Increase In Relative Expression Research Articles

Regulation of dye-decolorizing peroxidase gene expression in Pleurotus ostreatus grown on glycerol as the carbon source.

Dye-decolorizing peroxidases (DyPs) (E.C. 1.11.1.19) are heme peroxidases that catalyze oxygen transfer reactions similarly to oxygenases. DyPs utilize hydrogen peroxide (H2O2) both as an electron acceptor co-substrate and as an electron donor when oxidized to their respective radicals. The production of both DyPs and lignin-modifying enzymes (LMEs) is regulated by the carbon source, although less readily metabolizable carbon sources do improve LME production. The present study analyzed the effect of glycerol on Pleurotus ostreatus growth, total DyP activity, and the expression of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), via real-time RT-qPCR, monitoring the time course of P. ostreatus cultures supplemented with either glycerol or glucose and Acetyl Yellow G (AYG) dye. The results obtained indicate that glycerol negatively affects P. ostreatus growth, giving a biomass production of 5.31 and 5.62 g/L with respective growth rates (micra; m) of 0.027 and 0.023 h-1 for fermentations in the absence and presence of AYG dye. In contrast, respective biomass production levels of 7.09 and 7.20 g/L and growth rates (μ) of 0.033 and 0.047 h-1 were observed in equivalent control fermentations conducted with glucose in the absence and presence of AYG dye. Higher DyP activity levels, 4,043 and 4,902 IU/L, were obtained for fermentations conducted on glycerol, equivalent to 2.6-fold and 3.16-fold higher than the activity observed when glucose is used as the carbon source. The differential regulation of the DyP-encoding genes in P. ostreatus were explored, evaluating the carbon source, the growth phase, and the influence of the dye. The global analysis of the expression patterns throughout the fermentation showed the up- and down- regulation of the three Pleos-dyp genes evaluated. The highest induction observed for the control media was that found for the Pleos-dyp1 gene, which is equivalent to an 11.1-fold increase in relative expression (log2) during the stationary phase of the culture (360 h), and for the glucose/AYG media was Pleos-dyp-4 with 8.28-fold increase after 168 h. In addition, glycerol preferentially induced the Pleos-dyp1 and Pleos-dyp2 genes, leading to respective 11.61 and 4.28-fold increases after 144 h. After 360 and 504 h of culture, 12.86 and 4.02-fold increases were observed in the induction levels presented by Pleos-dyp1 and Pleos-dyp2, respectively, in the presence of AYG. When transcription levels were referred to those found in the control media, adding AYG led to up-regulation of the three dyp genes throughout the fermentation. Contrary to the fermentation with glycerol, where up- and down-regulation was observed. The present study is the first report describing the effect of a less-metabolizable carbon source, such as glycerol, on the differential expression of DyP-encoding genes and their corresponding activity.

The influence of silver nanoparticles on the process of epithelial transition in the context of cancer metastases.

Exposure to nanoparticles (NPs) can occur in a variety of occupational situations. Ultrafine particles of natural and anthropological origin toxicity has been described in epidemiological studies. Meanwhile, the risks associated with NPs exposure are not comprehensively assessed. A wide spectrum of NPs toxicity has been demonstrated, mainly through the induction of oxidative stress and inflammatory mediators. Among the newly described mechanisms of NPs toxicity is the induction of fibrosis via the epithelial-mesenchymal transition (EMT), which is also a key mechanism of cancer metastasis. The effect of NPs on EMT in the context of metastasis has not been sufficiently described so far, and the results of studies do not allow for the formulation of unambiguous conclusions. Therefore, the aim of the work was to determine the biological activity of silver NPs against MDA-MB-436 triple-negative breast cancer cells. Exposure to nanoparticles (NPs) can occur in a variety of occupational situations. Ultrafine particles of natural and anthropological origin toxicity has been described in epidemiological studies. Meanwhile, the risks associated with NPs exposure are not comprehensively assessed. A wide spectrum of NPs toxicity has been demonstrated, mainly through the induction of oxidative stress and inflammatory mediators. Among the newly described mechanisms of NPs toxicity is the induction of fibrosis via the epithelial-mesenchymal transition (EMT), which is also a key mechanism of cancer metastasis. The effect of NPs on EMT in the context of metastasis has not been sufficiently described so far, and the results of studies do not allow for the formulation of unambiguous conclusions. Therefore, the aim of the work was to determine the biological activity of silver NPs against MDA-MB-436 triple-negative breast cancer cells. Silver nanoparticles (AgNPs) cause a statistically significant increase in relative expression of all tested mesenchymal EMT markers - cadherin 2, vimentin, matrix metalloproteinase 2 and matrix metalloproteinase 9. At the same time, reduction of epithelial cadherin 1 expression was observed. The level of MDA-MB-436 migration and TGF-beta 1 secretion was slighty increased in AgNPs-treated cells, with no influence on invasion potential. Potentially prometastatic effect of AgNPs encourages further work on the safety of nanomaterials. Med Pr Work Health Saf. 2023;74(6):541-8.

