations in pressure, and increased with geomagnetic activity. The number of strokes increased with temperature, daily temperature range and geomagnetic activity. Detrimental effects on strokes of low pressure and falling pressure were observed. Relative risks of infarctions and strokes during geomagnetic storms were 1.29 (95% CI 1.19-1.40) and 1.25 (1.10-1.42), respectively. The number of strokes doubled during cold spells. The influence of barometric pressure on hospitalizations was relatively greater than the influence of geomagnetic activity, and the influence of temperature was greater than the influence of pressure. Brain strokes were more sensitive to inclement weather than myocardial infarctions. This paper provides quantitative estimates of the expected increases in hospital admissions on the worst days and can help to develop preventive health plans for cardiovascular diseases.

MAL/MRTF-A controls migration of non-invasive cells by upregulation of cytoskeleton-associated proteins.

Monomeric actin regulates gene expression through serum response factor (SRF) by inhibiting its transcriptional coactivator myocardin-related transcription factor (MAL/MRTF). Many affected genes encode cytoskeletal components. We have analysed the migratory effects of actin-MAL signalling and of new target genes in non-invasive highly adherent cells. Expression of active MAL impaired migration of both fibroblasts and epithelial cells, whereas dominant-negative constructs and partial knockdown of MAL/MRTF enhanced motility. Knockdown of three newly characterised G-actin-regulated MAL targets, integrin α5, plakophilin 2 (Pkp2) and FHL1, enhanced cell migration. All three were upregulated by external stimulation through actin-MAL-SRF signalling, and MAL and SRF were inducibly recruited to cis-regulatory elements of the integrin α5 and Pkp2 genes. Finally, the reduced migration of epithelial cells stably expressing MAL was partially reversed by knockdown of Pkp2 and FHL1. We conclude that the actin-MAL pathway promotes adhesive gene expression, including integrin α5, Pkp2 and FHL1, and that this is anti-motile for non-invasive cells harbouring high basal activity.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Epithelial Protein Lost in Neoplasm alpha (Eplin-alpha) is transcriptionally regulated by G-actin and MAL/MRTF coactivators.

Epithelial Protein Lost in Neoplasm alpha is a novel cytoskeleton-associated tumor suppressor whose expression inversely correlates with cell growth, motility, invasion and cancer mortality. Here we show that Eplin-alpha transcription is regulated by actin-MAL-SRF signalling. Upon signal induction, the coactivator MAL/MRTF is released from a repressive complex with monomeric actin, binds the transcription factor SRF and activates target gene expression. In a transcriptome analysis with a combination of actin binding drugs which specifically and differentially interfere with the actin-MAL complex (Descot et al., 2009), we identified Eplin to be primarily controlled by monomeric actin. Further analysis revealed that induction of the Eplin-alpha mRNA and its promoter was sensitive to drugs and mutant actins which stabilise the repressive actin-MAL complex. In contrast, the Eplin-beta isoform remained unaffected. Knockdown of MRTFs or dominant negative MAL which inhibits SRF-mediated transcription impaired Eplin-alpha expression. Conversely, constitutively active mutant actins and MAL induced Eplin-alpha. MAL and SRF were bound to a consensus SRF binding site of the Eplin-alpha promoter; the recruitment of MAL to this region was enhanced severalfold upon induction. The tumor suppressor Eplin-alpha is thus a novel cytoskeletal target gene transcriptionally regulated by the actin-MAL-SRF pathway, which supports a role in cancer biology.

Extreme temperature episodes and mortality in Yakutsk, East Siberia.

