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Sample records for affect blastocyst formation

  1. Inhibition of Rho-associated protein kinase increases the ratio of formation of blastocysts from single human blastomeres

    PubMed Central

    HUANG, SUNXING; DING, CHENHUI; MAI, QINGYUN; XU, YANWEN; ZHOU, CANQUAN

    2016-01-01

    Y-27632 is a specific inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho GTPase. The present study aimed to determine the effect of the specific ROCK inhibitor, Y-27632, on fresh human embryos and on single blastomeres obtained from discarded human embryos. A total of 784 poor-quality embryos were included, of which 526 were allocated to blastocyst culture directly and the remaining 258 were allocated to blastomere isolation. Embryos and single blastomeres were cultured either with, or without, Y-27632. Embryonic development was observed and recorded daily from day 5 onwards. Y-27632 did not affect the ratio of blastocyst formation or the quality of the human embryos. The duration of blastocyst formation was compared between the two groups in the embryo culture. On day 5, the blastocyst formation ratio in the experimental group was 11.4% (26/228), which was significantly (P=0.015) lower than the corresponding rate (19.7%; 44/223) in the control group. Survival analysis of the blastocyst formation duration showed that the median formation duration in the experimental group was significantly higher than that of the control group. The present study also obtained 1,192 blastomeres from 258 discarded day 3 embryos, and sibling blastomeres of similar sizes were equally allocated to experimental and control groups (n=596 in each). Treatment with Y-27632 increased the blastocyst formation ratio of human individual blastomeres, with 82 blastocysts of 596 blastomeres (13.8%), and 51 blastocysts of 596 blastomeres (8.6%) formed in the presence and absence of Y-27632, respectively (P=0.004). Compared with the control group, the mRNA and protein expression levels of E-cadherin in the blastocysts from blastomeres were enhanced by Y-27632 (P=0.022). In conclusion, the present study demonstrated that Y-27632 has different effects on the cleavage-stage of embryos and single blastomeres. Y-27632 increases the ratio of formation of blastocysts

  2. ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

    PubMed Central

    Kwon, Jeongwoo

    2016-01-01

    The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins. PMID:27077008

  3. The impact of food intake and social habits on embryo quality and the likelihood of blastocyst formation.

    PubMed

    Braga, Daniela Paes Almeida Ferreira; Halpern, Gabriela; Setti, Amanda S; Figueira, Rita Cássia S; Iaconelli, Assumpto; Borges, Edson

    2015-07-01

    The aim of this study was to evaluate the influence of patients' lifestyle factors and eating habits on embryo development. A total of 2659 embryos recovered from 269 patients undergoing intracytoplasmic sperm injection cycles were included. The frequency of intake of food items and social habits were registered and its influences on embryo development evaluated. The consumption of cereals, vegetables and fruits positively influenced the embryo quality at the cleavage stage. The quality of the embryo at the cleavage stage was also negatively correlated with the consumption of alcoholic drinks and smoking habits. The consumption of fruits influenced the likelihood of blastocyst formation, which was also positively affected by the consumption of fish. Being on a weight-loss diet and consumption of red meat had a negative influence on the likelihood of blastocyst formation. The likelihood of blastocyst formation was also negatively influenced by the consumption of alcoholic drinks and by smoking habits. The consumption of red meat and body mass index had a negative effect on the implantation rate and the likelihood of pregnancy. In addition, being on a weight-loss diet had a negative influence on implantation rate. Our evidence suggests a possible relationship between environmental factors and ovary biology. PMID:25982093

  4. Blastocyst rate of in vitro embryo production in sheep is affected by season.

    PubMed

    Mara, L; Sanna, D; Casu, S; Dattena, M; Muñoz, I M Mayorga

    2014-08-01

    It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 μg/ml estradiol-17β. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6-7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes. PMID:23458093

  5. Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality.

    PubMed

    Dovolou, Eleni; Periquesta, Eva; Messinis, Ioannis E; Tsiligianni, Theodora; Dafopoulos, Konstantinos; Gutierrez-Adan, Alfonso; Amiridis, Georgios S

    2014-03-01

    Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P < 0.05). In experiment 3, ghrelin significantly suppressed blastocysts yield. Significant differences were detected in various relative mRNA abundance, giving an overall final notion that embryos produced in the

  6. Stress exposure during the preimplantation period affects blastocyst lineages and offspring development.

    PubMed

    Burkuš, Ján; Kačmarová, Martina; Kubandová, Janka; Kokošová, Natália; Fabianová, Kamila; Fabian, Dušan; Koppel, Juraj; Čikoš, Štefan

    2015-01-01

    We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life. PMID:25985793

  7. Stress exposure during the preimplantation period affects blastocyst lineages and offspring development

    PubMed Central

    BURKUŠ, Ján; KAČMAROVÁ, Martina; KUBANDOVÁ, Janka; KOKOŠOVÁ, Natália; FABIANOVÁ, Kamila; FABIAN, Dušan; KOPPEL, Juraj; ČIKOŠ, Štefan

    2015-01-01

    We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life. PMID:25985793

  8. The mammalian blastocyst.

    PubMed

    Frankenberg, Stephen R; de Barros, Flavia R O; Rossant, Janet; Renfree, Marilyn B

    2016-01-01

    The blastocyst is a mammalian invention that carries the embryo from cleavage to gastrulation. For such a simple structure, it exhibits remarkable diversity in its mode of formation, morphology, longevity, and intimacy with the uterine endometrium. This review explores this diversity in the light of the evolution of viviparity, comparing the three main groups of mammals: monotremes, marsupials, and eutherians. The principal drivers in blastocyst evolution were loss of yolk coupled with evolution of the placenta. An important outcome of blastocyst development is differentiation of two extraembryonic lineages (trophoblast and hypoblast) that contribute to the placenta. While in many species trophoblast segregation is often coupled with blastocyst formation, in marsupials and at least some Afrotherians, these events do not coincide. Thus, many questions regarding the conservation of molecular mechanisms controlling these events are of great interest but currently unresolved. For further resources related to this article, please visit the WIREs website. PMID:26799266

  9. Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.

    PubMed

    Inaba, Yasushi; Miyashita, Satoshi; Somfai, Tamás; Geshi, Masaya; Matoba, Satoko; Dochi, Osamu; Nagai, Takashi

    2016-04-01

    This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells. PMID:26996887

  10. Supplementation of sperm cryopreservation media with cell permeable superoxide dismutase mimetic agent (MnTE) improves goat blastocyst formation.

    PubMed

    Forouzanfar, Mohsen; Abid, Abdolah; Hosseini, Sayyed Morteza; Hajian, Mehdi; Nasr Esfahani, Mohammad Hossein

    2013-12-01

    The aim of this study was to assess whether a cell permeable superoxide dismutase agent such as MnTE, can further improve the quality of frozen/thawed semen sample using a commercially optimized sperm cryopreservation media (Bioxcell). Bioxcell was supplemented with different concentration of MnTE. Sperm membrane integrity, motility, viability and acrosomal status were assessed after freezing. Optimized concentration of MnTE was defined and used to assess fertilization and developmental potential. 0.1 μM MnTE significantly improved membrane integrity while 0.01 μM MnTE significantly improved acrosomal integrity post thawing. Addition of 0.01 μM MnTE also improved blastocyst formation rate. Supplementation of commercially optimized cryopreservation media with MnTE further improves the quality of goat frozen semen sample and may have important consequence of future embryo development. This effect may be attributed to cell permeable behavior of this antioxidant which may protect sperm genome from ROS-induced DNA damage. PMID:23981864

  11. Liver receptor homolog 1 influences blastocyst hatching in pigs

    PubMed Central

    GUO, Jing; ZHAO, Ming-Hui; LIANG, Shuang; CHOI, Jeong-Woo; KIM, Nam-Hyung; CUI, Xiang-Shun

    2016-01-01

    Liver receptor homolog 1 (Lrh1, also known as Nr5a2) belongs to the orphan nuclear receptor superfamily and has diverse functions in development, metabolism, and cell differentiation and death. Lrh1 regulates the expression of Oct4, which is a key factor of early embryonic differentiation. However, the role of Lrh1 in early development of mammalian embryo is unknown. In the present study, the localization, Lrh1 mRNA expression, and LRH1 protein levels in porcine early parthenotes were examined by immunofluorescence and real-time reverse-transcription polymerase chain reaction. To determine the role of Lrh1 in porcine early embryo development, the parthenotes were treated with the specific LRH1 antagonist 505601. The immunofluorescence signal for LRH1 was only observed in the nucleus of blastocysts. The blastocyst developmental rate in the presence of 50 and 100 μM 505601 was significantly lower than that in the control group. The blastocyst hatching rate was also reduced in the presence of 50 and 100 μM 505601 than that under control conditions. The latter effect was possibly due to the decreased expression of hatching-related genes such as Fn1, Itgα5, and Cox2 upon the inhibition of Lrh1. Incubation with the LRH1 antagonist also increased the number of apoptotic cells among the blastocysts. Moreover, LRH1 inhibition enhanced the expression of the pro-apoptotic genes Bax and Casp3, and reduced the expression of the anti-apoptotic gene Bcl2. Lrh1 inhibition also led to significant decrease in the expression levels of Oct4 mRNA and octamer-binding transcription factor 4 (OCT4) protein in the blastocysts. In conclusion, Lrh1 affects blastocyst formation and hatching in porcine embryonic development through the regulation of OCT4 expression and cell apoptosis. PMID:26971889

  12. Expression of stemness markers in mouse parthenogenetic-diploid blastocysts is influenced by slight variation of activation protocol adopted.