Morphological evaluation of the feline placenta correlates with gene expression of vascular growth factors and receptors†.

Placental angiogenesis is critical for normal development. Angiogenic factors and their receptors are key regulators of this process. Dysregulated placental vascular development is associated with pregnancy complications. Despite their importance, vascular growth factor expression has not been thoroughly correlated with placental morphologic development across gestation in cats. We postulate that changes in placental vessel morphology can be appreciated as consequences of dynamic expression of angiogenic signaling agents. Here, we characterized changes in placental morphology alongside expression analysis of angiogenic factor splice variants and receptors throughout pregnancy in domestic shorthair cats. We observed increased vascular and lamellar density in the lamellar zone during mid-pregnancy. Immunohistochemical analysis localized the vascular endothelial growth factor A (VEGF-A) receptor KDR to endothelial cells of the maternal and fetal microvasculatures. PlGF and its principal receptor Flt-1 were localized to the trophoblasts and fetal vasculature. VEGF-A was found in trophoblast cells and associated with endothelial cells. We detected expression of two Plgf splice variants and four Vegf-a variants. Quantitative real-time polymerase chain reaction analysis showed upregulation of mRNAs encoding pan Vegf-a and all Vegf-a splice forms at gestational days 30-35. Vegf-A showed a marked relative increase in expression during mid-pregnancy, consistent with the pro-angiogenic changes seen in the lamellar zone at days 30-35. Flt-1 was upregulated during late pregnancy. Plgf variants showed stable expression during the first two-thirds of pregnancy, followed by a marked increase toward term. These findings revealed specific spatiotemporal expression patterns of VEGF-A family members consistent with pivotal roles during normal placental development.

Genome-Wide Identification and Evolution Analysis of the CYP76 Subfamily in Rice (Oryza sativa).

The CYP76 subfamily, a member of the CYP superfamily, plays crucial roles in the biosynthesis of phytohormones in plants, involving biosynthesis of secondary metabolites, hormone signaling, and response to environmental stresses. Here, we conducted a genome-wide analysis of the CYP76 subfamily in seven AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, and Oryza glumaepatula. These were identified and classified into three groups, and it was found that Group 1 contained the largest number of members. Analysis of cis-acting elements revealed a large number of elements related to jasmonic acid and light response. The gene duplication analysis revealed that the CYP76 subfamily expanded mainly in SD/WGD and tandem forms and underwent strong purifying selection during evolution. Expression pattern analysis of OsCYP76s in various developmental stages revealed that the majority of OsCYP76s exhibit relatively restricted expression patterns in leaves and roots. We further analyzed the expression of CYP76s in O. sativa, japonica, and O. sativa, indica under cold, flooding, drought, and salt abiotic stresses by qRT-PCR. We found that OsCYP76-11 showed a huge increase in relative expression after drought and salt stresses. After flooding stress, OsiCYP76-4 showed a greater increase in expression compared to other genes. CYP76 in japonica and indica showed different response patterns to the same abiotic stresses, revealing functional divergence in the gene family during evolution; these may be the key genes responsible for the differences in tolerance to indica japonica. Our results provide valuable insights into the functional diversity and evolutionary history of the CYP76 subfamily and pave the way for the development of new strategies for improving stress tolerance and agronomic traits in rice.

