Duplicate Experiments Research Articles

An Evaluation of Fibrin(ogen) Determinants of Thromboelastography

Abstract 2251Thromboelastography (TEG) was used in ∼200 studies published in 2009, is mainly used for global blood coagulation assessment, and is thrombin and fibrinogen concentration-dependent. Reflecting the time course of fibrin polymerization, TEG measures lag time, rate, and maximum amplitude (MA) of clot stiffness, and is substantially enhanced by platelets. To identify more specific fibrin(ogen) determinants potentially relevant to interpretation we investigated MA under different TEG conditions. The procedure utilizes a polymethylmethacrylate (hydrophobic surface) cup and pin set, the cup oscillating through 4° 45’ (6 cycles/minute). The pin (sensor) is suspended via a torsion wire, and transmits a tuning fork-like signal, displayed as a graph, as the clot links pin and cup. Clots cross-linked (by factor XIIIa) were shown insoluble in 6 M urea, and were obtained by adding thrombin to fibrinogen solutions, pH 7.4, containing 8 mM CaCl2 and either 50 nM factor XIII or 30% afibrinogenemic plasma with or without gel-sieved platelets (190,000/μl). Except where otherwise stated, 6 μM fibrinogen was employed. In three sets of experiments without platelets, increasing thrombin (0.001 to 2 U /ml) progressively increased MA. For example, levels of 0.005, 0.1, 0.3, and 0.5 U/ml the yielded respective MAs of 16.7, 49, 62.8, and 65.3 mm. TEG enhancement by platelets reflects mainly their binding of the fibrin(ogen) C-terminal γ dodecapeptide and ensuing clot retraction. Several sets of experiments at different fibrinogen concentrations disclosed that the lower the concentration the more pronounced the platelet enhancement. For example, at of 0.3, 1, 3, and 6 μM fibrinogen (thrombin 0.4 U/ml), the respective control MAs were 0, 2.9, 15.9, 30.4, and 44.9 mm. The presence of platelets increased these to 10.6, 30.4, 44.9, and 64.8 mm, respectively. To test the role of the fibrin(ogen) αC region, two coagulable fibrinogen isolates (termed des-αC) lacking major C-terminal segments of varying length from their αC were prepared as described (Mosesson et al, J Biol Chem 249:4656). They displayed <5% intact Aα chains by gel scanning densitometry (sodium dodecyl sulfate-polyacrylamide electrophoresis of reduced protein bands). Estimated from the size of their Aα core remnants, most des-αC molecules lack the αC domain (Aα392–610) part of αC. Their clots yielded MA <2% that of fibrin with intact αC, n=5. In the presence of platelets, however, the MA was only moderately decreased (e.g. 53% ± 11%, SD, n=4) of controls. Fibrinogen adsorption to hydrophobic surfaces is known to be characteristically tight and to displace other plasma proteins, requires its D region, reaches near maximum of 2–3 molecule thick coating, ∼600 ng/cm2 within 2 minutes, and enhances its coagulability (Koo et al, J Thromb Haemost, 8: 2727–35). Cup and pin (CP) were exposed to intact fibrinogen (100 μg/ml, 2 hours), and washed. In a duplicate experiment, addition and clotting of des-αC fibrinogen again yielded MA <2% of control. Coating with des-αC fibrinogen did not alter the MA of intact fibrin clots. Monoclonal IgG antibodies, anti-Aα518–584 (mAb1), and anti-Aα241–476 (mAb2), of which both moderately decrease clot turbidity but do not impair coagulability, were tested (3 mols/mol fibrin with intact αC, non-coated CP) in the presence of platelets. In two separate sets of experiments, mAb1 decreased MA to 11.6% and 9% and mAb2 to 0.6% and 1.7% of respective non-immune IgG controls. In two other separate experiments, coating of CP with incoagulable intact fibrinogen (Frankfurt XIII, homodimeric AαR16C), followed by addition and clotting normal intact fibrinogen yielded MAs of 31% and 38% of respective normal controls. To evaluate hydrophobic surface effects, the water contact angle was decreased from 68° to 13° by CP exposure to UV ozone plasma (5 minutes, n=2). MA of clots formed in treated CP was 41% and 36% of untreated controls. We conclude that fibrin(ogen) adsorption to the hydrophobic CP surface, coagulability of adsorbed fibrinogen, and the αC domain of adsorbed and non-adsorbed fibrin(ogen) are significant TEG determinants. The results also suggest a significant or auxiliary role by the αC domain in the platelet enhancement of TEG. The pronounced stiffness of clots formed on hydrophobic, relative to hydrophilic, surface is of possible relevance in atherothrombotic lesions and in medical implants with hydrophobic surface. Disclosures:No relevant conflicts of interest to declare.