    PubMed

    Bianchi, Enrica; Geremia, Raffaele; Sette, Claudio

    2010-07-01

    The importance of obtaining stem cells through alternative methods has increased progressively in the recent years due to the potential role that embryonic stem (ES) cells play in the field of regenerative medicine. In this regard, generation of parthenogenetic blastocysts allows the production of ethic-free ES cells without the need to manipulate normal embryos. Our work was aimed at clarifying whether variations in the method adopted to generate diploid parthenogenetic blastocysts could determine differences in the quality of blastocysts produced. In vitro development of mouse oocytes activated with three protocols, using Sr2+ and cytochalasin for different time, was compared with that of in vivo fertilized embryos. We have evaluated the efficiency of blastocyst formation and analysed the expression pattern of the stemness markers OCT4, CDX2, and NANOG. Our results indicate that the yield of diploid parthenogenotes and the segregation of the stemness marker OCT4 in the developing blastocyst are influenced by the parthenogenetic protocol adopted. Particularly, even if all methods tested allowed the production of blastocysts in vitro, the correct segregation of OCT4 occurred only in blastocysts developed from oocytes concomitantly treated for 4 h with Sr2+ and cytochalasin D. Our results indicate that the protocol employed to develop parthenogenetic blastocysts in vitro affects the quality of cells in the inner cell mass. PMID:20376706

  13. Effect of clinically-related factors on in vitro blastocyst development after equine ICSI.

    PubMed

    Choi, Young-Ho; Velez, Isabel C; Macías-García, Beatriz; Riera, Fernando L; Ballard, Catherine S; Hinrichs, Katrin

    2016-04-15

    after nitrogen tank failure did not produce blastocysts; exogenous activation after ICSI increased cleavage rate but did not yield blastocysts. These studies provide information on factors that may affect in vitro blastocyst formation after equine ICSI as it is performed in a clinical program. PMID:26777560

  14. PC7 and the related proteases Furin and Pace4 regulate E-cadherin function during blastocyst formation

    PubMed Central

    Mesnard, Daniel

    2015-01-01

    The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell–cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell–cell adhesion and fate allocation. PMID:26416966

  15. PC7 and the related proteases Furin and Pace4 regulate E-cadherin function during blastocyst formation.

    PubMed

    Bessonnard, Sylvain; Mesnard, Daniel; Constam, Daniel B

    2015-09-28

    The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell-cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell-cell adhesion and fate allocation. PMID:26416966

  16. The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals

    PubMed Central

    Hosseini, Sayyed-Morteza; Moulavi, Fariba; Tanhaie-Vash, Nima; Asgari, Vajihe; Ghanaei, Hamid-Reza; Abedi-Dorche, Maryam; Jafarzadeh, Naser; Gourabi, Hossein; Shahverdi, Abdol-Hossein; Dizaj, Ahmad Vosough; Shirazi, Abolfazl; Nasr-Esfahani, Mohammad-Hossein

    2016-01-01

    Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with

  17. The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals.

    PubMed

    Hosseini, Sayyed-Morteza; Moulavi, Fariba; Tanhaie-Vash, Nima; Asgari, Vajihe; Ghanaei, Hamid-Reza; Abedi-Dorche, Maryam; Jafarzadeh, Naser; Gourabi, Hossein; Shahverdi, Abdol-Hossein; Dizaj, Ahmad Vosough; Shirazi, Abolfazl; Nasr-Esfahani, Mohammad-Hossein

    2016-01-01

    Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with

  18. Heat shock decreases the embryonic quality of frozen-thawed bovine blastocysts produced in vitro

    PubMed Central

    MORI, Miyuki; HAYASHI, Takeshi; ISOZAKI, Yoshihiro; TAKENOUCHI, Naoki; SAKATANI, Miki

    2015-01-01

    In this study, the effect of heat shock on frozen-thawed blastocysts was evaluated using in vitro-produced (IVP) bovine embryos. In experiment 1, the effects of 6 h of heat shock at 41.0 C on fresh blastocysts were evaluated. HSPA1A expression as a reflection of stress was increased by heat shock (P < 0.05), but the expressions of the quality markers IFNT and POU5F1 were not affected. In experiment 2, frozen-thawed blastocysts were incubated at 38.5 C for 6 h (cryo-con) or exposed to heat shock at 41.0 C for 6 h (cryo-HS). Then, blastocysts were cultured at 38.5 C until 48 h after thawing (both conditions). Cryo-HS blastocysts exhibited a decreased recovery rate: HSPA1A expression was dramatically increased compared with that in fresh or cryo-con blastocysts at 6 h, and IFNT expression was decreased compared with that in cryo-con blastocysts at 6 h (both P < 0.05). Cryo-con blastocysts at 6 h also exhibited higher HSPA1A expression than fresh blastocysts (P < 0.05). At 48 h after thawing, the number of hatched blastocysts and blastocyst diameter were lower in cryo-HS blastocysts (P < 0.05). Cryo-con blastocysts showed lower POU5F1 levels at 48 h than fresh, cryo-con or cryo-HS blastocysts at 6 h (P < 0.05), but their POU5F1 levels were not different from those of cryo-HS blastocysts at 48 h. These results indicated that application of heat shock to frozen-thawed blastocysts was highly damaging. The increase in damage by the interaction of freezing-thawing and heat shock might be one reason for the low conception rate in frozen-thawed embryo transfer in summer. PMID:26096768

  19. Development of in vitro fertilized feline embryos in a modified Earle's balanced salt solution: influence of protein supplements and culture dishes on fertilization success and blastocyst formation.

    PubMed

    Kanda, M; Miyazaki, T; Kanda, M; Nakao, H; Tsutsui, T

    1998-04-01

    The effects of protein supplements and culture dish type on in vitro fertilization (IVF) and embryo development in culture were examined in the domestic cat. In Experiment I, follicular oocytes were fertilized and cultured in either 1) modified Earle's balanced salt solution, designated MK-1, supplemented with one of the following: 10% human serum (HS), 10% FCS or 0.4% BSA, or 2) Medium 199 (M-199) supplemented with 10% FCS. Fertilization rates were lower (P < 0.01) in MK-1 + BSA (74.4%), MK-1 + FCS (56.1%), and M-199 + FCS (51.4%) than in MK-1 + HS (94.7%). A greater (P < 0.01) percentage of blastocysts was obtained in MK-1 + HS (50.0%) than in other treatment groups (range, 4.3-17.2%). In Experiment II, the effect of dish type (tissue culture dish, TCD, versus suspension culture dish, SCD) on embryo development was evaluated in MK-1 supplemented with either HS or BSA. Significantly higher proportions of IVF-derived embryos developed to blastocysts at 120 and 144 hr post-insemination, respectively, when cultured in HS/SCD (47.2 and 71.7%) than in BSA/SCD (11.4 and 27.3%) or BSA/TCD (10.4 and 25.0%). At 120 hr post-insemination, there was a lower (P < 0.01) percentage of blastocysts in HS/TCD (22.2%) than in HS/SCD. In Experiment III, six embryos per cat were transferred to the uterine horns of 17 recipients at 144 hr after hCG treatment. Five of 7 recipients which received late morulae cultured in MK-1 + BSA (SCD) for 120 hr became pregnant (71.4%). Eight of 10 recipients which received early blastocysts cultured in MK-1 + HS (SCD) for 120 hr became pregnant (80.0%). We conclude that MK-1 containing HS is highly beneficial for overcoming the in vitro developmental block of IVF-derived feline embryos and increasing the success rate of IVF/ET. PMID:9592713

  20. Relationship between reactive oxygen species and autophagy in dormant mouse blastocysts during delayed implantation

    PubMed Central

    Shin, Hyejin; Choi, Soyoung

    2014-01-01

    Objective Under estrogen deficiency, blastocysts cannot initiate implantation and enter dormancy. Dormant blastocysts live longer in utero than normal blastocysts, and autophagy has been suggested as a mechanism underlying the sustained survival of dormant blastocysts during delayed implantation. Autophagy is a cellular degradation pathway and a central component of the integrated stress response. Reactive oxygen species (ROS) are produced within cells during normal metabolism, but their levels increase dramatically under stressful conditions. We investigated whether heightened autophagy in dormant blastocysts is associated with the increased oxidative stress under the unfavorable condition of delayed implantation. Methods To visualize ROS production, day 8 (short-term dormancy) and day 20 (long-term dormancy) dormant blastocysts were loaded with 1-µM 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). To block autophagic activation, 3-methyladenine (3-MA) and wortmannin were used in vivo and in vitro, respectively. Results We observed that ROS production was not significantly affected by the status of dormancy; in other words, both dormant and activated blastocysts showed high levels of ROS. However, ROS production was higher in the dormant blastocysts of the long-term dormancy group than in those of the short-term group. The addition of wortmannin to dormant blastocysts in vitro and 3-MA injection in vivo significantly increased ROS production in the short-term dormant blastocysts. In the long-term dormant blastocysts, ROS levels were not significantly affected by the treatment of the autophagy inhibitor. Conclusion During delayed implantation, heightened autophagy in dormant blastocysts may be operative as a potential mechanism to reduce oxidative stress. Further, ROS may be one of the potential causes of compromised developmental competence of long-term dormant blastocysts after implantation. PMID:25309857

  1. Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer.

    PubMed

    Park, Kyung-Mee; Lee, Joohyeong; Hussein, Kamal Hany; Hong, Seok-Ho; Yang, Se-Ran; Lee, Eunsong; Woo, Heung-Myong

    2016-05-01

    Transgenic porcine induced pluripotent stem (iPS) cells are attractive cell sources for the development of genetically engineered pig models, because they can be expanded without senescence and have the potential for multiple gene manipulation. They are also useful cell sources for disease modeling and treatment. However, the generation of transgenic porcine iPS cells is rare, and their embryonic development after nuclear transfer (NT) has not yet been reported. We report here the generation of liver-specific oncogenes (TGF-α/c-Myc)-overexpressing porcine iPS (T/M iPS)-like cells. They expressed stem cell characteristics and were differentiated into hepatocyte-like cells that express oncogenes. We also confirmed that NT embryos derived from T/M iPS-like cells successfully developed blastocysts in vitro. As an initial approach toward porcine transgenic iPS cell generation and their developmental competence after NT, this study provides foundations for the efficient generation of genetically modified porcine iPS cells and animal models. PMID:26725870

  2. Generation of liver-specific TGF-α/c-Myc-overexpressing porcine induced pluripotent stem-like cells and blastocyst formation using nuclear transfer

    PubMed Central

    PARK, Kyung-Mee; LEE, Joohyeong; HUSSEIN, Kamal Hany; HONG, Seok-Ho; YANG, Se-Ran; LEE, Eunsong; WOO, Heung-Myong

    2015-01-01

    Transgenic porcine induced pluripotent stem (iPS) cells are attractive cell sources for the development of genetically engineered pig models, because they can be expanded without senescence and have the potential for multiple gene manipulation. They are also useful cell sources for disease modeling and treatment. However, the generation of transgenic porcine iPS cells is rare, and their embryonic development after nuclear transfer (NT) has not yet been reported. We report here the generation of liver-specific oncogenes (TGF-α/c-Myc)-overexpressing porcine iPS (T/M iPS)-like cells. They expressed stem cell characteristics and were differentiated into hepatocyte-like cells that express oncogenes. We also confirmed that NT embryos derived from T/M iPS-like cells successfully developed blastocysts in vitro. As an initial approach toward porcine transgenic iPS cell generation and their developmental competence after NT, this study provides foundations for the efficient generation of genetically modified porcine iPS cells and animal models. PMID:26725870

  3. Mechanically loaded myotubes affect osteoclast formation.

    PubMed

    Juffer, Petra; Jaspers, Richard T; Klein-Nulend, Jenneke; Bakker, Astrid D

    2014-03-01

    In response to mechanical loading skeletal muscle produces numerous growth factors and cytokines that enter the circulation. We hypothesized that myotubes produce soluble factors that affect osteoclast formation and aimed to identify which osteoclastogenesis-modulating factors are differentially produced by mechanically stimulated myotubes. C2C12 myotubes were subjected to mechanical loading by cyclic strain for 1 h, and postincubated with or without cyclic strain for 24 h. The effect of cyclic strain on gene expression in myotubes was determined by PCR. Conditioned medium (CM) was collected from cultures of unloaded and loaded myotubes and from MLO-Y4 osteocytes. CM was added to mouse bone marrow cells containing osteoclast precursors, and after 6 days osteoclasts were counted. Compared to unconditioned medium, CM from unloaded osteocytes increased osteoclast formation, while CM from unloaded myotubes decreased osteoclast formation. Cyclic strain strongly enhanced IL-6 expression in myotubes. CM from cyclically strained myotubes increased osteoclast formation compared to CM from unloaded myotubes, but this effect did not occur in the presence of an IL-6 antibody. In conclusion, mechanically loaded myotubes secrete soluble factors, among others IL-6, which affect osteoclast formation. These results suggest that muscle could potentially affect bone homeostasis in vivo via production of growth factors and/or cytokines. PMID:24264813

  4. Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts.

    PubMed

    Akizawa, Hiroki; Nagatomo, Hiroaki; Odagiri, Haruka; Kohri, Nanami; Yamauchi, Nobuhiko; Yanagawa, Yojiro; Nagano, Masashi; Takahashi, Masashi; Kawahara, Manabu

    2016-06-01

    A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P < 0.05)-which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan-FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2-KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle. Mol. Reprod. Dev. 83: 516-525, 2016. © 2016 Wiley Periodicals, Inc. PMID:27060901

  5. Nitric oxide regulates blastocyst hatching in mice

    PubMed Central

    Pan, Xiaoyan; Wang, Xuenan; Wang, Xiyan; Sun, Zhanxuan; Zhang, Xue; Liang, Xuanxuan; Li, Zhixin; Dou, Zhaohua

    2015-01-01

    Objective: This study is to determine the regulatory role of nitric oxide in mouse blastocyst hatching. Methods: Kunming female mice were superovulated and then mated with mature male mice. On day 2.5 of their pregnancy, the pregnant mice were killed and morulae were flushed from their uterine horns with culture media. Morulae were cultured in media with different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), 8-Br-3’-5’-cyclic guanosine monophosphate (8-Br-cGMP) or the combination of L-NAME with SNP or 8-Br-cGMP for 48 h. The hatched blastocysts were examined on day 5 and the expressions of epithelial nitric oxide synthase (eNOS) and active cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope. Results: L-NAME significantly reduced the expression of eNOS in blastocyst cells. With the increase of the concentrations of L-NAME, SNP or 8-Br-cGMP, blastocyst hatching rate was significantly lowered. In addition, 5 mM L-NAME, 2 μM SNP and 2 μM 8-Br-cGMP completely inhibited blastocyst hatching. Low concentrations of SNP or 8-Br-cGMP in culture media containing 5 mM L-NAME significantly reversed the inhibition of blastocyst hatching and promoted hatching development. Moreover, 5 mM L-NAME and 2 μM 8-Br-cGMP had no significant influence on the expression of active caspase 3 in blastocyst cells. SNP (> 500 nM) significantly increased the expression of active caspase 3 in blastocyst cells. Conclusions: NO/cGMP pathway plays an important role in mouse blastocyst hatching. Excessive or depleted NO can interrupt blastocyst hatching. Excessive NO leads to apoptosis of blastocyst cells. PMID:26221236

  6. Epigenetic Dysregulation Observed in Monosomy Blastocysts Further Compromises Developmental Potential

    PubMed Central

    Denomme, Michelle M.; McCallie, Blair R.; Parks, Jason C.; Schoolcraft, William B.; Katz-Jaffe, Mandy G.

    2016-01-01

    Epigenetic mechanisms such as DNA methylation regulate genomic imprinting and account for the distinct non-equivalence of the parental genomes in the embryo. Chromosomal aneuploidy, a major cause of infertility, distorts this highly regulated disparity by the presence or absence of chromosomes. The implantation potential of monosomy embryos is negligible compared to their trisomy counterparts, yet the cause for this is unknown. This study investigated the impact of chromosomal aneuploidy on strict epigenetically regulated domains, specifically imprinting control regions present on aneuploid chromosomes. Donated cryopreserved human IVF blastocysts of transferable quality, including trisomy 15, trisomy 11, monosomy 15, monosomy 11, and donor oocyte control blastocysts were examined individually for DNA methylation profiles by bisulfite mutagenesis and sequencing analysis of two maternally methylated imprinting control regions (ICRs), SNRPN (15q11.2) and KCNQ1OT1 (11p15.5), and one paternally methylated imprinting control region, H19 (11p15.5). Imprinted genes within the regions were also evaluated for transcript abundance by RT-qPCR. Overall, statistically significant hypermethylated and hypomethylated ICRs were found in both the trisomy and monosomy blastocysts compared to controls, restricted only to the chromosome affected by the aneuploidy. Increased expression was observed for maternally-expressed imprinted genes in trisomy blastocysts, while a decreased expression was observed for both maternally- and paternally-expressed imprinted genes in monosomy blastocysts. This epigenetic dysregulation and altered monoallelic expression observed at imprinting control regions in aneuploid IVF embryos supports euploid embryo transfer during infertility treatments, and may specifically highlight an explanation for the compromised implantation potential in monosomy embryos. PMID:27271036

  7. Research on Blastocyst Implantation Essential Factors (BIEFs).

    PubMed

    Yoshinaga, Koji

    2010-06-01

    Blastocyst implantation is a process of interaction between embryo and the uterus. To understand this process, this review tries to summarize what blastocyst implantation essential factors (BIEFs) play what roles, as well as where in the uterus and at what stage of implantation process. Addition of more new data to this kind of compilation of information will help the development of diagnosis and treatment of infertility caused by implantation failure. The major, important cells of the endometrial cells that interact with invading blastocyst (trophoblast) are luminal epithelial cells, stromal cells (decidual cells) and resident immune cells. BIEFs regulate these cells to successfully maintain pregnancy. PMID:20455874

  8. Vitrified/warmed single blastocyst transfer in preimplantation genetic diagnosis/preimplantation genetic screening cycles

    PubMed Central

    Huang, Jin; Li, Rong; Lian, Ying; Chen, Lixue; Shi, Xiaodan; Qiao, Jie; Liu, Ping

    2015-01-01

    Objective: To investigate the single blastocyst transfer in preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles. Methods: 80 PGD/PGS cycles undergoing blastocyst biopsy were studied. There were 88 warming cycles during the study period. Only one warmed blastocyst was transferred per cycle. The outcomes were followed up to the infants were born. Results: The embryo implantation rate was 54.55% (48/88). The clinical pregnancy rate was 54.55% (48/88) per transfer cycle and 60% (48/80) per initial PGD/PGS cycle. There was no multi-pregnant in this study. The live birth rate was 42.05% (37/88) per transfer cycle and 46.25% (37/80) per initial PGD/PGS cycle. Conclusion: In PGD/PGS cycles, single blastocyst transfer reduces the multiple pregnancy rate without affecting the clinical outcomes. PMID:26885112

  9. Astrophysics: Variable snow lines affect planet formation

    NASA Astrophysics Data System (ADS)

    Matthews, Brenda

    2016-07-01

    Observations of the disk of dust and gas around a nascent star reveal that the distance from the star at which water in the disk forms ice is variable. This variation might hinder the formation of planets. See Letter p.258

  10. Factors Affecting Thermally Induced Furan Formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furan, a potential carcinogen, can be induced by heat from sugars and fatty acids. However, factors that contribute to its formation in foods are unclear. The objective of this research was to investigate the effects of pH, presence of phosphate, heating time and heating temperature on furan forma...

