Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovineleukemia virus (BLV) infection. However, commercially available kits test inSouth America detect only antibodies against the gp51 protein. With the aimto improve the sensitivity of the test, we developed here a two-step indirectdual ELISA test that included both proteins p24 and gp51, expressed andproduced in E. coli and baculovirus expression system respectively. Two hundredten BLV sera, stated as double positive or double negative by the combinationof commercial agar gel immunodiffusion (AGID) assay and a gp51-ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordancecorrelation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards.The statistical analysis demonstrated the value of sera correctly rankedhighest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.Fil: Larsen, Alejandra. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Corva, Santiago. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Panei, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Geisler, Christoph. University Of Wyoming; Estados UnidosFil: Mortola, Eduardo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentin

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51- ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.Facultad de Ciencias Veterinaria

Intensity of respiratory cortical arousals is a distinct pathophysiologic feature and is associated with disease severity in obstructive sleep apnea patients

Background: We investigated whether the number, duration and intensity of respiratory arousals (RA) on C3-electroencephalographic (EEG) recordings correlate with polysomnography (PSG)-related disease severity in obstructive sleep apnea (OSA) patients. We also investigated if every patient might have an individual RA microstructure pattern, independent from OSA-severity. Methods: PSG recordings of 20 OSA patients (9 female; age 27–80 years) were analyzed retrospectively. Correlation coefficients were calculated between RA microstructure (duration, EEG-intensity) and RA number and respiratory disturbance index (RDI), oxygen desaturation index (ODI) and arousal index (AI). Intraclass correlations (ICC) for both RA duration and intensity were calculated. Sleep stage-specific and apnea- and hypopnea-specific analyses were also done. The probability distributions of duration and intensity were plotted, interpolated with a kernel which fits the distribution. A Bayesian posterior distribution analysis and pair-wise comparisons of each patient with all other 19 patients were performed. Results: Of the analyzed 2600 RA, strong positive correlations were found between average RA intensity and both RDI and AI. The number of PSG-recorded RA was strongly positively correlated with RDI. Significant correlations between average RA intensity in REM, NREM2 and NREM3 sleep stages and total ODI were identified. No sleep stage-specific correlations of arousal microstructure with age, sex, RDI or AI were identified. Although between-subjects ICC values were 0.7 (all p < 0.05). While apnea-related RA duration did not differ from hypopnea-related RA duration, RA intensity was significantly higher (p = 0.00135) in hypopneas than in apneas. A clear individual pattern of arousal duration for each patient was made distinct. For arousal intensity, a Gaussian distribution was identified in most patients. The Bayesian statistics regarding the arousal microstructure showed significant differences between each pair of patients. Conclusions: Each individual patient with OSA might have an individual pattern of RA intensity and duration indicating a distinct individual pathophysiological feature. Arousal intensity was significantly higher in hypopneic than in apneic events and may be related causally to the diminished (compared to apneas) respiratory distress associated with hypopneas. RA intensity in REM, NREM2 and NREM3 strongly correlated with ODI

"Бегство от свободы" советских бэбибумеров: представления о социальном и экономическом благополучии послевоенного поколения в СССР

Исторические и социокультурные исследования бэбибумеров - первого послевоенного поколения - не теряют своей актуальности. В статье анализируются представления о социальном и экономическом благополучии послевоенного поколения в СССР сквозь призму трансформаций потребительских идеалов, социалистических ценностей, представлений о престижных видах деятельности и социальном статусе. Источниковую базу исследования составили материалы советской художественной кинематографии, позволяющие проследить исторически обусловленные различия социального протеста бэбибумеров в западноевропейских странах и в СССР

Improving the Baculovirus Expression Vector System with Vankyrin-Enhanced Technology

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex‐type N‐glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P‐vank‐1), which encodes an anti‐apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High FiveTM cells, and SfSWT‐4 cells, which can produce glycoproteins with complex‐type N‐glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N‐glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P‐vank‐1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT‐4 cells were infected with a vankyrin‐encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin‐expressing cells were combined with a vankyrin‐encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin‐encoding baculovirus vectors

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51- ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.Facultad de Ciencias Veterinaria

TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma

<p>Abstract</p> <p>Background</p> <p>Disseminated soft tissue sarcoma still represents a therapeutic dilemma because effective cytostatics are missing. Therefore we tested TRAIL and Tarolidine (TRD), two substances with apoptogenic properties on human fibrosarcoma (HT1080).</p> <p>Methods</p> <p>Viability, apoptosis and necrosis were visualized by TUNEL-Assay and quantitated by FACS analysis (Propidiumiodide/AnnexinV staining). Gene expression was analysed by RNA-Microarray and the results validated for selected genes by rtPCR. Protein level changes were documented by Western Blot analysis. NFKB activity was analysed by ELISA and proliferation assays (BrdU) were performed.</p> <p>Results and discussion</p> <p>The single substances TRAIL and TRD induced apoptotic cell death and decreased proliferation in HT1080 cells significantly. Gene expression of several genes related to apoptotic pathways (TRAIL: <it>ARHGDIA</it>, <it>NFKBIA</it>, <it>TNFAIP3</it>; TRD: <it>HSPA1A/B</it>, <it>NFKBIA</it>, <it>GADD45A</it>, <it>SGK</it>, <it>JUN</it>, <it>MAP3K14</it>) was changed. The combination of TRD and TRAIL significantly increased apoptotic cell death compared to the single substances and lead to expression changes in a variety of genes (<it>HSPA1A/B</it>, <it>NFKBIA</it>, <it>PPP1R15A</it>, <it>GADD45A</it>, <it>AXL</it>, <it>SGK</it>, <it>DUSP1</it>, <it>JUN</it>, <it>IRF1</it>, <it>MYC</it>, <it>BAG5</it>, <it>BIRC3</it>). NFKB activity assay revealed an antipodal regulation of the several subunits of NFKB by TRD and TRD+TRAIL compared to TRAIL alone.</p> <p>Conclusion</p> <p>TRD and TRAIL are effective to induce apoptosis and decrease proliferation in human fibrosarcoma. A variety of genes seems to be involved, pointing to the NFKB pathway as key regulator in TRD/TRAIL-mediated apoptosis.</p

Experimental evidence of near-wall reverse flow events in a zero pressure gradient turbulent boundary layer

This study reports on experimentally observed rare near-wall reverse flow events in a fully developed turbulent flat plate boundary layer at zero pressure gradient with Reynolds numbers between Re_\theta \approx 2500 and Re_\theta \approx 8000 (Re_\tau = 800-2400). The reverse flow events are captured using high magnification particle image velocimetry sequences with record lengths varying from 50 000 to 126 000 samples. Time resolved particle image sequences allow singular reverse flow events to be followed over several time steps whereas long records of nearly statistically independent samples provide a variety of single snapshots at a higher spatial resolution. The probability of occurrence lies in the order of 0.012-0.018% which matches predictions from direct numerical simulations (DNS). The typical size of the reverse flow bubble is about 30 wall units in length and 5 wall units in height which agrees well with similar observations made in existing DNS data

Identification and validation of potential new biomarkers for prostate cancer diagnosis and prognosis using 2D-DIGE and MS

This study was designed to identify and validate potential new biomarkers for prostate cancer and to distinguish patients with and without biochemical relapse. Prostate tissue samples analyzed by 2D-DIGE (two-dimensional difference in gel electrophoresis) and mass spectrometry (MS) revealed downregulation of secernin-1 (P < 0.044) in prostate cancer, while vinculin showed significant upregulation (P < 0.001). Secernin-1 overexpression in prostate tissue was validated using Western blot and immunohistochemistry while vinculin expression was validated using immunohistochemistry. These findings indicate that secernin-1 and vinculin are potential new tissue biomarkers for prostate cancer diagnosis and prognosis, respectively. For validation, protein levels in urine were also examined by Western blot analysis. Urinary vinculin levels in prostate cancer patients were significantly higher than in urine from nontumor patients (P = 0.006). Using multiple reaction monitoring-MS (MRM-MS) analysis, prostatic acid phosphatase (PAP) showed significant higher levels in the urine of prostate cancer patients compared to controls (P = 0.012), while galectin-3 showed significant lower levels in the urine of prostate cancer patients with biochemical relapse, compared to those without relapse (P = 0.017). Three proteins were successfully differentiated between patients with and without prostate cancer and patients with and without relapse by using MRM. Thus, this technique shows promise for implementation as a noninvasive clinical diagnostic technique