Abstract C203: Combined treatment of MK2206 (AKTi) and AZD6224 (MEKi) shows activity in triple-negative breast cancer cell line.

Abstract Background: Subsets of triple negative breast cancer (TNBC) such basal have activated signaling through the MAPK-ERK kinase (MEK) pathway. Use of MEK inhibitors in preclinical TNBC models demonstrates some cytostatic activity, but compensatory signaling through the PI3K/AKT pathway diminishes the antitumor effect of MEK inhibitor monotherapy in some TNBC cell lines. Combining a MEK inhibitor with an AKT inhibitor (horizontal blockade) may show increased antitumor activity. We sought to characterize the in vitro effects of the MEK inhibitor AZD6224 combined with the AKT inhibitor MK2206 in three different breast cancer cell lines. Methods: Cell Titer-Blue cell viability experiments were performed to test dilutions of AZD-6244 or MK-2206 alone or in combination at various molar ratios over a 72 hour time period in MCF-7 (ER+), SKBR3 (HER2+), and MBA-231 (TNBC) cell lines. Synergy was evaluated using Chou and Talalay combination index (CI) analyses (effective dose levels of 0.75, 0.9 and 0.95). Evaluation of MEK and AKT activity in the untreated, AZD6224, MK2206, and AZD+MK treated cells was performed using a BioRad (Hercules, CA) Bio-plex p-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) + pAKT (Ser473) phosphoprotein assay on a Luminex100. Results: Synergy was seen only in MDA-231 cells with a mean CI of .492 at a fixed dose ratio of 1:8. The mean fluorescence levels with standard errors of pERK1/2 and pAKT from duplicate experiments are shown in Table 1 in the four treatment groups. The greatest level of baseline MEK activation was seen in the MBA-231 cell line. A significant compensatory increase in p-AKT levels in the MBA-231 cells treated with AZD6224 alone was not observed, but this was seen in the SKBR3 cells. Conclusions: The combination of AZD6224+MK2206 demonstrates enhanced antitumor activity in the TNBC MBA-231 cell line over AZD6224 monotherapy. The strategy of combining a MEK inhibitor with an AKT inhibitor should be further explored in the treatment of TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C203.

Italian population data for the new ENFSI/EDNAP loci D1S1656, D2S441, D10S1248, D12S391, D22S1045. The GeFI collaborative exercise and concordance study

A collaborative exercise of the ISFG Italian Working Group GeFI was organised to investigate the five new ENFSI/EDNAP miniSTR loci D1S1656, D2S441, D10S1248, D12S391 and D22S1045. Allele frequencies were determined in a sample of 960 individuals collected by the nineteen participating laboratories. The concordance of the genotypes has been evaluated in duplicate experiments, using different kits. No discordant genotypes were revealed for the five ENFSI/EDNAP miniSTR markers. All the labs participating in the collaborative exercise correctly typed the two blind blood stains sent for proficiency testing.

Lack of teratological effects in rats exposed to 20 or 60 kHz magnetic fields.