INTRODUCTION: Although the health impacts of heat waves and, to a lesser extent, cold spells in big cities in moderate climates have been well documented, little is known about the same impacts in the circumpolar region. An epidemiological study in an Arctic town presents considerable difficulties for the statistician because of small population sizes. When daily mortality counts are mostly 0, 1 or 2, they are not normally distributed and do not fit the independence assumption. The aim of this study was to take these difficulties into account and assess the impacts of extreme temperature events on mortality rates in Yakutsk, a city with a strongly continental climate, situated near the north pole. METHOD: Long-term distributions of daily mean temperatures were analyzed for identification of heat waves and cold spells during the study period of 1999 to 2007. The authors investigated daily mortality from all non-accidental causes, coronary heart disease and cerebrovascular causes among the age groups 30-64 years and 65 years and over. Statistical analysis was in two steps. Step 1 involved Student's t-tests of samples, which consisted of cumulative mortalities during each heat wave. This provided a measure of the average health effect of all identified heat waves, and the same analysis was performed separately for cold spells. At Step 2, the authors compared the observed cumulative mortality during each individual temperature wave with expected seasonal mortality, using chi(2) tests. RESULTS: The analysis of the impacts of six heat waves and three cold spells provided sufficient evidence that cardiovascular and non-accidental mortalities increased in Yakutsk during both heat waves and cold spells. The magnitude of established health effects was approximately the same for heat and cold. No significant differences were found between the two analyzed age groups in terms of relative excess mortality. Coronary heart disease mortality increased more than two-fold during some of the identified temperature waves, while non-accidental mortality increased by approximately 50%. The time lags between the temperature wave and observed increase in mortality varied between 8 and 14 days, which indicated that the health effects of temperature extremes were delayed rather than immediate. The evidence obtained of the effects of temperature waves on cerebrovascular mortality was not conclusive. Addressing the methodological implications of dealing with small cities, the authors linked the sensitivity of the applied statistical tests to arithmetic means and relative standard deviations of daily death counts, and to the duration of temperature waves. CONCLUSIONS: The proposed methodology can be applied in other medium-sized towns (populations >200,000, approximately); however, only large relative increases in mortality will be statistically significant. For example, relative risks of less than 2.0 for coronary disease mortality and 1.4 for non-accidental mortality are likely to be non-significant.

Chromatin-Associated Membraneless Organelles in Regulation of Cellular Differentiation.

Membrane-free intracellular biocondensates are enclosures of proteins and nucleic acids that form by phase separation. Extensive ensembles of nuclear "membraneless organelles" indicate their involvement in genome regulation. Indeed, nuclear bodies have been linked to regulation of gene expression by formation of condensates made of chromatin and RNA processing factors. Important questions pertain to the involvement of membraneless organelles in determining cell identity through their cell-type-specific composition and function. Paraspeckles provide a prism to these questions because they exhibit striking cell-type-specific patterns and since they are crucial in embryogenesis. Here, we outline known interactions between paraspeckles and chromatin, and postulate how such interactions may be important in regulation of cell fate transitions. Moreover, we propose long non-coding RNAs (lncRNAs) as candidates for similar regulation because many form foci that resemble biocondensates and exhibit dynamic patterns during differentiation. Finally, we outline approaches that could ascertain how chromatin-associated membraneless organelles regulate cellular differentiation.

Temporal dynamics of fixation duration, saccade amplitude, and viewing trajectory.

The prospective goal of our study is a quantitative estimation of the contribution of various factors and mechanisms during image viewing. In this paper, experimental data about temporal dynamics of eye movement parameters and viewing trajectory are considered. Three images were presented to each subject (n = 12) under two experimental conditions: "free viewing of initial images" and "search for modified regions in previously presented images". Averaged fixation duration and saccade amplitude, as well as type of viewing trajectory were determined in each consequent period of trials having 30 fixation points. Viewing trajectories were classified into three types: (1) scanning, (2) grouped, and (3) mixed. In spite of individual variations (subject and image), several common peculiarities of temporal dynamics of image viewing were revealed. Specifically: (i) fixation duration in the first and last trial periods were less than during the second one; (ii) saccade amplitude had opposite dynamics; (iii) the scanning trajectories dominated in the first and last periods as compared with the second one; (iv) the mixed and grouped trajectories are more pronounced in the second period; (v) independent of their temporal consequence, the periods with maximal fixation duration differed from those with minimal duration by saccade amplitude and dominating viewing trajectories.

BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis.

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/ß-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.

Scanpaths of Complex Image Viewing: Insights From Experimental and Modeling Studies.

From the first works of Buswell, Yarbus, and Noton and Stark, the scan path for viewing complex images has been considered as a possible key to objective estimation of cognitive processes and their dynamics. However, evidences both pro and con were revealed in the modern research. In this article, the results supporting the Yarbus-Stark concept are presented. In psychophysical tests, two types of images (three paintings from Yarbus` works and four textures) were used with two instructions, namely, "free viewing" and "search for modified image regions." The focus of the analysis of experimental results and modeling has been given to local elements of the scan path. It was shown that each parameter used (square of viewing area, S; distance between center of mass of viewing area and image center, R; parameter Xi, based on duration of the current fixation and angle between preceding and following saccades), reflects the specificity of both visual task and image properties. Additionally, the return gaze fixations which have a set of specific properties and mainly address to the areas of interest on image were revealed. Evidently these facts can be formalized in an advanced mathematical model as additional instrument to study the mechanisms of complex image viewing.

