Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

Transcriptional elongation of most eukaryotic genes by RNA polymerase II requires the kinase activity of the positive transcription elongation factor b (P-TEFb). The catalytically active P-TEFb complex becomes inactive when sequestered into the large complex by the cooperative actions of 7SK snRNA and HEXIM1. In this study, we report that HEXIM1 forms oligomers in cells. This oligomerization is mediated by its predicted coiled-coil region in the C-terminal domain and 7SK snRNA that binds a basic region within the central part of HEXIM1. Alanine-mutagenesis of evolutionary conserved leucines in the coiled-coil region and the digestion of 7SK snRNA by RNase A treatment prevent this oligomerization. Importantly, mutations of the N-terminal part of the coiled-coil region abrogate the ability of HEXIM1 to bind and inhibit P-TEFb. Finally, the formation of HEXIM1 oligomers via the C-terminal part of the coiled-coil or basic regions is critical for the inhibition of transcription. Our results suggest that two independent regions in HEXIM1 form oligomers to incorporate P-TEFb into the large complex and determine the inhibition of transcriptional elongation

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans

[Pomen molekul MHC pri imunoterapiji tumorjev]

Tumour immunontherapy attempts to use the specificity and capability of the immune system to kill malignant cells with a minimum damage to normal tissue. Increasing knowledge of the identity of tumour antigens should help us design more effective therapeutic vaccines. Increasing evidence has demonstrated that MHC class II molecules and CD4+T cells play important roles in generating and maintaining antitumour immune responses in animal models. These data suggest that be necessary to involve both CD+ and CD+T cells for more effective antitumour therapy. Novel strategies have been developed for enhancing T cell responses against cancer by prolonging antigen prersentation of denritic cells to T cells, by the inclusion of MHC class II-restricted tumour antigens and by genetically modifying tumour cells to present antigen to T lymphocytes directly. Vaccines against cancers aim to induce tumour-specific effector T cells that can reduce tumour mass and induce development of tumour-specific T cell memory, that can control tumour relapse.Izhodišča. Imunoterapija tumorjev izrablja sposobnost imunskega sistema, da se specifično ubija tumorske celice, pri tem pa minimalno poškoduje normalno tkivo. Vedno večje poznavanje tumorskih celic, pri tem pa minimalno poškoduje normalno tkivo. Vedno večje poznavanje tumorskih antigenov prispeva k načrtovanju bolj učinkovitih terapevtskih cepiv. Študije narejene na živalskih modelih so pokazale, da so poleg molekul MHC I in celic CD8+ T pri nastanku in vzdrževanju imunskega odziva proti tumorjem pomembne tudi molekuleMHC II in celice CD4+ T. Rezultati nakazujejo, da bo za učinkovito protitumorsko cepivo potrebno aktivirati tako celice CD4+ kot tudi CD8+ T. V zadnjem času so se razvile nove strategije za okrepitev T celičnega odgovora proti raku, ki izrabljajo sposobnost dendritiènih celic, da delujejo kot antigeni. Z vključevanjem tumorskih antigenov specifičnih za molekule MHC II in z genetičnim spreminjanem tumorskih celic, da delujejo kot antigeni (antigen-predstavitvene celice), lahko podaljšamo predstavljanje antigenov celicam T preko dendritičnih celic. Zaključki. Z združitvijo različnih pristopov bi lahko naredili učinkovito protitumorsko cepivo, ki bi vzpodbudilo delovanje za tumor specifičnih celic T, te pa bi ubile tumorske celice. S tem bi zmanjšale obseg tumorja, obenem pa bi vzpodbudile tudi nastanek za tumor specifičnega celičnega spomina T, ki bi omejil ali preprečil ponovni nastanek tumorja

Setting up a gene expression study for tissue cells by method of quantitative real-time PCR

