Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

BACKGROUND: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. RESULTS: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types. CONCLUSION: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells

Software and hardware platform for testing of Automatic Generation Control algorithms

Development and implementation of new Automatic Generation Control (AGC) algorithms requires testing them on a model that adequately simulates primary energetic, information and control processes. In this article an implementation of a test platform based on HRTSim (Hybrid Real Time Simulator) and SCADA CK-2007 (which is widely used by the System Operator of Russia) is proposed. Testing of AGC algorithms on the test platform based on the same SCADA system that is used in operation allows to exclude errors associated with the transfer of AGC algorithms and settings from the test platform to a real power system. A power system including relay protection, automatic control systems and emergency control automatics can be accurately simulated on HRTSim. Besides the information commonly used by conventional AGC systems HRTSim is able to provide a resemblance of Phasor Measurement Unit (PMU) measurements (information about rotor angles, magnitudes and phase angles of currents and voltages etc.). The additional information significantly expands the number of possible AGC algorithms so the test platform is useful in modern AGC system developing. The obtained test results confirm that the proposed system is applicable for the tasks mentioned above

Lymphatic exosomes promote dendritic cell migration along guidance cues

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.Peer reviewe

Отсылочные нормы таможенного права в таможенных кодексах ТС/EAЭC и EC: сравнительный анализ практики применения

Актуальность темы исследования обусловлена необходимостью универсального изучения отсылочных норм, так как это является одним из неотъемлемых элементов научного анализа правоприменительного и правотворческого процессов.В процессе исследования была рассмотрена структура ТК ТС и ТК ЕАЭС,были выделены основные изменения, изучен правотворческий и правоприменительный процесс использования отсылочного способа изложения элементов норм права, проанализирована динамика и характер использования отсылочных норм права в таможенных кодексах ТС, ЕАЭС и ЕС 2000-х - 2010-х годов, были определены перспективы развития отсылочных норм права в таможенном законодательстве государств-членов ЕАЭС.The relevance of the research topic is due to the need for universal study of reference rules, as this is one of the integral elements of the scientific analysis of law enforcement and law-making processes. During the study, the structure of the CC of the CU and CC of the EAEU was considered, the main changes were identified, the law-making and law enforcement process of using the reference method of presenting the elements of the law, analyzed the dynamics and nature of the use of reference rules of law in the Customs Codes of the CU, EAEU and the EU of the 2000s-2010s, the prospects for the development of reference rules of law in the customs legislation of the EAEU member states were determined

Arp3 controls the podocyte architecture at the kidney filtration barrier

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion