Integration of aqueous (micellar) two-phase systems on the proteins separation

A two-step approach combining an aqueous two-phase system (ATPS) and an aqueous micellar two-phase system (AMTPS), both based on the thermo-responsive copolymer Pluronic L-35, is here proposed for the purification of proteins and tested on the sequential separation of three model proteins, cytochrome c, ovalbumin and azocasein. Phase diagrams were established for the ATPS, as well as co-existence curves for the AMTPS. Then, by scanning and choosing the most promising systems, the separation of the three model proteins was performed. The aqueous systems based on Pluronic L-35 and potassium phosphate buffer (pH = 6.6) proved to be the most selective platform to separate the proteins (SAzo/Cyt = 1667; SOva/Cyt = 5.33 e SAzo/Ova = 1676). The consecutive fractionation of these proteins as well as their isolation from the aqueous phases was proposed, envisaging the industrial application of this downstream strategy. The environmental impact of this downstream process was studied, considering the carbon footprint as the final output. The main contribution to the total carbon footprint comes from the ultrafiltration (~ 49%) and the acid precipitation (~ 33%) due to the energy consumption in the centrifugation. The ATPS step contributes to ~ 17% while the AMTPS only accounts for 0.30% of the total carbon footprint.publishe

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al.This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2012). Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS ONE. 7(12):1-11. https://doi.org/10.1371/journal.pone.0051271S111712Harness, J. V., Turovets, N. A., Seiler, M. J., Nistor, G., Altun, G., Agapova, L. S., … Keirstead, H. S. (2011). Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells. PLoS ONE, 6(1), e14499. doi:10.1371/journal.pone.0014499Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., … Zhou, Q. (2010). Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders. Journal of Assisted Reproduction and Genetics, 27(6), 285-291. doi:10.1007/s10815-010-9408-5Koh, C. J., Delo, D. M., Lee, J. W., Siddiqui, M. M., Lanza, R. P., Soker, S., … Atala, A. (2009). Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications. Methods, 47(2), 90-97. doi:10.1016/j.ymeth.2008.08.002Vrana, K. E., Hipp, J. D., Goss, A. M., McCool, B. A., Riddle, D. R., Walker, S. J., … Cibelli, J. B. (2003). Nonhuman primate parthenogenetic stem cells. Proceedings of the National Academy of Sciences, 100(Supplement 1), 11911-11916. doi:10.1073/pnas.2034195100Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., … Liu, L. (2009). Birth of Parthenote Mice Directly from Parthenogenetic Embryonic Stem Cells. Stem Cells, 27(9), 2136-2145. doi:10.1002/stem.158Sritanaudomchai, H., Ma, H., Clepper, L., Gokhale, S., Bogan, R., Hennebold, J., … Mitalipov, S. (2010). Discovery of a novel imprinted gene by transcriptional analysis of parthenogenetic embryonic stem cells. Human Reproduction, 25(8), 1927-1941. doi:10.1093/humrep/deq144Fang, Z. F., Gai, H., Huang, Y. Z., Li, S. G., Chen, X. J., Shi, J. J., … Sheng, H. Z. (2006). Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Experimental Cell Research, 312(18), 3669-3682. doi:10.1016/j.yexcr.2006.08.013Wang, S., Tang, X., Niu, Y., Chen, H., Li, B., Li, T., … Ji, W. (2007). Generation and Characterization of Rabbit Embryonic Stem Cells. Stem Cells, 25(2), 481-489. doi:10.1634/stemcells.2006-0226Piedrahita, J. A., Anderson, G. B., & BonDurant, R. H. (1990). On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos. Theriogenology, 34(5), 879-901. doi:10.1016/0093-691x(90)90559-cNaturil-Alfonso, C., Saenz-de-Juano, M. D., Peñaranda, D. S., Vicente, J. S., & Marco-Jiménez, F. (2011). Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos. Animal Reproduction Science, 127(3-4), 222-228. doi:10.1016/j.anireprosci.2011.08.005Besenfelder, U., Strouhal, C., & Brem, G. (1998). A Method for Endoscopic Embryo Collection and Transfer in the Rabbit. Journal of Veterinary Medicine Series A, 45(1-10), 577-579. doi:10.1111/j.1439-0442.1998.tb00861.xMehaisen, G. M. K., Viudes-de-Castro, M. P., Vicente, J. S., & Lavara, R. (2006). In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does. Theriogenology, 65(7), 1279-1291. doi:10.1016/j.theriogenology.2005.08.007Bilodeau-Goeseels, S., & Schultz, G. A. (1997). Changes in Ribosomal Ribonucleic Acid Content Within in Vitro-produced Bovine Embryos1. Biology of Reproduction, 56(5), 1323-1329. doi:10.1095/biolreprod56.5.1323Conesa, A., Gotz, S., Garcia-Gomez, J. M., Terol, J., Talon, M., & Robles, M. (2005). Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18), 3674-3676. doi:10.1093/bioinformatics/bti610Edgar, R. (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Research, 30(1), 207-210. doi:10.1093/nar/30.1.207Weltzien, F.-A., Pasqualini, C., Vernier, P., & Dufour, S. (2005). A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase. General and Comparative Endocrinology, 142(1-2), 134-142. doi:10.1016/j.ygcen.2004.12.019Llobat, L., Marco-Jiménez, F., Peñaranda, D., Saenz-de-Juano, M., & Vicente, J. (2011). Effect of Embryonic Genotype on Reference Gene Selection for RT-qPCR Normalization. Reproduction in Domestic Animals, 47(4), 629-634. doi:10.1111/j.1439-0531.2011.01934.xLiu, N., Enkemann, S. A., Liang, P., Hersmus, R., Zanazzi, C., Huang, J., … Liu, L. (2010). Genome-wide Gene Expression Profiling Reveals Aberrant MAPK and Wnt Signaling Pathways Associated with Early Parthenogenesis. Journal of Molecular Cell Biology, 2(6), 333-344. doi:10.1093/jmcb/mjq029Abdoon, A. S., Ghanem, N., Kandil, O. M., Gad, A., Schellander, K., & Tesfaye, D. (2012). cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos. Theriogenology, 77(6), 1240-1251. doi:10.1016/j.theriogenology.2011.11.004Labrecque, R., & Sirard, M.-A. (2011). Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation. Reproduction, Fertility and Development, 23(4), 591. doi:10.1071/rd10243Rizos, D., Clemente, M., Bermejo-Alvarez, P., de La Fuente, J., Lonergan, P., & Gutiérrez-Adán, A. (2008). Consequences ofIn VitroCulture Conditions on Embryo Development and Quality. Reproduction in Domestic Animals, 43, 44-50. doi:10.1111/j.1439-0531.2008.01230.xLonergan, P., Rizos, D., Kanka, J., Nemcova, L., Mbaye, A., Kingston, M., … Boland, M. (2003). Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction, 337-346. doi:10.1530/rep.0.1260337Memili, E., & First, N. L. (2000). Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote, 8(1), 87-96. doi:10.1017/s0967199400000861Latham, K. E. (2001). Embryonic genome activation. Frontiers in Bioscience, 6(3), d748-759. doi:10.2741/a639Niemann, H., & Wrenzycki, C. (2000). Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: Implications for subsequent development. Theriogenology, 53(1), 21-34. doi:10.1016/s0093-691x(99)00237-xCorcoran, D., Fair, T., Park, S., Rizos, D., Patel, O. V., Smith, G. W., … Lonergan, P. (2006). Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos. Reproduction, 131(4), 651-660. doi:10.1530/rep.1.01015Morison, I. M., Ramsay, J. P., & Spencer, H. G. (2005). A census of mammalian imprinting. Trends in Genetics, 21(8), 457-465. doi:10.1016/j.tig.2005.06.008Bischoff, S. R., Tsai, S., Hardison, N., Motsinger-Reif, A. A., Freking, B. A., Nonneman, D., … Piedrahita, J. A. (2009). Characterization of Conserved and Nonconserved Imprinted Genes in Swine1. Biology of Reproduction, 81(5), 906-920. doi:10.1095/biolreprod.109.078139Cruz-Correa, M., Zhao, R., Oveido, M., Bernabe, R. D., Lacourt, M., Cardona, A., … Giardiello, F. M. (2009). Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene. Epigenetics, 4(2), 114-118. doi:10.4161/epi.4.2.7954Park, C.-H., Uh, K.-J., Mulligan, B. P., Jeung, E.-B., Hyun, S.-H., Shin, T., … Lee, C.-K. (2011). Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos. PLoS ONE, 6(7), e22216. doi:10.1371/journal.pone.0022216Thurston, A., Taylor, J., Gardner, J., Sinclair, K. D., & Young, L. E. (2007). Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage. Reproduction, 135(1), 29-40. doi:10.1530/rep-07-0211Li, Y., & Sasaki, H. (2011). Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming. Cell Research, 21(3), 466-473. doi:10.1038/cr.2011.15Mamo, S., Gal, A., Polgar, Z., & Dinnyes, A. (2008). Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos. BMC Molecular Biology, 9(1), 67. doi:10.1186/1471-2199-9-67Navarrete Santos, A., Tonack, S., Kirstein, M., Pantaleon, M., Kaye, P., & Fischer, B. (2004). Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts. Reproduction, 128(5), 517-526. doi:10.1530/rep.1.0020

Foxa1 Reduces Lipid Accumulation in Human Hepatocytes and Is Down-Regulated in Nonalcoholic Fatty Liver

Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance

A Novel Bocavirus Associated with Acute Gastroenteritis in Australian Children

Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. Viruses such as rotavirus, adenovirus, and caliciviruses are a major cause of AGE, but in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we used molecular screening to investigate a cluster of undiagnosed cases that were part of a larger case control study into the etiology of pediatric AGE. Degenerate oligonucleotide primed (DOP) PCR was used to non-specifically amplify viral DNA from fecal specimens. The amplified DNA was then cloned and sequenced for analysis. A novel virus was detected. Elucidation and analysis of the genome indicates it is a member of the Bocavirus genus of the Parvovirinae, 23% variant at the nucleotide level from its closest formally recognized relative, the Human Bocavirus (HBoV), and similar to the very recently proposed second species of Bocavirus (HBoV2). Fecal samples collected from case control pairs during 2001 for the AGE study were tested with a bocavirus-specific PCR, and HBoV2 (sequence confirmed) was detected in 32 of 186 cases with AGE (prevalence 17.2%) compared with only 15 controls (8.1%). In this same group of children, HBoV2 prevalence was exceeded only by rotavirus (39.2%) and astrovirus (21.5%) and was more prevalent than norovirus genogroup 2 (13.4%) and adenovirus (4.8%). In a univariate analysis of the matched pairs (McNemar's Test), the odds ratio for the association of AGE with HBoV2 infection was 2.6 (95% confidence interval 1.2–5.7); P = 0.007. During the course of this screening, a second novel bocavirus was detected which we have designated HBoV species 3 (HBoV3). The prevalence of HBoV3 was low (2.7%), and it was not associated with AGE. HBoV2 and HBoV3 are newly discovered bocaviruses, of which HBoV2 is the thirdmost-prevalent virus, after rotavirus and astrovirus, associated with pediatric AGE in this study

Genetic Characterization of Human T-Cell Lymphotropic Virus Type 1 in Mozambique: Transcontinental Lineages Drive the HTLV-1 Endemic

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of Adult T-Cell Leukemia/Lymphoma (ATL), the Tropical Spastic Paraparesis/HTLV-1-associated Myelopathy (TSP/HAM) and other inflammatory diseases, including dermatitis, uveitis, and myositis. It is estimated that 2–8% of the infected persons will develop a HTLV-1-associated disease during their lifetimes, frequently TSP/HAM. Thus far, there is not a specific treatment to this progressive and chronic disease. HTLV-1 has means of three transmission: (i) from mother to child during prolonged breastfeeding, (ii) between sexual partners and (iii) through blood transfusion. HTLV-1 has been characterized in 7 subtypes and the geographical distribution and the clinical impact of this infection is not well known, mainly in African population. HTLV-1 is endemic in sub-Saharan Africa. Mozambique is a country of southeastern Africa where TSP/HAM cases were reported. Recently, our group estimated the HTLV prevalence among Mozambican blood donors as 0.9%. In this work we performed a genetic analysis of HTLV-1 in blood donors and HIV/HTLV co-infected patients from Maputo, Mozambique. Our results showed the presence of three HTLV-1 clusters within the Cosmopolitan/Transcontinental subtype/subgroup. The differential rates of HIV-1/HTLV-1 co-infection in the three HTLV-1 clusters demonstrated the dynamic of the two viruses and the need for implementation of control measures focusing on both retroviruses

Biocomposite films based on κ-carrageenan/locust bean gum blends and clays : physical and antimicrobial properties

The aims of this work were to evaluate the physical and antimicrobial properties of biodegradable films composed of mixtures of κ-carrageenan (κ-car) and locust bean gum (LBG) when organically modified clay Cloisite 30B (C30B) was dispersed in the biopolymer matrix. Film-forming solutions were prepared by adding C30B (ranging from 0 to 16 wt.%) into the κ-car/LBG solution (40/60 wt.%) with 0.3 % (w/v) of glycerol. Barrier properties (water vapour permeability, P vapour; CO2 and O2 permeabilities), mechanical properties (tensile strength, TS, and elongation-at-break, EB) and thermal stability of the resulting films were determined and related with the incorporation of C30B. Also, X-ray diffraction (XRD) was done in order to investigate the effect of C30B in film structure. Antimicrobial effects of these films against Listeria monocytogenes, Escherichia coli and Salmonella enterica were also evaluated. The increase of clay concentration causes a decrease of P vapour (from 5.34 × 10−11 to 3.19 × 10−11 g (m s Pa)−1) and an increase of the CO2 permeability (from 2.26 × 10−14 to 2.91 × 10−14 g (m s Pa)−1) and did not changed significantly the O2 permeability for films with 0 and 16 wt.% C30B, respectively. Films with 16 wt.% clay exhibited the highest values of TS (33.82 MPa) and EB (29.82 %). XRD patterns of the films indicated that a degree of exfoliation is attained depending on clay concentration. κ-car/LBG–C30B films exhibited an inhibitory effect only against L. monocytogenes. κ-car/LBG–C30B composite films are a promising alternative to synthetic films in order to improve the shelf life and safety of food products.J. T. Martins, A. I. Bourbon, A. C. Pinheiro and M. A. Cerqueira gratefully acknowledge the Fundacao para a Ciencia e Tecnologia (FCT, Portugal) for their fellowships (SFRH/BD/32566/2006, SFRH/BD/73178/2010, SFRH/BD/48120/2008 and SFRH/BPD/72753/2010, respectively), and B. W. S. Souza acknowledges the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil)

Cardiac Complications in Patients with Community-Acquired Pneumonia: A Systematic Review and Meta-Analysis of Observational Studies

Vicente Corrales-Medina and colleagues report estimates of the risk of cardiac complications among patients with community-acquired pneumonia from a systematic review and meta-analysis