BACKGROUND: A risk assessment of magnetic field (MF) exposure conducted by the World Health Organization indicated the need for biological studies on primary hazard identification and quantitative risk evaluation of intermediate frequency (300 Hz–100 kHz) MFs. Because induction heating cookers generate such MFs for cooking, reproductive and developmental effects are a concern due to the close proximity of the fields' source to a cook's abdomen. METHODS: Pregnant Crl:CD(SD) rats (25/group) were exposed to a 20 kHz, 0.2 mT(rms) or 60 kHz, 0.1 mT(rms) sinusoidal MF or sham-exposed for 22 hr/day during organogenesis, and their fetuses were examined for malformations on gestation day 20. All teratological evaluations were conducted in a blind fashion, and experiments were duplicated for each frequency to confirm consistency of experimental outcomes. RESULTS: No exposure-related changes were found in clinical signs, gross pathology, or number of implantation losses. The number of live fetuses and low-body-weight fetuses as well as the incidence of external, visceral, and skeletal malformations in the fetuses did not indicate significant differences between MF-exposed and sham-exposed groups. Although some fetuses showed isolated changes in sex ratio and skeletal variation and ossification, such changes were neither reproduced in duplicate experiments nor were they common to specific field frequencies. CONCLUSIONS: Exposure of rats to MFs during organogenesis did not show significant reproducible teratogenicity under experimental conditions. Present findings do not support the hypothesis that intermediate frequency MF exposure after implantation carries a significant risk for developing mammalian fetuses. Birth Defects Res (Part B) 92:469–477, 2011. © 2011 Wiley Periodicals, Inc.

Two different docetaxel resistant MCF-7 sublines exhibited different gene expression pattern

The objective of the present study was to investigate gene expression pattern of two docetaxel resistant MCF-7 breast carcinoma sublines step wisely selected in 30 and 120nM docetaxel. Cell proliferation assay was performed in order to demonstrate development of docetaxel resistance. cDNA microarray analysis was performed using Affymetrix(®) Human Genome U133 Plus 2.0 Arrays in duplicate experiments. Quantitative and semi-quantitative gene expression analysis was also performed to confirm gene expression analysis for selected genes. XTT results demonstrated that 30 (MCF-7/30nM DOC) and 120nM (MCF-7/120nM DOC) docetaxel selected cells were 13- and 47-fold resistant, respectively. cDNA microarray analysis demonstrated that expression profiles of MCF-7 and MCF-7/30nM DOC were more similar to each other where expression profile of MCF-7/120nM DOC was different as examined by line graphs and scatter plots. 2,837 and 4,036 genes were significantly altered in 30 and 120nM docetaxel resistant sublines, respectively. Among these, 849 genes were altered in common in two docetaxel resistant sublines. Antiapoptotic gene expression (e.g., Bcl-2 and APRIL) were noticeably altered in MCF-7/30nM DOC. However, docetaxel resistance in MCF-7/120nM DOC were more complicated with the involvement of ECM related gene expression, cytokine and growth factor signaling, ROS metabolism and EMT related gene expression together with higher level of MDR1 expression. Expression profiles in 30 and 120nM docetaxel resistant sublines changed gradually with increasing resistance index. Drug resistance development seems to be step wise event in MCF-7 cells.

Experimental Investigation of Reactive Absorption of Ammonia and Carbon Dioxide by Carbonated Ammonia Solution

In this work, reactive absorption of gases in aqueous electrolyte solutions has been investigated resulting in the development of a procedure in order to calculate the concentrations of ionic and molecular species in the liquid phase. Two duplicate experiments were conducted to investigate simultaneous reactive absorption of ammonia and carbon dioxide in partially carbonated ammonia solutions. The experiments were carried out employing an absorption pilot plant. The compositions of the electrolytes (ammonia and carbon dioxide groups) have been determined using principle knowledge of electrolyte solutions. The results revealed that the concentrations of ionic and molecular species in the liquid phase drastically influence the absorption rates of ammonia and carbon dioxide.

MicroRNA expression patterns in lobular neoplasia.