The immunomodulatory activity of the betulonic acid based compound.

The immunomodulatory activity of a betulonic acid-based compound with furocoumarin (BABCF; 2-azido, 9-N-methylpiperazinomethyl oreozelone) has been investigated. Male C57BL/6 mice (aged 3 months) treated with the cytostatic agent cyclophosphamide (CP) and intact individuals served as experimental models. The expression of genes was studied in bone marrow (IL-12, IL-10, IL-1β, TNF-α, TGF-β, M-CSF, GM-CSF) or in the suspension of peritoneal cells (IL-12, IL-10; as the injection site). The surface markers of T-lymphocytes (CD3, CD4, and CD8) in fractions of venous blood mononuclear cells (MNCs) were determined by means of flow cytometry using antibodies. Histological and morphometric studies were performed to assess the impact of CP and BABCF on the thymus. BABCF caused a pronounced (about 3-fold) increase in relative expression of the GM-KSF gene. BABCF caused a local increase in the expression of IL-12 in the peritoneal cavity cells and restored the relative content of T-lymphocytes in the blood of CP-treated mice treated affecting mainly CD3⁺CD4⁺ lymphocytes. This substance reduced the tissue density of the thymic cortex and thymic medulla in CP-treated mice. Thus, results of this study suggest that BABCF exhibits a stimulating effect on the cellular link of immunity and promotes maintenance of the number of T-lymphocytes in the blood due to their migration from the central organs of the immune system.

Magnetopriming Actuates Nitric Oxide Synthesis to Regulate Phytohormones for Improving Germination of Soybean Seeds under Salt Stress.

In this study, the role of the signalling molecule nitric oxide (NO) in magnetopriming-mediated induction of salinity tolerance in soybean seeds is established. The cross-talk of NO with germination-related hormones gibberellic acid (GA), abscisic acid (ABA) and auxin (IAA) for their ability to reduce the Na+/K+ ratio in the seeds germinating under salinity is highlighted. Salt tolerance index was significantly high for seedlings emerging from magnetoprimed seeds and sodium nitroprusside (SNP, NO-donor) treatment. The NO and superoxide (O2•−) levels were also increased in both of these treatments under non-saline and saline conditions. NO generation through nitrate reductase (NR) and nitric oxide synthase-like (NOS-like) pathways indicated the major contribution of NO from the NR-catalysed reaction. The relative expression of genes involved in the NO biosynthetic pathways reiterated the indulgence of NR in NO in magnetoprimed seeds, as a 3.86-fold increase in expression was observed over unprimed seeds under salinity. A 23.26-fold increase in relative expression of NR genes by the NO donor (SNP) was observed under salinity, while the NR inhibitor (sodium tungstate, ST) caused maximum reduction in expression of NR genes as compared to other inhibitors [L-NAME (N(G)-nitro-L-arginine methyl ester; inhibitor of nitric oxide synthase-like enzyme) and DPI (diphenylene iodonium; NADPH oxidase inhibitor)]. The ratio of ABA/GA and IAA/GA decreased in magnetoprimed and NO donor-treated seeds, suggesting homeostasis amongst hormones during germination under salinity. The magnetoprimed seeds showed low Na+/K+ ratio in all treatments irrespective of NO inhibitors. Altogether, our results indicate that a balance of ABA, GA and IAA is maintained by the signalling molecule NO in magnetoprimed seeds which lowers the Na+/K+ ratio to offset the adverse effects of salinity in soybean seeds.