  11. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

    Zarkoob, Hoda; Bodduluri, Sandeep; Ponnaluri, Sailahari V.; Selby, John C.; Sander, Edward A.

    2015-01-01

    Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned

  12. How the cosmological constant affects gravastar formation

    SciTech Connect

    Chan, R.; Silva, M.F.A. da; Rocha, P. E-mail: mfasnic@gmail.com

    2009-12-01

    Here we generalized a previous model of gravastar consisted of an internal de Sitter spacetime, a dynamical infinitely thin shell with an equation of state, but now we consider an external de Sitter-Schwarzschild spacetime. We have shown explicitly that the final output can be a black hole, a ''bounded excursion'' stable gravastar, a stable gravastar, or a de Sitter spacetime, depending on the total mass of the system, the cosmological constants, the equation of state of the thin shell and the initial position of the dynamical shell. We have found that the exterior cosmological constant imposes a limit to the gravastar formation, i.e., the exterior cosmological constant must be smaller than the interior cosmological constant. Besides, we have also shown that, in the particular case where the Schwarzschild mass vanishes, no stable gravastar can be formed, but we still have formation of black hole.

  13. Diverse Roles of Prostaglandins in Blastocyst Implantation

    PubMed Central

    2014-01-01

    Prostaglandins (PGs), derivatives of arachidonic acid, play an indispensable role in embryo implantation. PGs have been reported to participate in the increase in vascular permeability, stromal decidualization, blastocyst growth and development, leukocyte recruitment, embryo transport, trophoblast invasion, and extracellular matrix remodeling during implantation. Deranged PGs syntheses and actions will result in implantation failure. This review summarizes up-to-date literatures on the role of PGs in blastocyst implantation which could provide a broad perspective to guide further research in this field. PMID:24616654

  14. Comparison of vitrified outcomes between human early blastocysts and expanded blastocysts.

    PubMed

    Song, Wen-Yan; Wang, Xue-Gai; Jin, Hai-Xia; Yao, Gui-Dong; Zhang, Xiang-Yang; Shi, Sen-Lin; Yang, Hong-Yi; Peng, Zhao-Feng; Sun, Ying-Pu

    2016-05-01

    We compared the vitrified outcomes between early and expanded blastocysts with or without laser drilling. The grade III embryos from the patients undergoing in vitro fertilization-embryo transfer (IVF-ET) in our reproductive center from September 2009 to February 2015 were incubated into early blastocysts and expanded blastocysts. The early blastocysts and expanded blastocysts were, respectively, divided into laser group (vitrification after laser drilling), non-laser group (direct vitrification), and control group (fresh non-vitrified blastocysts). After thawing, the blastular anabiosis rate, expansion rate, hatching rate, and apoptosis were observed in each group and then were compared amongst groups. This study indicated that the blastular expansion rate (all P < 0.01) and hatching rate (all P < 0.01) were significantly lower, but the blastular apoptosis (all P < 0.05) was significantly higher in both laser and non-laser groups than in the control group in the early blastocysts. In the expanded blastocysts, the blastular anabiosis rate was significantly higher in the laser group than in the non-laser group (P < 0.01), and the blastular expansion rate was significantly higher, but the blastular apoptosis was significantly lower in both laser group and control group than in the non-laser group (all P < 0.05). The blastular expansion rate (all P < 0.01) and hatching rate (all P < 0.01) were significantly higher, but the blastular apoptosis (all P < 0.05) was significantly lower in the expanded laser group than in both early laser and early non-laser groups. We conclude that vitrification for laser-drilling expanded blastocysts can achieve the best outcomes. PMID:26956359

  15. Effects of partial or complete laser-assisted hatching on the hatching of mouse blastocysts and their cell numbers

    PubMed Central

    2013-01-01

    Background It is still debatable whether a full-thickness assisted hatching (AH) is better than the partial zona thinning. In this research, we used a mouse model to study the effect of partial and complete laser-AH on the rate of completely hatched blastocyst and their cell numbers. Methods In experiment 1, mouse morulae had 0, 1, 2 or 3 full-thickness openings of 10 microns created in the zona pellucida with an infrared laser beam. In the second experiment, 0, 1 and 2 openings of 20 microns were studied. In the third experiment, a full-thickness opening of 20 microns or quarter-thinning of the zonal circumference to a depth of 90% was compared with non-AH controls. Results No difference in blastocyst formation was found in laser-treated groups and in the controls. In experiment 1, the rate of completely hatched blastocysts was significantly lower than the controls. In experiment 2 when the size of the opening was increased, blastocysts completely hatched at a significantly higher rate than that in the controls. In experiment 3, the rate of completely hatched blastocysts was the highest in the full-thickness group. Cell numbers in completely hatched blastocysts from both AH groups were significantly fewer than those in the controls. Conclusions Full-thickness opening resulted in a higher rate of completely hatched blastocysts than quarter zonal-thinning and controls, but the cell numbers were significantly decreased. PMID:23510434

  16. Hypomethylation and Genetic Instability in Monosomy Blastocysts May Contribute to Decreased Implantation Potential

    PubMed Central

    McCallie, Blair R.; Parks, Jason C.; Patton, Alyssa L.; Griffin, Darren K.; Schoolcraft, William B.; Katz-Jaffe, Mandy G.

    2016-01-01

    DNA methylation is a key epigenetic mechanism responsible for gene regulation, chromatin remodeling, and genome stability, playing a fundamental role during embryonic development. The aim of this study was to determine if these epigenetic marks are associated with chromosomal aneuploidy in human blastocysts. Surplus, cryopreserved blastocysts that were donated to research with IRB consent were chosen with varying chromosomal aneuploidies and respective implantation potential: monosomies and trisomies 7, 11, 15, 21, and 22. DNA methylation analysis was performed using the Illumina Infinium HumanMethylation450 BeadChip (~485,000 CpG sites). The methylation profiles of these human blastocysts were found to be similar across all samples, independent of chromosome constitution; however, more detailed examination identified significant hypomethylation in the chromosome involved in the monosomy. Real-time PCR was also performed to determine if downstream messenger RNA (mRNA) was affected for genes on the monosomy chromosome. Gene dysregulation was observed for monosomy blastocysts within significant regions of hypo-methylation (AVEN, CYFIP1, FAM189A1, MYO9A, ADM2, PACSIN2, PARVB, and PIWIL3) (P < 0.05). Additional analysis was performed to examine the gene expression profiles of associated methylation regulators including: DNA methyltransferases (DNMT1, DNMT3A, DNMT3B, DNMT3L), chromatin modifying regulators (CSNK1E, KDM1, PRKCA), and a post-translational modifier (PRMT5). Decreased RNA transcription was confirmed for each DNMT, and the regulators that impact DNMT activity, for only monosomy blastocysts (P < 0.05). In summary, monosomy blastocysts displayed hypomethylation for the chromosome involved in the error, as well as transcription alterations of associated developmental genes. Together, these modifications may be contributing to genetic instability and therefore be responsible for the limited implantation potential observed for full monosomy blastocysts. PMID

  17. Effect of blastocoel fluid reduction before vitrification on gene expression in mouse blastocysts.

    PubMed

    Kazemi, Parinaz; Dashtizad, Mojtaba; Shamsara, Mehdi; Mahdavinezhad, Forough; Hashemi, Ehsan; Fayazi, Samaneh; Hajarian, Hadi

    2016-08-01

    Artificial collapse of the blastocoel cavity before vitrification can improve the quality of warmed embryos, yet how reduction of blastocoel fluid impacts formation of the blastocyst cell lineages is not clear. The present study assessed the effect of pre-vitrification blastocoel fluid reduction on the survival, hatching rate, and the expression of genes related to apoptosis (Tp53), pluripotency (Pou5f1, Nanog), and differentiation (Cdx2, Eomes, Gata6) in mouse blastocysts. In vivo-produced blastocysts were randomly divided into three groups: The first group was vitrified and warmed; the second group underwent artificial collapse of the blastocoel cavity prior to vitrification and warming; the third group served as the control, in which neither vitrification or artificial collapse was performed. The survival rate of treatment groups was similar to the control group, whereas the hatching rate of artificial collapse/vitrified blastocysts was significantly higher than vitrified blastocysts. Quantitative reverse-transcription PCR analysis revealed a considerable reduction in the expression of Cdx2, Eomes, Gata6, Grb2, and Tp53 transcripts following artificial collapse/vitrification in comparison to the vitrification-alone group; the abundance of Pou5f1 and Nanog, however, did not change. These results suggest that artificial collapse of the blastocoel cavity before vitrification leads to relatively normal expression of apoptosis and development-related genes plus higher hatching rates. Mol. Reprod. Dev. 83: 735-742, 2016 © 2016 Wiley Periodicals, Inc. PMID:27409768

  18. A retrospective study of single frozen-thawed blastocyst transfer

    PubMed Central

    Ryu, Eun Kyung; Song, Seung Hyun; Yoon, San Hyun; Lim, Kyung Sil; Lee, Won Don; Lim, Jin Ho