10551 Background: Lobular carcinoma in situ (LCIS) is a marker of increased risk for subsequent development of invasive breast cancer. Molecular studies have identified a clonal relationship between LCIS and concurrent invasive lobular carcinoma (ILC), suggesting that LCIS may be a precursor lesion of ILC. We hypothesized that differences in miRNA expression patterns might exist in LCIS lesions with concurrent ILC compared with LCIS lesions found alone. Methods: RNA was collected from matched frozen and archival formalin fixed paraffin embedded (FFPE) breast tissue using TRIZOL extractions. MiRNA expression profiling was performed using the Illumina miRNA DASL assay, in duplicate experiments, allowing simultaneous profiling of 735 miRNAs. MiRNAs of interest were further validated and analyzed by quantitative RT-PCR (qrt-PCR) in triplicate. To verify our data by localizing specific miRNA sequences of interest, in situ hybridization was performed on FFPE sections using a one-day protocol with locked nucleic acid (LNA) detection probes for miR-375 and miR-182, the U6 small nuclear RNA probe as a positive control and a scrambled miRNA probe as a negative control. Results: qrt-PCR analysis of the miR-183/182/96 cluster and miR-375 was performed using normal breast tissue, LCIS alone, and matched synchronous LCIS and ILC. A trend of increasing miRNA expression correlating with a model of histological progression toward invasive disease was observed. Results of in-situ hybridization confirmed this trend, showing the weakest signal intensity for both miR-375 and miR-182 in normal breast tissue, a stronger signal in LCIS alone, and the strongest signal in LCIS associated with synchronous ILC. Localization of signal to the nucleus was observed in normal breast tissue and LCIS alone, while a more diffuse cytoplasmic localization pattern was observed in LCIS associated with ILC, possibly suggesting nucleocytoplasmic transport for further processing of these miRNAs during progression to a more invasive phenotype. Conclusions: Upregulation of miR-375 and the miR-183/182/96 cluster in LCIS may correlate with risk for progression to ILC. These novel biomarkers may also be potential targets for treatment.

High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

BackgroundLaser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti.ResultsTotal RNA was isolated from Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group) of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives (Bouin's, Carnoy's, Formoy's, Cal-Rite, 4% formalin, 10% neutral buffered formalin, or zinc formalin) to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol® method. The results indicated that RNA from Carnoy's and Bouin's fixative samples was comparable to that of fresh frozen midguts (control) in duplicate experiments. When Carnoy's and Bouin's were used to fix the midguts for the LMM procedure, however, Carnoy's-fixed RNA clearly showed much less degradation than Bouin's-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoy's treated LMM RNA than Bouin's-fixed RNA in quantitative real-time PCR (qRT-PCR) assays, suggesting there were more intact target mRNAs in the Carnoy's fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 106 μm2 in the LMM procedure.ConclusionsCarnoy's fixative was found to be highly compatible with LMM, producing high quality RNA from Ae. aegypti midguts while inactivating viral pathogens. Our findings suggest that LMM in conjunction with Carnoy's fixation can be applied to studies in Ae. aegypti infected with arboviruses without compromising biosafety and RNA quality. This LMM method should be applicable to other mosquito vector studies.

PLGF expression in coronary artery smooth muscle cells is sensitive to changes in shear stress

Hemodynamic forces (stretch and shear stress) have a profound effect on the vasculature by regulating collateral artery remodeling (arteriogenesis) and development of atherosclerotic plaque. Placental growth factor (PLGF) stimulates arteriogenesis via monocyte recruitment. However, little is known about the effects of hemodynamic forces on PLGF. Based on a previous finding that arterial ligation increases PLGF in downstream collaterals, we hypothesized that PLGF is regulated by shear stress. To test this hypothesis, human coronary artery smooth muscle cells (HCASMC) were grown on the bottom of porous inserts and human coronary endothelial cells (HCAEC) were grown on the top. The HCAEC layer was then exposed to 3 levels of shear for 2 h in a cone and plate device (time‐averaged shear of 10 dyn/cm2 mimicking normal coronary artery flow; 2.5 dyn/cm2recirculatory shear simulating turbulent flow post‐stenosis; or no shear) (N=5 experiments in duplicate). Shear did not affect PLGF mRNA in HCAEC (qPCR). Interestingly, however, PLGF mRNA in HCASMC was increased by ~50% by normal shear, compared to recirculatory shear (p&lt;0.05), although HCASMC were not directly exposed to shear. Preliminary PLGF protein analysis (ELISA) was consistent with the RNA data. These findings suggest that PLGF expression in HCASMC may be regulated by hemodynamic forces in an endothelium‐dependent manner. Funding: NIH R01 HL084494 (PL).