Local delivery of usnic acid loaded Rhamnolipid vesicles by gelatin / tragacanth gum / montmorillonite/ vanillin cryogel scaffold for expression of osteogenic biomarkers and antimicrobial activity

Treatment of osteomyelitis as a destructive disease with strong, systemic, and high-dose antibiotics always faces tough challenges and severe side effects. The present study aimed to design dual-purpose cryogel nanocomposite scaffolds with antibacterial and bone regenerative properties. The gelatin/tragacanth gum/montmorillonite/vanillin cryogel scaffold (GTM/V) was prepared and enriched with Usnic acid (UA) encapsulated in Rhamnolipid (RH) biosurfactant nanoparticles as a dispersing agent with antioxidant and antimicrobial properties. The physicochemical properties of drug loaded nanoparticles, mechanical, rheological, and biological properties of the cryogel were evaluated. Biological evaluations, including cell proliferation (by MTT assay), cell attachment (by SEM), osteogenic activity (by Alizarin Red staining and alkaline phosphatase assay), and osteogenic gene expression (by quantitative real-time polymerase chain reaction) were studied using MG-63 cells. The antibiofilm effect of the cryogel was studied on Staphylococcus aureus. The results showed the mechanical profile of UA/RH loaded GTM/V scaffold was 3.08 ± 0.20 MPa compressive strength at 70% strain and the elastic modulus was 19.69 ± 1.57 MPa. Biocompatible UA/RH containing cryogel scaffolds showed osteogenic effect through increasing the ALP activity, and the Alizarin-red staining on days 7 and 14 compared to the control group (P < 0.05). The relative increase in expression of bone repair markers, including COL1A1, BGLAP, and SPP1F were shown by RT-PCR. The scaffold had strong antibacterial effects on S. aureus, with an inhibition zone of 1.40 ± 0.21 cm and antibiofilm activity of 43.76 ± 1.65%. In conclusion, our findings support the application of UA/RH loaded GTM/V scaffold for the treatment of osteomyelitis and bone regeneration.

The effect of constant light and melatonin on urethane-induced lung carcinogenesis in mice and expression of clock genes and proteins

Introduction. Circadian rhythms are an adaptational mechanism to day-night cycle. At the cellular level rhythms are supported by oscillations of transcription of Bmal1, 2, Clock and Npas2, Per1-3, Cry1, 2 clock genes; in the organism rhythms are synchronized with melatonin, the "hormone of darkness". Reliable data have been obtained that disruption of biological "clocks" at the cellular and/or organism level are associated with carcinogenesis, but the experimental results in this direction remain incomplete.&#x0D; Aim. Assessment of the continuous lighting and melatonin administration effects on chemically induced carcinogenesis in mice and expression of clock genes and proteins in normal and tumor lung tissue.&#x0D; Materials and methods. Lung tumors were induced by urethane in 120 male SHR mice. Animals were kept at constant (LL) or standard (LD) light; half of the animals received melatonin (MT) daily at 20 mg/l at night with water for 45 weeks of experiment. At the end of the experiment, the frequency and multiplicity of lung tumours of different size were assessed. Expression of clock genes Clock, Bmal1, Cry1 was assessed in tumor samples and normal lung tissue by real-time PCR; protein content of BMAL1, CLOCK, CRY1 and PER2 was determined by immunohistochemistry. Statistical data were processed using the programs MS Excel 2007, GraphPad Prism 6.0 with commonly used statistical criteria.&#x0D; Results. The number of animals with lung tumors was between 89 and 100% in different groups. Large tumors (&gt;2 mm) were more frequently observed in LL group (62 tumors out of 294, 21,1%) than in LD group (59 tumors out of 405, 14,6%, p=0,0245). Administration of exogenous melatonin at constant lighting statistically significantly reduced the frequency of large tumors (28 tumors out of 320, 8.8%, p=0,0001 in comparison with LL). In normal lung tissue of animals kept at constant lighting (LL) the increase in relative expression of clock genes in comparison with LD group was revealed: Bmal1 by 3,1 times (p=0,02) and Cry1 by 3,6 times (p=0,0002). No such differences were found for Clock gene. The relative content of BMAL1 and CLOCK proteins in all experimental conditions was higher in adenomas and adenocarcinomas compared to normal tissue.&#x0D; Conclusion. Constant lighting promotes the development of chemically induced lung tumors, and melatonin administration inhibits carcinogenesis under constant lighting.&#x0D; The content of BMAL1 and CLOCK transcription activator proteins in lung tumors was found to increase in comparison with normal tissue, whereas no increase in the expression level of corresponding genes in tumors was observed.

