Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

<p>Abstract</p> <p>Background</p> <p>Treatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells). We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells.</p> <p>Results</p> <p>The activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC₅₀= 78 μM). UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS). This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin) and BDNF (brain derived neurotrophic factor).</p> <p>Conclusion</p> <p>The present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein kinase cascade.</p

Совершенствование управления персоналом с использованием программных систем на примере Нефтеюганского филиала АО «ССК»

Объектом исследования является: программная система управления персоналом организации Нефтеюганского филиала АО "Сибирская Сервисная Компания". Цель работы – выявление особенностей системы программного обеспечения в управление персоналом в НФ АО "ССК" и разработка мероприятий по повышению эффективности работы с персоналом с использованием нового программного обеспечения. В результате исследования:предложена программная система "Босс-кадровик" для совершенствования процесса управления персоналом на анализируемом предприятии. Актуальность темы состоит в оптимизации процесса управления персоналом, за счет внедрения программной системы для исследуемого предприятия Нефтеюганского филиала АО "ССК".The subject of the study is: a software system for personnel management of the Nefteyugansk branch of JSC Siberian Service Company. The purpose of the work is to identify the features of the software system in personnel management in the NF of "SSK" and to develop measures to improve the efficiency of work with personnel using new software. As a result of the research: the "Boss-kadrovik" software system was proposed to improve the process of personnel management at the analyzed enterprise. The topicality of the topic is to optimize the process of personnel management, through the introduction of a software system for the company under study in the Nefteyugansk branch of SSK

Genomic landscape, polymorphism and possible LINE-associated delivery of G-Quadruplex motifs in the bovine genes

ABSTRACTG-Quadruplex structures are non-B DNA structures that occur in regions carrying short runs of guanines. They are implicated in several biological processes including transcription, translation, replication and telomere maintenance as well as in several pathological conditions like cancer and thus they have gained the attention of the scientific community. The rise of the –omics era significantly affected the G-quadruplex research and the genome-wide characterization of G-Quadruplexes has been rendered a necessary first step towards applying genomics approaches for their study. While in human and several model organisms there is a considerable number of works studying genome-wide the DNA motifs with potential to form G-quadruplexes (G4-motifs), there is a total absence of any similar studies regarding livestock animals. The objectives of the present study were to provide a detailed characterization of the bovine genic G4-motifs’ distribution and properties and to suggest a possible mechanism for the delivery of G4 motifs in the genes. Our data indicate that the distribution of G4s within bovine genes and the annotation of said genes to Gene Ontology terms are similar to what is already shown for other organisms. By investigating their structural characteristics and polymorphism, it is obvious that the overall stability of the putative quadruplex structures is in line with the current notion in the G4 field. Similarly to human, the bovine G4s are overrepresented in specific LINE repeat elements, the L1_BTs in the case of cattle. We suggest these elements as vehicles for delivery of G4 motifs in the introns of the bovine genes. Lastly, it seems that a basis exists for connecting traits of agricultural importance to the genetic variation of G4 motifs, thus, cattle could become an interesting new model organism for G4-related genetic studies.</jats:p

Immunomodulatory Properties of Sweet Whey-Derived Peptides in THP-1 Macrophages

Sweet whey (SW), a by-product of cheese production, has potential immunomodulatory properties that could be beneficial in preventing inflammation-related diseases. This study investigated the effects of SW derived from bovine, caprine, ovine, or an ovine/caprine mixture of milk on inflammation-related gene expression in THP-1-derived macrophages, both with and without LPS stimulation. Cells were treated with SW-D-P3 (a fraction smaller than 3 kDa produced by in vitro digestion), and the expression of inflammation-related genes was assessed using quantitative PCR. Results showed that the expression of TLR2 and ICAM1 was attenuated in non-LPS-stimulated macrophages treated with SW-D-P3, regardless of animal origin. Moreover, the expression of TLR4, IL1B, and IL6 was decreased and the expression of an NF-&kappa;B subunit RELA and CXCL8 was elevated in a subset of samples treated with SW-D-P3, depending on the milk source. In LPS-challenged cells, the expression of CXCL8 was upregulated and the expression of IRF5 and TNFRSF1A was downregulated in SW-D-P3-treated cells, regardless of animal origin. On the other hand, a number of inflammation-related genes were differentially expressed depending on the animal origin of the samples. Moreover, the higher IL10 expression observed in cells treated with ovine/caprine SW-D-P3 compared to those treated with SW-D-P3 of bovine, caprine, or ovine origin suggests an anti-inflammatory response, in which alternatively activated macrophages (M2 polarization phenotype) may participate. Overall, these findings suggest that incorporating SW into the food industry, either as a standalone ingredient or supplement, may help to prevent inflammation-related diseases

Effect of Milk Origin and Seasonality of Yogurt Acid Whey on Antioxidant Activity before and after In Vitro Gastrointestinal Digestion