Experimental solidification of an andesitic melt by cooling

Solidification experiments at (a) five different cooling rates (25, 12.5, 3, 0.5 and 0.125 °C/min) between 1300 and 800 °C, and (b) variable quenching temperatures (1100, 1000, 900 and 800 °C) at a fixed cooling rate of 0.5 °C/min were performed on an andesitic melt (SiO 2 = 58.52 wt.% and Na 2O + K 2O = 4.43 wt.%) at air conditions from high superheating temperature. The results show that simultaneous and duplicated experiments with Pt-wire or Pt-capsule produce identical run-products. Preferential nucleation on Pt-containers or bubbles is lacking. Plagioclase and Fe–Ti oxide crystals nucleate firstly from the melt. Clinopyroxene crystals form only at lower cooling rates (0.5 and 0.125 °C/min) and quenching temperatures (900 and 800 °C). At higher cooling rates (25, 12.5 and 3 °C/min) and quenching temperature (1100 °C), plagioclase and Fe–Ti oxide crystals are embedded in a glassy matrix; by contrast, at lower cooling rates (0.5 and 0.125 °C/min) and below 1100 °C they form an intergrowth texture. The crystallization of plagioclase and Fe–Ti oxide starts homogeneously and then proceeds by heterogeneous nucleation. The crystal size distribution (CSD) analysis of plagioclase shows that crystal coarsening increases with decreasing cooling rate and quenching temperature. At the same time, the average growth rate of plagioclases decreases from 2.1 × 10 − 6 cm/s (25 °C/min) to 5.7 × 10 − 8 cm/s (0.125 °C/min) and crystals tend to be more equant in habit. Plagioclases and Fe–Ti oxides depart from their equilibrium compositions with increasing cooling rate; plagioclases shift from labradorite–andesine to anorthite–bytownite. Therefore, kinetic effects due to cooling significantly change the plagioclase composition with remarkable petrological implications for the solidification of andesitic lavas and dikes. The glass-forming ability (GFA) of the andesitic melt has been also quantified in a critical cooling rate ( R c ) of ~ 37 °C/min. This value is higher than those measured for latitic ( R c ~ 1 °C/min) and trachytic ( R c < 0.125 °C/min) liquids demonstrating that little changes of melt composition are able to significantly shift the initial nucleation behavior of magmas and the following solidification paths.

A microarray based expression profiling of paclitaxel and vincristine resistant MCF-7 cells

Resistance to the broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. Drug resistant sublines to paclitaxel (MCF-7/Pac) and vincristine (MCF-7/Vinc) that were developed from sensitive MCF-7 cells (MCF-7/S) were used. cDNA microarray analysis was performed for the RNA samples of sensitive and resistant cells in duplicate experiments. GeneSpring GX 7.3.1 Software was used in data analysis. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of the paclitaxel and vincristine drug resistance. Additionally the upregulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant downregulation of apoptotic genes (i.e. PDCD2/4/6/8) and upregulation of some cell cycle regulatory genes ( CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Drug resistant cancer cells exhibit different gene expression patterns depending on drug treatment, and each drug resistance phenotype is probably genetically different. Further functional studies are needed to demonstrate the complete set of genes contributing to the drug resistance phenotype in breast cancer cells.

A proteome‐scale study on in vivo protein Nα‐acetylation using an optimized method

Protein N-terminal acetylation (N(α) -acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α) -modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α) -modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α) -modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α) -modified peptides. We employed a duplicate experiment using 80 μg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α) -acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.

Source water quality effects on monochloramine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus

There is a need for more information regarding monochloramine disinfection efficacy for viruses in water. In this study, monochloramine disinfection efficacy was investigated for coxsackievirus B5 (CVB5), echovirus 11 (E11), murine norovirus (MNV), and human adenovirus 2 (HAdV2) in one untreated ground water and two partially treated surface waters. Duplicate disinfection experiments were completed at pH 7 and 8 in source water at concentrations of 1 and 3 mg/L monochloramine at 5 and 15 °C. The Efficiency Factor Hom (EFH) model was used to calculate CT values (mg-min/L) required to achieve 2-, 3-, and 4-log 10 reductions in viral titers. In all water types, monochloramine disinfection was most effective for MNV, with 3-log 10 CT values at 5 °C ranging from 27 to 110. Monochloramine disinfection was least effective for HAdV2 and E11, depending on water type, with 3-log 10 CT values at 5 °C ranging from 1200 to 3300 and 810 to 2300, respectively. Overall, disinfection proceeded faster at 15 °C and pH 7 for all water types. Inactivation of the study viruses was significantly different between water types, but there was no indication that overall disinfection efficacy was enhanced or inhibited in any one water type. CT values for HAdV2 in two types of source water exceeded federal CT value recommendations in the US. The results of this study demonstrate that water quality impacts the inactivation of viruses and should be considered when developing chloramination plans.