Effect of constant 2:1 calcium to non-phytate phosphorus ratio over a range of concentrations during starter-grower and finisher phases on performance of broiler chicken

This experiment investigated the effect of 2:1 ratio of calcium (Ca) to non-phytate phosphorus (NPP) over a range of their concentrations on performance of broilers in phase 1 (1–24 days), phase 2 (25–38 days) and the whole period. A total of 900 male broilers were allocated into 60 pens. In phase 1, dietary treatments were high (H, 9.6 g/kg Ca and 4.8 g/kg NPP), moderate (M, 7.6 g/kg Ca and 3.8 g/kg NPP), low (L, 5.6 g/kg Ca and 2.8 g/kg NPP) and very low (VL, 3.6 g/kg Ca and 1.8 g/kg) level of Ca and NPP. H and M diets showed higher average daily feed intake (ADFI) and serum Ca than those of L and VL diets. There was a linear decrease in tibia Ca and serum P and a linear increase in relative expression of SLC20A1 and CALB1 and retention of Ca and P when concentration of Ca and NPP was reduced. H diet resulted in the highest tarsometatarsus strength and feed conversion ratio (FCR). M and L diets showed higher average daily gain (ADG) than those of H and VL diets. VL diet demonstrated the lowest tibia ash. On day 25, treatments H, M, L and VL received, respectively, 4, 3, 2 and 1 types of finisher diets (H, M, L and VL) during phase 2, resulting in a total of 10 treatments; HH, HM, HL, HVL, MM, ML, MVL, LL, LVL and VLVL. HH vs. HM vs. HL vs. HVL showed a linear decrease of ADFI, ADG, serum P, tibia ash, tarsometatarsus strength and a linear increase of FCR. HH vs. MM vs. LL vs. VLVL demonstrated a linear increase of relative expression of CALB1 and SLC20A1 and a linear decrease of serum P, and in terms of growth performance, MM showed the best results generally. MM vs. ML vs. MVL showed a linear decrease of ADFI and ADG and a linear increase of FCR. HM vs. ML vs. LVL demonstrated a linear decrease of ADG, serum Ca and P and relative expression of CALB1 and a linear increase of FCR (during phase 2). In conclusion, reducing Ca and NPP level over finisher phase, similarly for all combinations of treatments, resulted in poorer performance either in finisher phase or the whole period. Generally, 7.6 g/kg Ca and 3.8 g/kg NPP over the whole period maximized broiler’s performance without negative effects on bone ash and strength.

Secoiridoids Metabolism Response to Wounding in Common Centaury (Centaurium erythraea Rafn) Leaves.

Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression pattern was observed for CeBIS1, imposing itself as the TF that is prominently involved in wound-induced regulation of SGs biosynthesis genes. The high degree of positive correlations between and among the biosynthetic genes and targeted TFs expressions indicate the transcriptional regulation of SGs biosynthesis in response to wounding with a significant role of CeBIS1, which is a known component of the jasmonic acid (JA) signaling pathway.

Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer

To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO3− g−1 dry soil d−1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+ → NH2OH → NO → NO2- → NO3−) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.

Vascular endothelial growth factor in tumor cells in platinum-containing chemotherapy of advanced ovarian cancer

Aim. Study of the relationship of vascular endothelial growth factor expression in tumor cells of ascites and primary tumor in patients with ascitic form of ovarian cancer undergoing neoadjuvant chemotherapy according to the AP scheme (doxorubicin + cisplatin).&#x0D; Methods. The level of vascular endothelial growth factor messenger ribonucleic acid (mRNA) was assessed using PCR-RT in epithelial cells of ascites and primary tumor tissue obtained intraoperatively after 2–4 courses of neoadjuvant chemotherapy according to the AP scheme from 25 patients with a verified diagnosis of ovarian cancer (serous adenocarcinoma) at stage III–IV according to the International Federation of Gynecology and Obstetrics. Processing of the results was performed using the software package Statistica 10.&#x0D; Results. An increase in the expression of vascular endothelial growth factor mRNA in epithelial cells of ascites in 60% of samples was established. In the intraoperatively obtained tumor tissue after 2–4 courses of neoadjuvant chemotherapy according to the AP scheme, vascular endothelial growth factor expression was increased in 73% of the samples. At the same time, the increase of relative expression in the tumor after neoadjuvant chemotherapy did not depend on the degree of therapeutic pathomorphosis but was related to early relapse and increased level of cancer antigen-125 (CA-125). Median of relapse-free survival in vascular endothelial growth factor-positive ovarian cancer was less than 5 months.&#x0D; Conclusion. Neoadjuvant chemotherapy according to the AP scheme increases the expression of vascular endothelial growth factor in the tumor tissue in advanced ovarian cancer; the degree of vascular endothelial growth factor overexpression in ovarian tumor is related to the presence of early relapse and an increase in the serum CA-125 level.