Gene expression analysis by quantitative reverse transcription polymerase chain reaction (RT-qPCR) allows accurate and sensitive measurment of gene expression levels. However, a series of steps needs to be taken to ensure the relevance, accuracy, correct interpretation and repeatability of the RT-qPCR experiment. We describe here a simple experiment of determining relative gene expression for caspase 8 gene in chicken chondrocytes treated with an apoptosis inducing compound 5-fluorouracil. We use this example to point out some important guidelines in setting up a gene expression study in tissue cells, analyzing and interpreting the results and reporting on the findings.Analiza izražanja genov z metodo reverznega pomnoževanja in kvantitativne verižne reakcije s polimerazo v realnem času (RT-qPCR) omogoča natančno in občutljivo določanje nivoja izražanja tarčnih genov. Vendar je za zagotavljanje točnosti, natančne interpretacije in ponovljivosti eksperimenta, predvsem pa relevantnosti rezultatov, pomembno natančno poznavanje korakov postopka. V tem prispevku opisujemo enostaven poskus, v katerem smo v celični liniji kokošjih hondrocitov, ki smo jih tretirali s spojino 5-fluorouracil, ki sproži apoptozo, določili relativno izražanje gena, ki kodira encim kaspazo 8.Ta primer smo uporabili za izpostavitev nekaterih pomembnih smernic pri oblikovanju študije izražanja genov v tkivnih celicah, analizi in interpretaciji rezultatov ter oblikovanju zaključkov

Comparison of methods for relative quantification of gene expression using real-time PCR

Quantitative real-time PCR (qPCR) has become a widely used tool for quantifying gene expression. Several methods for relative quantification have been developed, enabling rapid and reliable detection and quantification of specific nucleic acids. These methods, based on qPCR include: the standard curve method, the efficiency calibrated method and the 2[-deltadelta]Cq method. Here we analyzed if these three methods generate comparable results. To evaluate their performance, we analyzed the expression of the nuclease gene MS53_0284 from Mycoplasma synoviae type strain WVU 1853 during in vitro infection of CEC-32 cells, using qPCR. As determined, all three methods generated comparable and reliable results when all necessary conditions were fulfiled. Also, the efficiency calibrated and the standard curve methods were more suitable for quantifying small differences in relative gene expression than the 2[-deltadelta]Cq method.Metoda kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) je postala najpogosteje uporabljen način analize izražanja genov. Razvitih je bilo več metod za relativno kvantifikacijo genske ekspresije, ki omogočajo hitro in zanesljivo detekcijo ter kvantifikacijo specifičnih nukleinskih kislin. Mednje spadajo: metoda z umeritveno krivuljo, metoda z upoštevanjem učinkovitosti pomnoževanja in metoda po enačbi 2[-deltadelta]Cq. V tej študiji smo z analizirajem izražanja gena MS53_0284, ki kodira nukleazo pri bakteriji Mycoplasma synoviae WVU 1853, po okužbi celic CEC-32 in vitro s qPCR preverili primerljivost rezultatov, dobljenih z uporabo omenjenih metod. Pokazali smo, da z upoštevanjem potrebnih pogojev z omenjenimi metodami pridobimo primerljive rezultate ter da sta metoda z umeritveno krivuljo in metoda z upoštevanjem učinkovitosti pomnoževanja primernejši za ugotavljanje majhnih razlik v izražanju genov kot metoda po enačbi 2[-deltadelta]Cq

Phenotypic Characterization of Mycoplasma synoviae Induced Changes in the Metabolic and Sensitivity Profile of In Vitro Infected Chicken Chondrocytes

In infectious synovitis caused by Mycoplasma synoviae chicken chondrocytes (CCH) may come into direct contact with these bacteria that are also capable of invading CCH in vitro. In this study, phenotype microarrays were used to evaluate the influence of Mycoplasma synoviae on the global metabolic activity of CCH. Therefore, CCH were cultured in the presence of 504 individual compounds, spotted in wells of 11 phenotype microarrays for eukaryotic cells, and exposed to Mycoplasma synoviae membranes or viable Mycoplasma synoviae. Metabolic activity and sensitivity of normal cells versus infected cells were evaluated. Metabolic profiles of CCH treated with viable Mycoplasma synoviae or its membranes were significantly different from those of CCH alone. CCH treated with Mycoplasma synoviae membranes were able to use 48 carbon/nitrogen sources not used by CCH alone. Treatment also influenced ion uptake in CCH and intensified the sensitivity to 13 hormones, 5 immune mediators, and 29 cytotoxic chemicals. CCH were even more sensitive to hormones/immune mediators when exposed to viable Mycoplasma synoviae. Our results indicate that exposure to Mycoplasma synoviae or its membranes induces a wide range of metabolic and sensitivity modifications in CCH that can contribute to pathological processes in the development of infectious synovitis