Discovery and Quantification of Small RNA Molecules In Acute Myeloid Leukaemia Using Solexa High-Throughput Clonal Sequencing

AbstractAbstract 847It was recently established by real-time PCR that the expression level of microRNAs (miRNAs) provides molecular signatures characteristic of the major translocation-mediated gene fusion events in acute myeloid leukaemia (AML). In particular we showed that t(15;17) leukaemias are associated with the up regulation of miRNAs located in the 14q32 imprinted domain. However results were restricted to a fraction of known miRNAs. We report the results of high-throughput clonal sequencing (Illumina) applied to systematically characterise and quantify the small non-coding (snc)RNA transcriptome of 38 libraries of size-fractionated RNA obtained from 36 cytogenetically and clinically distinct cases of AML and 2 normal bone marrow from healthy donors.Sequences were aligned to the genome of reference and to miRNA and sncRNA databases using Novoalign. To account for imperfect DICER processing overhang at both 5′- and 3′-end was allowed. A location was recorded where >=2 reads mapped in any sample. Reads were excluded that did not map uniquely. In order to determine the association of the pattern of expression of miRNAs and sncRNAs with the karyotypic leukaemic subgroups, samples were first normalised. The mean of total number of reads was calculated for each sample. One profile was used as reference for scaling factor. The values of each of the other samples were divided by the scaling factors. ANOVA was applied to search for miRNAs and sncRNAs associated with AML cytogenetic subtypes. The genomic context of unidentified tags was screened with RNAfold from the Vienna package to identify potential new miRNA.The sequencing approach proved highly reproducible, correlation coefficients as high as 0.99 were calculated for duplicate experiments, and showed unbiased quantitative features when compared with the real-time PCR measurements previously obtained for known miRNAs. The average number of reads per sample was 11,212,898. The distribution of sncRNAs and miRNAs was determined for each sample. A total of 621 miRNAs and 1525 different species of sncRNAs were expressed in the 38 samples. The miRNAs represented up to 70% of total reads. The C/D Box small nucleolar RNAs (snoRNAs) were highly represented among the remaining sncRNAs. An unsupervised hierarchical cluster analysis of the sncRNA reads for 38 samples revealed molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. To find sncRNAs with statistically significant differences in expression level among the major cytogenetic groups, an ANOVA test was applied to the 38 AML samples, including the two normal bone marrows. One hundred seventy five sncRNAs passed a 0.5% false discovery rate (FDR) filter. We identified a set of sncRNAs, called CD/Box, differentially expressed in imprinted regions. Certain small RNAs showed expression only in one leukaemia subtype, e.g. the majority of miRNAs located at 14q32, 41 out of 51, were exclusively expressed in the t(15;17) leukaemia samples, thus confirming and extending previous findings from this laboratory. We also noted the high expression of a group of small nucleolar RNAs (snoRNAs) located in the same region. We identified 36 potential new miRNAs expressed in AML. We validated by real-time PCR the expression of one of the new miRNAs located on chromosome 11 which showed clear differential read counts between the samples. We also observed a high degree of variation in length in 80% of the miRNAs, particularly evident at the 3′ end. Some of the length variants were consistent with leukemia sub-typetype.We have demonstrated the potential of using high-throughput sequencing to uncover novel aspects of the disease aetiology by combining the global miRNA expression pattern with the detection of multiple miRNA variants and new hairpin molecules. Disclosures:No relevant conflicts of interest to declare.