Assessment of TCF7L2 expression after bariatric surgery.

ObjectiveTo assess the influence of bariatric surgery on transcription factor 7-like 2 (TCF7L2) expression and its association with body mass index (BMI) and Type 2 diabetes mellitus (T2DM).MethodsProspective study performed between 2016 and 2018, where 26 obese patients undergoing bariatric surgery were divided into two subgroups: diabetics and non-diabetics. The RNAs were extracted from peripheral blood samples that were obtained from each patient in two different moments: before surgery and after 12 months of follow-up. The relative expression of TCF7L2 was determined according to the delta-Ct method.ResultsThe linear regression model of BMI x delta-Ct showed a positive correlation (p = 0.037). In the subgroups, an inversely proportional relationship was found between delta-Ct and BMI in the diabetic group and a directly proportional relationship in the non-diabetic group (p>0.05 in both). In the postoperative period, the regression model was similar to the preoperative, except when analyzing the subgroups, where diabetic patients showed a directly proportional relationship (p>0.05). The relative expression of TCF7L2 showed an average of 1.16 ± 0.91, CI-95% 0.79–1.53. There was an increase in relative expression of 48% in the non-diabetic group (p = 0.021), and a decrease of 27% in the T2DM group (p>0.05) in the postoperative. There was a positive correlation between a greater decrease in BMI and increased relative expression (p = 0.027).ConclusionOur results showed that generally, the TCF7L2 expression increase with a decrease in BMI, however, for patients with T2DM, it exhibits an inverse pattern, which is normalized one year after bariatric surgery.

Establishment and characterization of a human embryonic stem cell line, NERCe002-A-3, with inducible 14-3-3ζ overexpression

NERCe002-A-3 cells were generated from the normal human embryonic stem cell line NERCe002-A. NERCe002-A-3 cells overexpressed 14-3-3ζ after exposure to doxycycline. 14-3-3ζ protein have the ability to bind a multitude of functionally diverse signalling proteins. The NERCe002-A-3 cell line is considered a model for functional studies of the 14-3-3ζ protein in hESC self-renewal and cell differentiation. Doxycycline-treated NERCe002-A-3 cells showed a>27-fold increase in relative expression of 14-3-3ζ as compared with un-induced cells. Characterization assays proved that NERCe002-A-3 cells express typical markers of pluripotency and have the ability to form the three germ layers in vivo.

Expression Analysis of Cell Surface Markers: Cluster of Differentiation 4, Chemokine Receptor 5 and C-X-C Chemokine Receptor Type 4 in Different Cell Lines after Infection with HIV

The host cell infection by Human Immunodeficiency Virus (HIV) is initiated through the entry of the virus into the cell. The major target cells of HIV are cluster of differentiation 4 (CD4)+ T lymphocytes, which express co-receptors, chemokine receptor 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4). HIV-1 envelope glycoprotein 120 (Gp120) binds to its primary receptor CD4, a member of the immunoglobulin superfamily enhances T-Cell Receptor (TCR)-mediated signaling, is absolutely required for the infection. Recent reports on cell surface marker expression levels by infecting CD4+ T cells with HIV-1 viruses gave an insight into host cell responses. In our study, we infected Jurkat, SupT1 cells and PBMCs with HIV-1 and analyzed the expression of cell surface markers gp120, CD4, CCR5 and CXCR4 levels by quantitative real time PCR (qRT-PCR) at different time intervals of post infection. Our study shows that there is ~ 5-fold increase in expression of gp120 1 h post infection and 1.5-fold increase in relative expression of CD4 and CCR5 at 2 h post infection, whereas CXCR4 showed differential up-regulation in different cell lines which needs further analysis.