The Combination of Serum and Oral Fluid Cortisol Levels and Welfare Quality Protocol® for Assessment of Pig Welfare on Intensive Farms

Animal welfare is important; therefore, veterinarians and other animal welfare experts try to use different tools for pig welfare assessment. Several welfare protocols are available for pig welfare assessment, and one of the most used is Welfare Quality (WQ) protocol®. Elevated values of cortisol can be indicative of stress and, therefore, poor welfare. Our aim was to assess the correlation between serum cortisol levels from individual samples and oral fluid cortisol levels in group samples with the grades received for pig welfare using the WQ protocol®. Samples were taken at six different commercial pig farms. Animals were divided into age-dependent categories: 5 weeks old (w/o); 7 w/o; 9 w/o; 11 w/o weaners; fatteners; and breeding sows (10 pigs/category). Cortisol was determined in individual sera and group samples of oral fluid (OF), and was compared to values considered to be physiological. Based on WQ protocol® answers, five farms’ welfare level was deemed acceptable, and one was enhanced. Four out of 29 sera and 5 out of 30 OF samples were considered physiological, while in most other samples it was elevated. The correlation between cortisol levels in sera, OF, and WQ protocol® scores was not statistically significant. The cortisol level in OF should be just one of the welfare indicators, i.e., alongside the WQ protocol® filled out by a welfare expert

Haematological profiles of pigs of different age in relation to the presence or absence of porcine reproductive and respiratory virus, porcine circovirus type 2 and hepatitis E virus

This study was aimed to assessing haematological parameters in pigs of different age categories from six farms differing in the presence or absence of selected pathogens. The following categories were included: 5 age groups of growers, fatteners and breeding sows. Individual blood samples for determining complete blood count and white blood cell differential count were taken. Group samples of oral fluid and faeces were collected from each farm and tested for detection of Porcine Reproductive and Respiratory Virus (PRRSV), Porcine Circovirus Type 2 (PCV2), and Hepatitis E Virus (HEV) using PCR, RT-PCR, and qRT-PCR protocols. On farms free of PRRSV, PCV2 and HEV, the age of pigs had significant effect on: white blood cell count (WBC), haemoglobin concentration (Hb), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, platelet count (PLT), percentage of neutrophils, lymphocytes and eosinophils and absolute numbers of neutrophils, lymphocytes, monocytes, basophils and ‘large unstained cells’ (LUCs). On positive farms all observed blood parameters except the percentage of LUCs were significantly affected by the age. The percentages of lymphocytes, mean corpuscular volume (MCV) and haematocrit were significantly lowered by PRRSV, while WBC, PLT, percentage and absolute numbers of neutrophils, basophils and LUCs were increased. Nine-week-old pigs with PCV2 had significantly lower red blood cell count, PLT, percentage and absolute numbers of monocytes and LUCs, and significantly higher MCV, MCH, percentage of neutrophils and percentage and absolute number of eosinophils than 9-week-old pigs with PCV2 and HEV. Age-related changes in haematological parameters occurred in PCR negative and positive farms.HIGHLIGHTS Age-related changes in haematological parameters were present in PCR negative farms and in PCR positive farms in relation to selected pathogens. Values for several haematological parameters in 9-week-old pigs with a monoinfection differed significantly from those in pigs of the same age with two viral infections. To interpret results of haematological analyses correctly, it is important to consider the haematological analyser used, the pig age and, health history, and clinical data