    2016-01-01

    Objective To study the clinical outcomes of single frozen-thawed blastocyst transfer cycles according to the hatching status of frozen-thawed blastocysts. Methods Frozen-thawed blastocysts were divided into three groups according to their hatching status as follows: less-than-expanded blastocyst (≤EdB), hatching blastocyst (HgB), and hatched blastocyst (HdB). The female age and infertility factors of each group were evaluated. The quality of the single frozen-thawed blastocyst was also graded as grade A, tightly packed inner cell mass (ICM) and many cells organized in the trophectoderm epithelium (TE); grade B, several and loose ICM and TE; and grade C, very few ICM and a few cells in the TE. The clinical pregnancy and implantation rate were compared between each group. The data were analyzed by either t-test or chi-square analysis. Results There were no statistically significant differences in average female ages, infertility factors, or the distribution of blastocyst grades A, B, and C in each group. There was no significant difference in the clinical pregnancy and implantation rate of each group according to their blastocyst grade. However, there was a significant difference in the clinical pregnancy and implantation rate between each group. In the HdB group, the clinical pregnancy and implantation rate were similar regardless of the blastocyst quality. Conclusion There was an effect on the clinical outcomes depending on whether the blastocyst hatched during single frozen-thawed blastocyst transfer. When performing single frozen-thawed blastocyst transfer, the hatching status of the frozen-thawed blastocyst may be a more important parameter for clinical outcomes than the quality of the frozen-thawed blastocyst. PMID:27358829

  19. Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

    PubMed Central

    Wang, Kai; Sengupta, Satyaki; Magnani, Luca; Wilson, Catherine A.; Henry, R. William; Knott, Jason G.

    2010-01-01

    During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development. PMID:20485553

  20. Short-term urea feeding decreases in vitro hatching of bovine blastocysts.

    PubMed

    Ferreira, F A; Gomez, R G G; Joaquim, D C; Watanabe, Y F; de Castro e Paula, L A; Binelli, M; Rodrigues, P H M

    2011-07-15

    Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL ± 1.13 vs 22.12 mg/dL ± 0.86; mean ± SEM), neither the number of COCs recovered (8.8 ± 1.0 vs 9.2 ± 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes. PMID:21496898

  1. Assessment of actin cytoskeleton and nuclei in bovine blastocysts developed under different culture conditions using a novel computer program.

    PubMed

    Kuzmany, A; Havlicek, V; Brem, G; Walter, I; Besenfelder, U

    2011-02-01

    This study was performed to investigate the effects, in terms of nuclear material and actin cytoskeleton quantities (fluorescent pixel counts), of four different bovine blastocyst culturing techniques (in vitro, stepwise in vitro-to-in vivo, or purely in vivo). Cumulus oocyte complexes from abattoir-sourced ovaries were matured in vitro and allocated to four groups: IVP-group embryos developed up to blastocyst stage in vitro. Gamete intra-fallopian transfer (GIFT)-group oocytes were co-incubated with semen for 4 h before transfer to oviducts of heifers. Following in vitro fertilization, cleaved embryos (day 2 of embryo development, day 2-7 group) were transferred into oviducts on day 2. Multiple ovulation embryo transfer (MOET)-group embryos were obtained by superovulating and inseminating heifers; the heifers' genital tracts were flushed at day 7 of blastocyst development. Within each group, ten blastocysts were selected to be differentially dyed (for nuclei and actin cytoskeleton) with fluorescent stains. A novel computer program (ColorAnalyzer) provided differential pixel counts representing organelle quantities. Blastocysts developed only in vivo (MOET group) showed significantly more nuclear material than did blastocysts produced by any other technique. In terms of actin cytoskeleton quantity, blastocysts produced by IVP and by day 2-7 transfer did not differ significantly from each other. Gamete intra-fallopian transfer- and MOET-group embryos showed significantly larger quantities of actin cytoskeleton when compared with any other group and differed significantly from each other. The results of this study indicate that culturing under in vitro conditions, even with part time in vivo techniques, may adversely affect the quantity of blastocyst nuclear material and actin cytoskeleton. The software employed may be useful for culture environment evaluation/developmental competence assessment. PMID:20477985

  2. Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro

    PubMed Central

    Salilew-Wondim, Dessie; Fournier, Eric; Hoelker, Michael; Saeed-Zidane, Mohammed; Tholen, Ernst; Looft, Christian; Neuhoff, Christiane; Besenfelder, Urban; Havlicek, Vita; Rings, Franca; Gagné, Dominic; Sirard, Marc-André; Robert, Claude; A. Shojaei Saadi, Habib; Gad, Ahmed; Schellander, Karl; Tesfaye, Dawit

    2015-01-01

    Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and

  3. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology

    NASA Astrophysics Data System (ADS)

    Tenzer, Stefan; Docter, Dominic; Kuharev, Jörg; Musyanovych, Anna; Fetz, Verena; Hecht, Rouven; Schlenk, Florian; Fischer, Dagmar; Kiouptsi, Klytaimnistra; Reinhardt, Christoph; Landfester, Katharina; Schild, Hansjörg; Maskos, Michael; Knauer, Shirley K.; Stauber, Roland H.

    2013-10-01

    In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.

  4. The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification

    PubMed Central

    Kim, Hye Jin; Lee, Ki Hwan; Park, Sung Baek; Choi, Young Bae

    2015-01-01

    Objective The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. Methods In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. Results The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). Conclusion The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice. PMID:26473108

  5. The spatiotemporal hormonal orchestration of human folliculogenesis, early embryogenesis and blastocyst implantation.

    PubMed

    Atwood, Craig S; Vadakkadath Meethal, Sivan

    2016-07-15

    The early reproductive events starting with folliculogenesis and ending with blastocyst implantation into the uterine endometrium are regulated by a complex interplay among endocrine, paracrine and autocrine factors. This review examines the spatiotemporal integration of these maternal and embryonic signals that are required for successful reproduction. In coordination with hypothalamic-pituitary-gonadal (HPG) hormones, an intraovarian HPG-like axis regulates folliculogenesis, follicular quiescence, ovulation, follicular atresia, and corpus luteal functions. Upon conception and passage of the zygote through the fallopian tube, the contribution of maternal hormones in the form of paracrine secretions from the endosalpinx to embryonic development declines, with autocrine and paracrine signaling becoming increasingly important as instructional signals for the differentiation of the early zygote/morula into a blastocyst. These maternal and embryonic signals include activin and gonadotropin-releasing hormone 1 (GnRH1) that are crucial for the synthesis and secretion of the 'pregnancy' hormone human chorionic gonadotropin (hCG). hCG in turn signals pre-implantation embryonic cell division and sex steroid production required for stem cell differentiation, and subsequent blastulation, gastrulation, cavitation and blastocyst formation. Upon reaching the uterus, blastocyst hatching occurs under the influence of decreased activin signaling, while the attachment and invasion of the trophoblast into the endometrium appears to be driven by a decrease in activin signaling, and by increased GnRH1 and hCG signaling that allows for tissue remodeling and the controlled invasion of the blastocyst into the uterine endometrium. This review demonstrates the importance of integrative endocrine, paracrine, and autocrine signaling for successful human reproduction. PMID:27045358

  6. Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats.

    PubMed

    Romaguera, R; Moll, X; Morató, R; Roura, M; Palomo, M J; Catalá, M G; Jiménez-Macedo, A R; Hammami, S; Izquierdo, D; Mogas, T; Paramio, M T

    2011-07-01

    Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality. PMID:21295839

  7. How does tidal flow affect pattern formation in mussel beds?

    PubMed

    Sherratt, Jonathan A; Mackenzie, Julia J

    2016-10-01

    In the Wadden Sea, mussel beds self-organise into spatial patterns consisting of bands parallel to the shore. A leading explanation for this phenomenon is that mussel aggregation reduces losses from dislodgement and predation, because of the adherence of mussels to one another. Previous mathematical modelling has shown that this can lead to spatial patterning when it is coupled to the advection from the open sea of algae-the main food source for mussels in the Wadden Sea. A complicating factor in this process is that the advection of algae will actually oscillate with the tidal flow. This has been excluded from previous modelling studies, and the present paper concerns the implications of this oscillation for pattern formation. The authors initially consider piecewise constant ("square-tooth") oscillations in advection, which enables analytical investigation of the conditions for pattern formation. They then build on this to study the more realistic case of sinusoidal oscillations. Their analysis shows that future research on the details of pattern formation in mussel beds will require an in-depth understanding of how the tides affect long-range inhibition among mussels. PMID:27343625

  8. Alteration of the langerin oligomerization state affects Birbeck granule formation.

    PubMed

    Chabrol, Eric; Thépaut, Michel; Dezutter-Dambuyant, Colette; Vivès, Corinne; Marcoux, Julien; Kahn, Richard; Valladeau-Guilemond, Jenny; Vachette, Patrice; Durand, Dominique; Fieschi, Franck

    2015-02-01

    Langerin, a trimeric C-type lectin specifically expressed in Langerhans cells, has been reported to be a pathogen receptor through the recognition of glycan motifs by its three carbohydrate recognition domains (CRD). In the context of HIV-1 (human immunodeficiency virus-1) transmission, Langerhans cells of genital mucosa play a protective role by internalizing virions in Birbeck Granules (BG) for elimination. Langerin (Lg) is directly involved in virion binding and BG formation through its CRDs. However, nothing is known regarding the mechanism of langerin assembly underlying BG formation. We investigated at the molecular level the impact of two CRD mutations, W264R and F241L, on langerin structure, function, and BG assembly using a combination of biochemical and biophysical approaches. Although the W264R mutation causes CRD global unfolding, the F241L mutation does not affect the overall structure and gp120 (surface HIV-1 glycoprotein of 120 kDa) binding capacities of isolated Lg-CRD. In contrast, this mutation induces major functional and structural alterations of the whole trimeric langerin extracellular domain (Lg-ECD). As demonstrated by small-angle x-ray scattering comparative analysis of wild-type and mutant forms, the F241L mutation perturbs the oligomerization state and the global architecture of Lg-ECD. Correlatively, despite conserved intrinsic lectin activity of the CRD, avidity property of Lg-ECD is affected as shown by a marked decrease of gp120 binding. Beyond the change of residue itself, the F241L mutation induces relocation of the K200 side chain also located within the interface between protomers of trimeric Lg-ECD, thereby explaining the defective oligomerization of mutant Lg. We conclude that not only functional CRDs but also their correct spatial presentation are critical for BG formation as well as gp120 binding. PMID:25650933

  9. Effect of hyaluronan on developmental competence and quality of oocytes and obtained blastocysts from in vitro maturation of bovine oocytes.