Analysis of the Fluoroquinolone Antibiotic Resistance Mechanism of Salmonella enterica Isolates.

Quinolone-resistant Salmonella strains were isolated from patient samples, and several quinolone-sensitive strains were used to analyze mutations in the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE and to screen for plasmid-mediated quinolone resistance. Among the 21 strains that showed resistance to nalidixic acid and ciprofloxacin (MIC 0.125-2.0 μg/ml), 17 strains had a mutation in QRDR codon 87 of gyrA, and 3 strains had a single mutation (Ser83 → Phe). Another cause of resistance, efflux pump regulation, was studied by examining the expression of acrB, ramA, marA, and soxS. Five strains, including Sal-KH1 and Sal-KH2, showed no increase in relative expression in an analysis using the qRT-PCR method (p < 0.05). In order to determine the genes involved in the resistance, the Sal-9 isolate that showed decreased susceptibility and did not contain a mutation in the gyrA QRDR was used to make the STM (MIC 8 μg/ml) and STH (MIC 16 μg/ml) ciprofloxacin-resistant mutants. The gyrA QRDR Asp87 → Gly mutation was identified in both the STM and STH mutants by mutation analysis. qRT-PCR analysis of the efflux transporter acrB of the AcrAB-TolC efflux system showed increased expression levels in both the STM (1.79-fold) and STH (2.0-fold) mutants. In addition, the expression of the transcriptional regulator marA was increased in both the STM (6.35-fold) and STH (21.73-fold) mutants. Moreover, the expression of soxS was increased in the STM (3.41-fold) and STH (10.05-fold) mutants (p < 0.05). Therefore, these results indicate that AcrAB-TolC efflux pump activity and the target site mutation in gyrA are involved in quinolone resistance.

103 OVARIAN STIMULATION OF ANESTROUS CATS WITH FOLLICLE-STIMULATING HORMONE IMPROVES OOCYTE QUALITY AND DEVELOPMENTAL CAPABILITY AFTER PARTHENOGENETIC ACTIVATION

The domestic cat is a valuable model for developing assisted reproductive technologies in endangered felids. However, the in vitro production of cat embryos is negatively affected during the anestrous season. It seems that a low FSH level or the few FSH receptors in the granulosa cells might affect oocyte quality. The objective of this research was to evaluate the effect of ovarian stimulation of anestrous cats with FSH on oocyte quality and developmental capability after parthenogenetic activation. For this, two experimental groups were made. The first (FSH group) consisted of 9 anestrous cats treated with a subcutaneous dose of 5 mg of porcine FSH, every 24 h, for 4 days. The second (control group) consisted of 10 untreated anestrous cats. The ovaries were recovered by ovariohysterectomy. Cumulus-oocyte complexes (COCs) were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. Grade I and II COCs were matured in vitro during 30 h and matured oocytes were used for parthenogenetic activation. Matured oocytes were activated using 7% ethanol for 5 min, followed by incubation in 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 5 h. Presumed zygotes were in vitro cultured in SOF for 8 days, and blastocyst and hatching blastocyst rates were assessed. The relative expression of pluripotency (OCT4, SOX2, and NANOG) and differentiation markers (CDX2 and GATA6) were evaluated by RT-qPCR in the blastocysts. The results were normalized using the geometric mean of GAPDH and SDHA. Statistical analysis was performed using the Wilcoxon non-parametric test. A total (mean ± s.d.) of 741 COCs (82.33 ± 36.61 per cat) were recovered from the FSH group and 660 COCs (66 ± 20.67 per cat) from the control group (P &gt; 0.05). The FSH group had a higher rate of grade I COCs and a lower rate of grade III and IV COCs (22.28% and 50.62%, respectively) than the control group (13.2% and 60.08%, respectively; P &lt; 0.05). However, no significant differences were found in grade II COCs between FSH (26.21%) and control groups (27.01%; P &gt; 0.05). Thereafter, 190 matured oocytes were activated in the FSH group and 198 in the control group. The FSH group had higher blastocyst and hatching blastocyst rates (30.52% and 13.15%, respectively) than the control group (13.13% and 1.01% respectively; P &lt; 0.05). The blastocysts from the FSH group had a higher cell number (± s.d.) than their counterparts from the control group (199.9 ± 53.6 and 121 ± 34.91, respectively; P &lt; 0.05). Furthermore, the blastocysts from FSH group had an increased relative expression of OCT4 and GATA6 (P &lt; 0.05). In conclusion, ovarian stimulation of anestrous cats with FSH improves oocyte quality and embryo developmental capability, which agrees with an increase in relative expression of OCT4 and GATA6.