Proteolysis, lipolysis, volatile compounds, texture, and flavor of Hispánico cheese made using frozen ewe milk curds pressed for different times

Hispánico cheese is manufactured in Spain from a mixture of cow and ewe milk. Production of ewe milk varies throughout the year, with a peak in spring and a valley in summer and autumn. To overcome this seasonal shortage, curd from spring ewe milk may be frozen and used for cheese manufacture some months later. In the present work, ewe milk curds pressed for 15, 60, or 120min were held at −24°C for 4 mo, thawed, cut to 1-mm pieces, and mixed with fresh cow milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized cow and ewe milk in the same (80:20) proportion. Cheeses, made in duplicate experiments, were analyzed throughout a 60-d ripening period. No significant differences between cheeses were found for lactic acid bacteria counts, dry matter content, hydrophilic peptides, 47 out of 68vol.tile compounds, texture, and flavor characteristics. On the other hand, differences of minor practical significance between experimental and control cheeses, unrelated to the use of frozen ewe milk curd or the pressing time of ewe milk curd, were found for pH value, aminopeptidase activity, proteolysis, hydrophobic peptides, free amino acids, free fatty acids, and the remaining 21vol.tile compounds. It may be concluded that the use of frozen ewe milk curd in the manufacture of Hispánico cheese does not alter its main characteristics.

Vaccination Against Strongyloides venezuelensis with Homologue Antigens Using New Immunomodulators

Control of Strongyloides stercoralis infection is based on antihelminthic treatment. However, the development of an efficient vaccine is an important goal for more effectively managing this disease. Different Strongyloides spp. antigen preparations have been previously tested but without satisfactory results. In the present study, we evaluated whether the doxycholic acid extract of Strongyloides venezuelensis stage 3 larvae was able to protect CD1 mice against a homologue infection. Moreover, we included saponins from Quillaja saponaria (Qs) and immunomodulatory substances, i.e., Phlebodium pseudoaureum hydroalcoholic extract (PAL) or the amino alcohol AA0029, which has an aliphatic chain of 14 carbon atoms with a hydrophilic amino alcohol head, where the amino group is protected with a butoxycarbonyl group. The DOCSv antigen, together with the adjuvant components, was emulsified in a non-mineral water/oil emulsion. We performed duplicate experiments with each of the 2 immunomodulators. The DOCSv antigen used with Qs+PAL induced high levels of protection in terms of fecal egg count reduction (93.2-99.4%), 86-88% in adult worm recovery, and reduction in swelling of the small intestine. Mice vaccinated with the DOCSv antigen, together with Qs+AA0029, showed a 73.7-97.0% reduction in fecal egg count, with reduction in inflammation of the small intestine. High levels of IgG and IgG2a were observed in mice vaccinated with Qs+AA0029+DOCSv, indicating a Th1 immune response. Also, vaccinated mice recognized bands of 34, 39, 43, 95, and 170 kDa in a Western blot.

Collagen type XI α1 may be involved in the structural plasticity of the vertebral column in Atlantic salmon (Salmo salar L.)

Atlantic salmon (Salmo salar L.) vertebral bone displays plasticity in structure, osteoid secretion and mineralization in response to photoperiod. Other properties of the vertebral bone, such as mineral content and mechanical strength, are also associated with common malformations in farmed Atlantic salmon. The biological mechanisms that underlie these changes in bone physiology are unknown, and in order to elucidate which factors might be involved in this process, microarray assays were performed on vertebral bone of Atlantic salmon reared under natural or continuous light. Eight genes were upregulated in response to continuous light treatment, whereas only one of them was upregulated in a duplicate experiment. The transcriptionally regulated gene was predicted to code for collagen type XI alpha1, a protein known to be involved in controlling the diameter of fibrillar collagens in mammals. Furthermore, the gene was highly expressed in the vertebrae, where spatial expression was found in trabecular and compact bone osteoblasts and in the chordoblasts of the notochordal sheath. When we measured the expression level of the gene in the tissue compartments of the vertebrae, the collagen turned out to be 150 and 25 times more highly expressed in the notochord and compact bone respectively, relative to the expression in the trabecular bone. Gene expression was induced in response to continuous light, and reduced in compressed vertebrae. The downregulation in compressed vertebrae was due to reduced expression in the compact bone, while expression in the trabecular bone and the notochord was unaffected. These data support the hypothesis that this gene codes for a presumptive collagen type XI alpha1, which may be involved in the regulatory pathway leading to structural adaptation of the vertebral architecture.