    PubMed

    Opiela, Jolanta; Romanek, Joanna; Lipiński, Daniel; Smorąg, Zdzisław

    2014-01-01

    The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P < 0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P < 0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure. PMID:24689043

  10. Mouse embryo motion and embryonic development from the 2-cell to blastocyst stage using mechanical vibration systems.

    PubMed

    Asano, Yuka; Matsuura, Koji

    2014-06-01

    We investigated the effect of mechanical stimuli on mouse embryonic development from the 2-cell to blastocyst stage to evaluate physical factors affecting embryonic development. Shear stress (SS) applied to embryos using two mechanical vibration systems (MVSs) was calculated by observing microscopic images of moving embryos during mechanical vibration (MV). The MVSs did not induce any motion of the medium and the diffusion rate using MVSs was the same as that under static conditions. Three days of culture using MVS did not improve embryonic development. MVS transmitted MV power more efficiently to embryos than other systems and resulted in a significant decrease in development to the morula or blastocyst stage after 2 days. Comparison of the results of embryo culture using dynamic culture systems demonstrated that macroscopic diffusion of secreted materials contributes to improved development of mouse embryos to the blastocyst stage. These results also suggest that the threshold of SS and MV to induce negative effects for mouse embryos at stages earlier than the blastocyst may be lower than that for the blastocyst, and that mouse embryos are more sensitive to physical and chemical stimuli than human or pig embryos because of their thinner zona pellucida. PMID:23697534

  11. Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

    PubMed

    Veshkini, Arash; Khadem, Ali Akbar; Mohammadi-Sangcheshmeh, Abdollah; Alamouti, Ali Asadi; Soleimani, Masoud; Gastal, Eduardo L

    2016-08-01

    The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 μM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 μM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 μM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 μM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 μM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 μM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression. PMID:26584822

  12. Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos.

    PubMed

    Feugang, Jean-Magloire; Donnay, Isabelle; Mermillod, Pascal; Marchandise, Joelle; Lequarre, Anne-Sophie

    2005-07-01

    Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos. PMID:15806560

  13. Changes in tri-methylation profile of lysines 4 and 27 of histone H3 in bovine blastocysts after cryopreservation.

    PubMed

    Maldonado, Mariângela Bueno Cordeiro; Penteado, João Carlos Torrente; Faccio, Bianca Maria Campanelli; Lopes, Flavia Lombardi; Arnold, Daniel Robert

    2015-12-01

    Pregnancy rates from cryopreserved embryos remain lower than non-cryopreserved counterparts, even though these embryos appear morphologically normal. How epigenetic events, such as histone modifications, are affected by cryopreservation of embryos remains unknown. The current study evaluated the effect of conventional freezing/thawing of in vitro produced bovine blastocyst embryos on histone modifications, H3K4me3 and H3K27me3. At day 7 of in vitro culture, blastocyst stage embryos were either frozen by conventional freezing method (-0.5 °C/min in 1.5 M ethylene glycol; F/T group) or remained in culture for an additional 18 h (Ctrl). Frozen embryos were stored in liquid N2 for 14 days, thawed and placed in culture for 36 h for recovery. Control and re-expanded frozen-thawed blastocysts from both groups were fixed in 4% paraformaldehyde and stored in PBS +0.1% triton-X at 4 °C. Immunofluorescence, utilizing antibodies against H3K4me3 and H3K27me3, was conducted and staining intensity was analyzed as percentage of total DNA. Day 7 blastocyst development rate was 35.55% (352/990) with blastocyst recovery at 54.23% (77/142) 36 h post-thawing. Total cell numbers per blastocyst were not different amongst groups (117.8 ± 12.49 and 116.1 ± 14.69, F/T and Ctrl groups respectively). Global staining for the active mark, H3K4me3, was lower in F/T blastocysts compared to Ctrl (17.24 ± 2.80% vs. 34.95 ± 3.77%; P < 0.01). However, staining for the inhibitory mark, H3K27me3, was nearly 2-fold higher in F/T blastocysts (40.41 ± 3.83% vs. 21.29 ± 3.92%; P < 0.01). These results suggest that bovine blastocysts, subjected to conventional freezing methods, have altered histone modifications that may play a role in poor pregnancy rates. PMID:26408849

  14. Successful vitrification of bovine blastocysts on paper container.

    PubMed

    Kim, Y M; Uhm, S J; Gupta, M K; Yang, J S; Lim, J-G; Das, Z C; Heo, Y T; Chung, H-J; Kong, I-K; Kim, N-H; Lee, H T; Ko, D H

    2012-09-15

    Cryopreservation of bovine embryos can be performed by a variety of methods with variable degree of success. Here, we report a new, easy to perform, simple, inexpensive, and successful method for vitrification of bovine blastocysts. In vitro produced bovine blastocysts were exposed to vitrification solution (5.5 m ethylene glycol, 10% serum and 1% sucrose) in one single step for 20 s, loaded on a paper container prepared from commonly available non-slippery, absorbent writing paper, and then were directly plunged into liquid nitrogen for storage. Vitrified blastocysts were warmed by serial rinsing in 0.5, 0.25 and 0.125 m sucrose solution for 1 min each. Results showed that one step exposure of bovine blastocysts to cryoprotective agents was sufficient to achieve successful cryopreservation. Under these conditions, more than 95% of blastocysts survived the vitrification-warming on paper containers which was significantly higher than those obtained from other containers, such as electron microscope (EM) grid (78.1%), open pulled straw (OPS; 80.2%), cryoloop (76.2%) or plastic straw (73.9%). Embryo transfer of blastocysts vitrified-warmed on paper container resulted in successful conception (19.3%) and full-term live birth of offspring (12.3%) which were lower (P < 0.05) than those obtained from non-vitrified blastocysts (38.0 and 32.7%) but were comparable (P > 0.05) to those obtained from blastocysts vitrified-warmed on EM grid (23.3 and 14.2%). Our results, therefore, suggest that paper may be an inexpensive and useful container for the cryopreservation of animal embryos. PMID:22763071

  15. Comparison of Cryotop and micro volume air cooling methods for cryopreservation of bovine matured oocytes and blastocysts

    PubMed Central

    PUNYAWAI, Kanchana; ANAKKUL, Nitira; SRIRATTANA, Kanokwan; AIKAWA, Yoshio; SANGSRITAVONG, Siwat; NAGAI, Takashi; IMAI, Kei; PARNPAI, Rangsun

    2015-01-01

    This study was designed to compare the efficiency of the Cryotop method and that of two methods that employ a micro volume air cooling (MVAC) device by analyzing the survival and development of bovine oocytes and blastocysts vitrified using each method. In experiment I, in vitro-matured (IVM) oocytes were vitrified using an MVAC device without direct contact with liquid nitrogen (LN2; MVAC group) or directly plunged into LN2 (MVAC in LN2 group). A third group of IVM oocytes was vitrified using a Cryotop device (Cryotop group). After warming, vitrified oocytes were fertilized in vitro. There were no significant differences in cleavage and blastocyst formation rates among the three vitrified groups, with the rates ranging from 53.1% to 56.6% and 20.0% to 25.5%, respectively; however, the rates were significantly lower (P < 0.05) than those of the fresh control group (89.3% and 43.3%, respectively) and the solution control group (87.3% and 42.0%, respectively). In experiment II, in vitro-produced (IVP) expanded blastocysts were vitrified using the MVAC, MVAC in LN2 and Cryotop methods, warmed and cultured for survival analysis and then compared with the solution control group. The rate of development of vitrified-warmed expanded blastocysts to the hatched blastocyst stage after 24 h of culture was lower in the MVAC in LN2 group than in the solution control group; however, after 48–72 h of culture, the rates did not significantly differ between the groups. These results indicate that the MVAC method without direct LN2 contact is as effective as the standard Cryotop method for vitrification of bovine IVM oocytes and IVP expanded blastocysts. PMID:26119929

  16. Thyroid hormones alter the transcriptome of in vitro-produced bovine blastocysts.

    PubMed

    Ashkar, Fazl A; Revay, Tamas; Rho, NaYoung; Madan, Pavneesh; Dufort, Isabelle; Robert, Claude; Favetta, Laura A; Schmidt, Chris; King, W Allan

    2016-04-01

    Thyroid hormones (THs) have been shown to improve in vitro embryo production in cattle by increasing blastocyst formation rate, and the average cell number of blastocysts and by significantly decreasing apoptosis rate. To better understand those genetic aspects that may underlie enhanced early embryo development in the presence of THs, we characterized the bovine embryonic transcriptome at the blastocyst stage, and examined differential gene expression profiles using a bovine-specific microarray. We found that 1212 genes were differentially expressed in TH-treated embryos when compared with non-treated controls (>1.5-fold at P < 0.05). In addition 23 and eight genes were expressed uniquely in control and treated embryos, respectively. The expression of genes specifically associated with metabolism, mitochondrial function, cell differentiation and development were elevated. However, TH-related genes, including those encoding TH receptors and deiodinases, were not differentially expressed in treated embryos. Furthermore, the over-expression of 52 X-chromosome linked genes in treated embryos suggested a delay or escape from X-inactivation. This study highlights the significant impact of THs on differential gene expression in the early embryo; the identification of TH-responsive genes provides an insight into those regulatory pathways activated during development. PMID:26099992

  17. Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells.