Regulation of acetylcholine receptors during differentiation of bone mesenchymal stem cells harvested from human reaming debris

Acetylcholine (ACh) is an important signaling molecule in non-neuronal systems where it is involved in regulation of viability, proliferation, differentiation and migration of mesenchymal stem cells (MSC) that are capable to differentiate into osteoblasts, chondrocytes and adipocytes. Patients with the systemic disease osteoporosis show altered MSC properties, reduced bone formation and mineral density followed by increased bone fragility and high fracture incidence. Here we asked whether nicotinic and muscarinic acetylcholine receptors (AChR) are expressed in osteoblasts, adipocytes and chondrocytes differentiated from bone MSC extracted from human reaming debris (RDMSC) that was harvested during surgery of long bone diaphyseal fractures.Using RT-PCR, AChR were detected in RDMSC, osteoblasts, chondrocytes and adipocytes of male and female bone-healthy and of female osteoporotic donors. An up-regulation in multiplicity and occurrence of AChR subtypes was found in female compared to male donors and in osteoblast of male donors compared to adipocytes. Real-time RT-PCR analysis resulted in a significant increase in relative expression of nAChR α9 in chondrocytes compared to adipocytes of healthy female donors. The nAChR subunit α10 was significantly up-regulated in osteoblasts of healthy compared to osteoporotic donors as well as the mAChR M5 that is additionally decreased in osteoporotic osteoblasts compared to MSC and chondrocytes of osteoporotic donors.In summary, the gene expression of AChR during differentiation of RDMSC and its regulation in cells of osteoporotic donors lead to the assumption that AChR signaling is involved in bone formation and might be utilized to stimulate bone remodeling processes.

Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

Plasma miR-21 and miR-31 as Predictive Biomarkers for Evaluation of Therapeutic Efficacy in Metastatic Colorectal Cancer

Summary Introduction. Colorectal cancer (CRC) is the third most common cancer worldwide. Evaluation of patient response to the applied therapy regime is still challenging. Routine laboratory tests during follow-up do not provide necessary information on therapy efficacy. To improve life expectancy and quality of life for patient with CRC ongoing researches is concentrated to discovering new, reliable biomarkers which could contribute to define patients prognosis and choice of therapy. One of topical research field for searching new biomarkers is mi-RNS. Aim of the study. The aim of this study was to analyze relative expression changes of miR-21 and miR-31 as potential biomarkers for evaluation of therapy efficacy in mCRC. Material and methods. In the present study seven patients with mCRC diagnosis were included. After surgery all patients received first line therapy with oxaliplatin. Blood samples for present study were collected every 2-3 weeks. Relative expression of miR-31 and miR-21 assessed with real time PCR. Data analysis carried out by delta delta Ct (ΔΔCt) algorithm. Correlation analysis was performed by R programme ver. 3.1.2 using Pearson correlation coefficient. Results. Our results in the correlation analysis for CEA serum level and relative expression level of miR-21 and miR-31 did not confirm significant correlation. MiR-31 showed increased expression soon after administration of therapy with a drop of relative expression closer to the CDP I. Acquired results revealed trend of increase in relative expression of miR-21 in PFS II compared to PFS I. Correlation analysis for miR-31 and miR-21 did not reach statistical reliability. Conclusions. The relative expression alteration pattern of miR-31 and miR-21 in plasma during the therapy is promising biomarker for evaluation of therapy efficacy. These findings need to be validated in large cohort sample set.