Yogurt acid whey (YAW) is a by-product of Greek strained yogurt production. The disposal of YAW constitutes an environmental problem, and given the increasing demand of Greek yogurt worldwide, its handling is a challenge. However, whey-derived peptides, resulting from microbial fermentation as well as those resulting from further hydrolysis during the digestion process, have been linked to enhanced biological activities. In this study, the antioxidant capacity of 33 samples of YAW obtained from Greek dairy companies of bovine, ovine or caprine origin was investigated using both cell-free and cell-based assays. The YAW samples, their in vitro digestion products (YAW-Ds) and a fraction of the digests (less than 3 kDa; YAW-D-P3) were assessed using four biochemical assays, namely ORAC, ABTS, FRAP and P-FRAP. Our data revealed a higher antioxidant capacity for digested samples compared with undigested samples, with all four methods. ORAC values after in vitro digestion were higher for the ovine samples compared to their bovine (YAW-D and YAW-D-P3) and caprine (YAW-D-P3) counterparts. Furthermore, the YAW-D-P3 fraction derived from samples collected in the summer months exhibited higher ORAC values when compared to the respective fraction from the winter months&rsquo; samples. The cellular antioxidant activity of ovine YAW-D-P3 was improved in H2O2-treated HT29 cells compared to the control H2O2-treated cells. However, YAW-D-P3 could not trigger either the pathways involving the transcription factors NF-&kappa;B or NFE2L2 or the gene expression of SOD1, CAT and HMOX1 in LPS-challenged THP-1-derived macrophages. These results suggest that YAW, and particularly YAW from ovine origin, could be used as a natural source for its antioxidant potential in human and animal nutrition

Evaluation of In Vitro Antihypertensive and Anti-Inflammatory Properties of Dairy By-Products

Sweet whey (SW) and yogurt acid whey (YAW) are dairy by-products of the cheese-making process and Greek-style yogurt production, respectively. Both of them are considered pollutants with huge volumes of SW and YAW produced due to the growing demand for dairy products worldwide. Moreover, whey-derived peptides, resulting from fermentation as well as from further hydrolysis during digestion, have been associated with various biological activities. In the present study, the angiotensin-converting enzyme (ACE)-inhibitory activity of 48 SW samples and 33 YAW samples from bovine, ovine, caprine, and ovine/caprine milk obtained were evaluated. Additionally, the SW and YAW digestates and two of their fractions (smaller than 10 kDa, SW-D-P10 and YAW-D-P10, and smaller than 3 kDa, SW-D-P3 and YAW-D-P3), which were obtained after in vitro digestion and subsequent ultrafiltration, were also subjected to evaluation. Our data indicated that the D-P10 and D-P3 fractions exhibited higher ACE-inhibitory activity compared to the corresponding values before digestion. The ACE-inhibitory capacity after in vitro digestion was higher for the ovine SW samples compared to their bovine and caprine counterparts. The effect of the D-P3 fraction on the inhibition of nitric oxide (NO) production and the expression of a selected panel of immune-response-related genes in LPS-stimulated RAW 264.7 macrophages was also evaluated. Fractions from both dairy by-products inhibited NO production in LPS-stimulated RAW 264.7 cells. Especially, ovine SW-D-P3 showed a strong NO inhibitory activity and suppressed inducible nitric oxide synthase (Nos2) mRNA levels. However, YAW-D-P3 could not trigger neither the gene expression of inflammatory macrophage mediators Nos2 and cyclooxygenase-2 (Ptgs2) nor tumor necrosis factor-&alpha; (Tnf) and interleukin 6 (Il6) in LPS-stimulated murine macrophages regardless of animal origin. These findings suggest that in vitro digestion could enhance the production of ACE-inhibitory peptides in both dairy by-products, while SW from ovine origin displays higher potential as an anti-inflammatory agent, effectively preventing excessive NO production

aniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin

AbstractSpecific capture of chromatin fractions with distinct and well-defined features has emerged as both challenging and a key strategy towards a comprehensive understanding of genome biology. In this context, we developed aniFOUND (accelerated native isolation of factors on unscheduled nascent DNA), an antibody-free method, which can label, capture, map and characterise nascent chromatin fragments that are synthesized in response to specific cues outside S-phase. We used the ‘unscheduled’ DNA synthesis (UDS) that takes place during the repair of UV-induced DNA lesions and coupled the captured chromatin to high-throughput analytical technologies. By mass-spectrometry we identified several factors with no previously known role in UVC-DNA damage response (DDR) as well as known DDR proteins. We experimentally validated the repair-dependent recruitment of the chromatin remodeller RSF1 and the cohesin-loader NIPBL at sites of UVC-induced photolesions. Developing aniFOUND-seq, a protocol for mapping UDS activity with high resolution, allowed us to monitor the landscape of UVC repair-synthesis events genome wide. We further resolved repair efficacy of the rather unexplored repeated genome, in particular rDNA and telomeres. In summary, aniFOUND delineates the proteome composition and genomic landscape of chromatin loci with specific features by integrating state-of-the-art ‘omics’ technologies to promote a comprehensive view of their function.</jats:p