Myocardin related transcription factors are required for coordinated cell cycle progression.

Myocardin related transcription factors A and B (MRTFs) activate serum response factor-driven transcription in response to Rho signaling and changes in actin dynamics. Myocardin and MRTFs have been implicated in anti-proliferative effects on a range of cell types. The precise mechanisms, however, remained elusive. We employed double knockdown of MRTF-A and MRTF-B in NIH 3T3 fibroblasts to evaluate its effects on cell cycle progression and proliferation. We show that transient depletion of MRTFs conveys a modest anti-proliferative effect and impinges on normal cell cycle progression, resulting in significantly shortened G 1 phase and slightly extended S and G 2 phase under normal growth conditions. Under serum-starved conditions we observed aberrant entry into the S and G 2 phases without subsequent cell division. This was accompanied by downregulation of cyclin-CDK inhibitors p27Kip1, p18Ink4c and 19Ink4d as well as upregulation of p21Waf1 and cyclin D1. Extended knockdown led to increased formation of micronuclei, while cells stably depleted of MRTFs tend to become aneuploid and polyploid. Thus, MRTFs are required for accurate cell cycle progression and maintenance of genomic stability in fibroblast cells.

Myocardin-related transcription factor A regulates expression of Bok and Noxa and is involved in apoptotic signalling.

The myocardin-related transcription factor MAL/MRTF-A relates changes in G-actin to SRF-mediated gene expression. The set of G-actin-controlled SRF target genes includes components of the cytoskeleton and cell migration machinery as well as various signal transducers. Our previous screen for G-actin regulated genes identified a number of targets with well-established anti-proliferative and proapoptotic functions. Here, we report that the two proapoptotic Bcl-2 family members Bok and Noxa/Pmaip are directly transcriptionally induced by activated MAL and upon activation of the actin-MAL-SRF pathway. This activation depends on MRTFs and is sensitive to the actin drug latrunculin but insensitive to p53 depletion. A cis-regulatory element comprising a CArG-like box in the Bok promoter is required and inducibly recruits MAL and SRF. Moreover, MAL/MRTF-dependent transcriptional activity and target gene expression is upregulated upon stimulation of fibroblasts with TNF and staurosporin. This is accompanied by nuclear accumulation of MRTFs. The results suggest a role for MAL in TNF signaling and implicate the MAL-SRF transcription module in regulating the proapoptotic Bcl-2 family network.

The OPCML tumor suppressor functions as a cell surface repressor-adaptor, negatively regulating receptor tyrosine kinases in epithelial ovarian cancer.

UNLABELLED: Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy, and its molecular basis is poorly understood. We previously demonstrated that opioid binding protein cell adhesion molecule (OPCML) was frequently epigenetically inactivated in epithelial ovarian cancers, with tumor suppressor function in vitro and in vivo. Here, we further show the clinical relevance of OPCML and demonstrate that OPCML functions by a novel mechanism in epithelial ovarian cancer cell lines and normal ovarian surface epithelial cells by regulating a specific repertoire of receptor tyrosine kinases: EPHA2, FGFR1, FGFR3, HER2, and HER4. OPCML negatively regulates receptor tyrosine kinases by binding their extracellular domains, altering trafficking via nonclathrin-dependent endocytosis, and promoting their degradation via a polyubiquitination-associated proteasomal mechanism leading to signaling and growth inhibition. Exogenous recombinant OPCML domain 1-3 protein inhibited the cell growth of epithelial ovarian cancers cell in vitro and in vivo in 2 murine ovarian cancer intraperitoneal models that used an identical mechanism. These findings demonstrate a novel mechanism of OPCML-mediated tumor suppression and provide a proof-of-concept for recombinant OPCML protein therapy in epithelial ovarian cancers. SIGNIFICANCE: The OPCML tumor suppressor negatively regulates a specific spectrum of receptor tyrosine kinases in ovarian cancer cells by binding to their extracellular domain and altering trafficking to a nonclathrin, caveolin-1­associated endosomal pathway that results in receptor tyrosine kinase polyubiquitination and proteasomal degradation. Recombinant OPCML domain 1-3 recapitulates this mechanism and may allow for the implementation of an extracellular tumor-suppressor replacement strategy.