Drug resistant breast cancer cells overexpress ETS1 gene

Purpose Multidrug resistance (MDR) is resistance to wide range of structurally unrelated anticancer agents. MDR is a serious limitation to the effective chemotherapy. Involvement of ETS1 overexpression in upregulation of MDR1 gene expression is implicated. In the present study the aim was to assess the involvement of ETS1 and the genes, which encode the proteins interacting with ETS1 in drug resistant MCF-7 breast cancer cells. Methods Drug resistant sublines to paclitaxel (MCF-7/Pac), docetaxel (MCF-7Doc), vincristine (MCF-7/Vinc) and doxorubicin (MCF-7/Dox) that were developed from sensitive MCF-7 cells (MCF-7/S) were used. cDNA microarray analysis was performed for the RNA samples of sensitive and resistant cells in duplicate experiments. GeneSpring GX 7.3.1 Software was used in data analysis. Microarray data was supported by immunocytochemistry and western blot for drug resistance protein, P-gp, encoded by MDR1 gene. Results According to microarray data MDR1 and ETS1 genes were highly overexpressed in all of the resistant sublines. Matrix metalloproteinase-1 gene ( MMP-1) was also tremendously upregulated only in vincristine resistant cells. Immunocytochemistry and western blot results confirmed that P-gp was highly overexpressed in resistant sublines compared to original MCF-7 cells. Conclusion High ETS1 expression levels in all resistant MCF-7 sublines may lead to the upregulation of the transcription of MDR1 gene. Overexpression of ETS1 gene in resistant cells may have contributed to the development of resistance in the cells. Furthermore, the significant upregulation of MMP1 and MMP9 in MCF-7/Vinc may also be related to an acquired invasive behavior of MCF-7 cell line due to vincristine treatment.

Cyclic AMP-dependent Protein Kinase Signaling Modulates Pregnane x Receptor Activity in a Species-specific Manner

Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, beta-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.

Improved manometric setup for the accurate determination of supercritical carbon dioxide sorption

An improved version of the manometric apparatus and its procedures for measuring excess sorption of supercritical carbon dioxide are presented in detail with a comprehensive error analysis. An improved manometric apparatus is necessary for accurate excess sorption measurements with supercritical carbon dioxide due to the difficulties associated with the high sensitivity of density for pressure and temperature changes. The accuracy of the apparatus is validated by a duplicate measurement and a comparison with literature data. Excess sorption and desorption of CO(2) on Filtrasorb 400 at 318.11 K up to 17 069 mole/m(3) (15.474 MPa) were selected for this validation. The measured excess sorption maximums are 7.79+/-0.04 mole/kg at 2253 mole/m(3) for the first sorption isotherm and 7.91+/-0.05 mole/kg at 2670 mole/m(3) for its subsequent desorption isotherm. The sorption and desorption peaks of the duplicate experiment are 7.92+/-0.04 mole/kg at 2303 mole/m(3) and 8.10+/-0.05 mole/kg at 2879 mole/m(3), respectively. Both data sets show desorption data being higher than the sorption data of the same data set. The maximum discrepancy between the desorption and sorption isotherms of one data set is 0.15 mole/kg. The discrepancy between the two excess sorption isotherms is 0.12 mole/kg or less. The a priori error of the excess sorption measurements is between 0.02 and 0.06 mole/kg. The error due to He contamination is between 0.01 and 0.05 mole/kg. The difference between the a priori uncertainty and the observed maximum discrepancies is considered to be acceptable. The sorption isotherms show identical qualitative behavior as data in the literature. The quantitative behavior is similar but the peak height and the linear decrease in excess sorption at high gas densities are 10% higher. A plot of the excess sorption versus the density can be used to obtain the sorbed phase density and the specific micropore volume. These sorbed phase densities are in excellent agreement with the data in the literature. Furthermore, the excess sorption data scaled to this specific micropore volume in this work and in the literature collapse on a single curve when plotted versus gas density.