    PubMed

    Hara, Hiromasa; Goto, Teppei; Takizawa, Akiko; Sanbo, Makoto; Jacob, Howard J; Kobayashi, Toshihiro; Nakauchi, Hiromitsu; Hochi, Shinichi; Hirabayashi, Masumi

    2016-04-01

    Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs. PMID:26990947

  18. Progesterone and its downstream molecules as blastocyst implantation essential factors.

    PubMed

    Yoshinaga, Koji

    2014-08-01

    This review is to update the previous review (Am J Reprod Immunol, 63, 2010 and 413) on the research on blastocyst implantation essential factors (BIEFs). Focus of the current review is on progesterone and its downstream molecules in the process of blastocyst implantation. To understand the process of implantation, we need to know where and when the BIEFs are expressed and what they do. Progress in this research area is rapid, and its update is indeed necessary. The basic concept of BIEFs is that they have dual functions, one physiological and the other immunological (J Reprod Dev, 58, 2012 and 196). As we are still exploring the mechanism of implantation, available data are incomplete and human data are few. Thus, I will use information obtained through research on animal models, in vitro studies, cell lines, and some human studies where available. The ultimate goal of the review is to understand human blastocyst implantation. PMID:24754263

  19. Selective single blastocyst transfer study: 604 cases in 6 years

    PubMed Central

    Sadasivam, Nirmala; Sadasivam, Narayanan M.

    2008-01-01

    AIM: To evaluate the credibility of single blastocyst transfer (SBT) method in selected group of patients. SETTINGS AND DESIGN: Retrospective analysis of SBT cases based on computerized data in a private Fertility research centre. MATERIALS AND METHODS: A total of 604 cases of SBTs, done during June 2000 to June 2006, have been analyzed retrospectively to assess the credibility of the method as a method of choice in selective high fertile group of patients. Women between 28 and 42 years have been included in the retrospective analysis, who had adequate number of eggs for fertilization, between 6 and 12. RESULTS AND CONCLUSIONS: Grade I blastocyst transfer resulted in 46.6% of clinical pregnancy and grade II blastocyst transfer resulted in 17.4% of clinical pregnancy rates. Overall pregnancy rate was 64%. Pregnancy loss, as early and late fetal wastages, was 11.06%. PMID:19562057

  20. Choline inhibition of amino acid transport in preimplantation mouse blastocysts

    SciTech Connect

    Campione, A.L.; Haghighat, N.; Gorman, J.; Van Winkle, L.J.

    1987-05-01

    Addition of 70 mM choline chloride to Brinster's medium (140 mM Na/sup +/) inhibited uptake of approx. 1 ..mu..M (/sup 3/H)glycine, leucine, lysine and alanine in blastocysts by about 50% each during a five-minute incubation period at 37/sup 0/C, whereas 70 mM LiCl, sodium acetate and NaCl or 140 mM mannitol had no effect. They attribute the apparent linear relationship between Gly transport in blastocysts and the square of the (Na/sup +/), observed when choline was substituted for Na/sup +/ in Brinster's medium, to concomitant, concentration-dependent enhancement and inhibition of transport by Na/sup +/ and choline, respectively. As expected, Gly uptake and the (Na/sup +/) were linearly related up to 116 mM Na/sup +/, when Na/sup +/ was replaced with Li/sup +/. The rates of Na/sup +/-independent Gly and Ala uptake were <5% and <2% of the total, respectively, and similar when either Li/sup +/ or choline replaced Na/sup +/. Therefore, neither Li/sup +/ nor choline appears to substitute for Na/sup +/ in supporting Na/sup +/-dependent transport in blastocysts. Na/sup +/-independent Leu uptake was 20 times faster than Gly or Ala uptake and appeared to be inhibited by choline in blastocysts since it was about 37% slower when choline instead of Li/sup +/ was substituted for Na/sup +/. In contrast to blastocysts, choline had no effect on amino acid transport in cleavage-stage mouse embryos. The unexpected sensitivity of transport to choline in blastocysts underscores the importance of testing the effects of this substance when it is used to replace Na/sup +/ in new transport studies.

  1. Blastocyst Elongation, Trophoblastic Differentiation and Embryonic Pattern Formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular basis behind elongation and concomitant gastrulation in ungulates that occurs during pre-implantation is still poorly understood. In-depth transcriptome analysis of the elongating porcine conceptus at specific stages has demonstrated that protein synthesis, protein trafficking, cell g...

  2. How Temperature and Water levels affect Polar Mesospheric Cloud Formation

    NASA Astrophysics Data System (ADS)

    Smith, L. L.; Randall, C. E.; Harvey, V.

    2012-12-01

    Using the Cloud Imaging and Particle Size (CIPS) instrument data, which is part of the Aeronomy in the Mesosphere (AIM) mission, we compare the albedo and ice water content measurements of CIPS with the Navy Operation Global Atmospheric Prediction System - Advanced Level Phyiscs and High Altitude (NOGAPS-ALPHA) temperature and water vapor data in order to derive a greater understanding of cloud formation and physics. We particularly focus on data from June 2007 and July 2007 in this case study because of particular cloud structures and formations during this time period for future studies.

  3. Dynamical effects during compaction band formation affecting their spatial periodicity

    NASA Astrophysics Data System (ADS)

    Cecinato, Francesco; Gajo, Alessandro

    2014-10-01

    Compaction bands (CBs) are responsible for significant anisotropy alterations of permeability in geological materials; hence, understanding their formation conditions appears of key importance to all applications involving fluid extraction/injection from/into the ground. While most of the available models to understand CB formation are focused on interpreting the onset of a single CB, little effort has been so far dedicated to understand the documented periodicity of CBs. In this paper, the role of dynamical effects in inducing the post onset evolution of CBs is analyzed by means of a dedicated model for porous media with compressible constituents, with reference to a horizontal layer of sandy, water-saturated material. Elastic waves are generated as a first CB occurs due to sudden, localized volumetric collapse. If the waves are reflected at the interface with a softer material or with a previously formed CB, they produce significant local effective stress concentrations, which can promote the formation of further CBs in a cascade fashion, according to a regular geometric pattern. The spatial distribution of dynamically generated CBs, as well as the extent of the phenomenon, depends on the geometry of the domain and on the material's permeability. Sensitivity analysis is also performed to assess the key properties that promote dynamical CB in situ formation, identifying as the most influential conditions large stratum stiffness (increasing with depth) and the presence of softer layers. In contrast, the presence of less permeable and/or stiffer layers is not believed to play a major role in the proposed mechanism.

  4. Should we be promoting embryo transfer at blastocyst stage?

    PubMed

    Maheshwari, Abha; Hamilton, Mark; Bhattacharya, Siladitya

    2016-02-01

    Improved laboratory standards and better culture media have made extended culture to blastocyst stage a reality to identify embryos with maximum implantation potential. The strategy of extended culture has become more popular across the world at a time when regulatory bodies have emphasized the need to increase the uptake of elective single embryo transfer, minimize complications associated with multiple births and aim for a healthy singleton live-birth as the preferred outcome in IVF. New data on perinatal outcomes suggest that pregnancies after embryo transfer at blastocyst stage are associated with a higher risk of preterm delivery, large for gestational age babies, monozygotic twins and altered sex ratio compared with those following embryo transfers at cleavage stage. In addition, concerns have been raised of increased congenital anomalies and epigenetic modifications with embryo transfer at blastocyst stage. Twenty-four years on from the first embryo transfer at blastocyst stage, we examine the reasons for extended embryo culture, evaluate the risks and benefits of this strategy and suggest the need to reconsider this policy in the interests of fetal safety. PMID:26673100

  5. Factors Affecting the Formation of Food Preferences in Preschool Children.

    ERIC Educational Resources Information Center

    Alles-White, Monica L.; Welch, Patricia

    1985-01-01

    Identifies and discusses factors that affect the development of food preferences in preschool children, including familiarity, age, parents, peers, teachers, and programs designed to influence food habits. Makes recommendations to preschool and day care programs for creating an atmosphere conducive to trying new foods. (Author/DST)

  6. Reproductive outcomes of retransferring retained embryos in blastocyst transfer cycles

    PubMed Central

    Yi, Hyun Jeong; Koo, Hwa Seon; Cha, Sun Hwa; Kim, Hye Ok; Park, Chan Woo

    2016-01-01

    Objective To determine the incidence of embryo retention (ER) in the transfer catheter following embryo transfer (ET) in blastocyst transfer and investigate whether retransferring retained embryos has an impact on reproductive outcomes in patients undergoing in vitro fertilization-ET. Methods We retrospectively analyzed the records of 1,131 blastocyst transfers, which comprised 223 single blastocyst transfer (SBT) and 908 double blastocyst transfer (DBT) cycles. Each SBT and DBT group was classified depending on whether ET was performed without retained embryos in the catheter during the first attempt (without-ER group) or whether any retained embryos were found following ET (ER group) for the purpose of comparing reproductive outcomes in a homogenous population. Results The overall incidence of finding retained embryos was 2.8% (32/1,131). There were no retained embryos in SBT cycles. In DBT cycles, implantation rates (30.0% vs. 26.6%), positive β-hCG rates (57.2% vs. 56.2%), clinical pregnancy rates (45.3% vs. 46.9%), and live birth rates (38.9% vs. 43.8%) were not significantly different between the without-ER and ER groups. There were no significant differences in the mean birth weight (g) 2,928.4±631.8 vs. 2,948.7±497.8 and the mean gestational age at birth (269.3±17.2 days vs. 264.2±25.7 days). A total of nine cases of congenital birth defects were found in this study population. Eight were observed in the without-ER group and one in the ER group. Conclusion Our results suggest that retransfer of retained embryos does not have any adverse impact on reproductive outcomes in blastocyst transfer cycles. Furthermore, our results support finding that SBT might be advantageous for decreasing the incidence of retained embryos in catheters. PMID:27358833

  7. Conductivity affects nanosecond electrical pulse induced pressure transient formation

    NASA Astrophysics Data System (ADS)

    Roth, Caleb C.; Barnes, Ronald A.; Ibey, Bennett L.; Beier, Hope T.; Glickman, Randolph D.

    2016-03-01

    Nanoporation occurs in cells exposed to high amplitude short duration (< 1μs) electrical pulses. The biophysical mechanism(s) responsible for nanoporation is unknown although several theories exist. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. Our group has shown that mechanical forces of substantial magnitude are also generated during nsEP exposures. We hypothesize that these mechanical forces may contribute to pore formation. In this paper, we report that alteration of the conductivity of the exposure solution also altered the level of mechanical forces generated during a nsEP exposure. By reducing the conductivity of the exposure solutions, we found that we could completely eliminate any pressure transients normally created by nsEP exposure. The data collected for this proceeding does not definitively show that the pressure transients previously identified contribute to nanoporation; however; it indicates that conductivity influences both survival and pressure transient formation.

  8. Effect of knockout serum replacement supplementation to culture medium on porcine blastocyst development and piglet production.

    PubMed

    Sakurai, Masahiro; Suzuki, Chie; Yoshioka, Koji

    2015-03-01

    We have previously developed chemically defined media suitable for in vitro production (IVP) of porcine embryos and subsequently generated piglets by nonsurgical embryo transfer. In this study, to further improve the culture conditions for IVP of porcine embryos, we evaluated the effect of knockout serum replacement (KSR), a substitute for serum or albumin, on the viability and development of porcine blastocysts. The addition of 5% (v:v) KSR to porcine blastocyst medium (PBM) on Day 5 (Day 0 = IVF) significantly increased the survival and hatching rates of blastocysts and the total cell number of Day-7 blastocysts compared with those in cultures without KSR or addition of 10% fetal bovine serum. Furthermore, the number of cells in the trophectoderm of Day-6 blastocysts and the ATP content of Day-7 blastocysts cultured with 5% KSR were significantly higher than those of blastocysts cultured without KSR. The mRNA expression of a rate-limiting enzyme in β-oxidation, carnitine palmitoyltransferase 1, in Day-6 blastocysts, and a serine proteinase, urokinase-type plasminogen activator, in Day-7 blastocysts cultured in 5% KSR-PBM was significantly higher than that of blastocysts cultured in PBM alone. Four of eight recipients (50%), in which Day-5 blastocysts treated with 5% KSR were transferred nonsurgically, became pregnant. However, the efficiency of piglet production (percentage of piglets born based on the number of embryos transferred) was similar to recipients with transferred blastocysts treated without KSR. The present study demonstrated that the addition of KSR to PBM enhanced the in vitro viability of porcine blastocysts. In addition, our data suggest that KSR improved development to the hatching stage and blastocyst quality by increasing ATP content and hatching-related mRNA expression of blastocysts. PMID:25434774

  9. Ginsenoside Rg1 Improves In vitro-produced Embryo Quality by Increasing Glucose Uptake in Porcine Blastocysts.

    PubMed

    Kim, Seung-Hun; Choi, Kwang-Hwan; Lee, Dong-Kyung; Oh, Jong-Nam; Hwang, Jae Yeon; Park, Chi-Hun; Lee, Chang-Kyu

    2016-08-01

    Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 (20 μg/mL) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions. PMID:26954154

  10. Comparison of Blastocyst and Sage Media for In Vitro Maturation of Human Immature Oocytes

    PubMed Central

    Pongsuthirak, Pallop; Songveeratham, Sorramon

    2015-01-01

    In vitro maturation (IVM) of human oocytes is an attractive alternative to conventional assisted reproductive technology (ART) treatment, as it involves no or minimal ovarian stimulation. Currently, commercialized media specifically designed for IVM are often used. These media are expensive, have limited shelf life, and must be ordered in advance. If standard culture media can be used in place of the specialized IVM media, it would simplify management and make IVM more feasible and more widely employed in ART centers around the world, especially in developing countries where resources are scarce. This study was, therefore, conducted to test the hypothesis that blastocyst medium was as good as commercial IVM medium to support maturation and developmental competence of human immature oocytes as previously shown in the mouse system. Immature oocytes were obtained by needle aspiration from 89 pregnant women during cesarean deliveries between April 2012 and February 2013. Sibling oocytes were allocated to Sage IVM media (512 oocytes) or blastocyst medium (520 oocytes) and assessed for maturation 36 hours later. Mature oocytes were inseminated by intracytoplasmic sperm injection and cultured up to 144 hours. There was no difference in maturation rate (65.0% vs 68.7%; P = .218) or fertilization rate (66.9% vs 66.4%; P = .872) of oocytes matured in vitro in both media. There was also no difference in the formation of good-quality blastocysts (46.6% vs 45.9%; P = .889) in the 2 groups. Further study should be done to ascertain implantation and pregnancy potential of these embryos. PMID:25015901

  11. Morphokinetics of cloned mouse embryos treated with epigenetic drugs and blastocyst prediction.

    PubMed

    Mallol, Anna; Piqué, Laia; Santaló, Josep; Ibáñez, Elena

    2016-03-01

    Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success. PMID:26621919

  12. Cochlear otosclerosis: does bone formation affect cochlear implant surgery?

    PubMed

    Fayad, J; Moloy, P; Linthicum, F H

    1990-05-01

    This study aimed to demonstrate that new bone formation in the scala tympani of patients deaf from otosclerosis does not preclude cochlear implant surgery. In seven temporal bones from patients with otosclerosis, we measured the extent of new bone from the round window to the distal part of the new growth. We compared results to surgical data on the extent of drilling and depth and ease of placement of the electrode in 20 patients deaf from otosclerosis. We also examined clinical performance and voltage requirements for long-term implant use in patients with and patients without ossification of the scala tympani. Findings in our limited sample of patients and bones show that obstruction of the basal turn, which occurs in some otosclerotic patients, does not preclude implant surgery. The dynamic range in the studied sample was relatively stable long-term and clinical performance did not differ between groups with and without an ossified scala tympani. PMID:2188511

  13. Histone octamer acetylation affects the free energy of nucleosome formation

    NASA Astrophysics Data System (ADS)

    Mooney, Alex; Manohar, Mridula; Edon, Annick; Nakkula, Robin; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Nucleosomes, histone octamer-DNA complexes, form the fundamental repeating units of eukaryotic chromatin. Numerous post-translational modifications of histone octamers are found in vivo and are known to play roles in gene regulation and DNA repair, but the molecular functions of these modifications are not well understood. In this study we consider the effects of acetylating histone protein H3 residues Lys^115 and Lys^122. These modifications reduce the positive surface charge of the histone octamer at contact points with the negatively charged DNA phosphate backbone and add steric bulk in the dyad region. We report results from competitive reconstitutions that show the free energy of nucleosome formation between wild-type and modified histone octamer binding to a strong nucleosome positioning sequence is reduced. These results suggest that these modifications may be involved in nucleosome assembly and disassembly.

  14. Vitrification of oocytes, embryos and blastocysts.

    PubMed

    Mukaida, Tetsunori; Oka, Chikahiro

    2012-12-01

    In assisted reproductive technology, cryopreservation of human oocytes and embryos has been significantly improved by refined slow-cooling and the new vitrification method. The slow-cooling method requires a programmed cryo-machine, and usually takes several hours. It is, however, difficult to eliminate injuries resulting from ice formation completely. Vitrification has become a reliable strategy because it is simple, can lead to high survival rates and viability, and has better clinical outcome. Vitrification transforms cells into an amorphous glassy state inside and outside the vitrified cell with ultra-rapid cooling and warming steps by plunging the oocytes and embryos into liquid nitrogen, instead of ice-crystal formation. Over the past decade, several advances in vitrification technologies have improved clinical efficiency and outcome. In this chapter, we focus on vitrification technologies for cryopreservation in human assisted reproductive technology. PMID:22940094

  15. TIMESCALES ON WHICH STAR FORMATION AFFECTS THE NEUTRAL INTERSTELLAR MEDIUM

    SciTech Connect

    Stilp, Adrienne M.; Dalcanton, Julianne J.; Weisz, Daniel R.; Williams, Benjamin F.; Warren, Steven R.; Skillman, Evan; Ott, Juergen; Dolphin, Andrew E.

    2013-08-01

    Turbulent neutral hydrogen (H I) line widths are often thought to be driven primarily by star formation (SF), but the timescale for converting SF energy to H I kinetic energy is unclear. As a complication, studies on the connection between H I line widths and SF in external galaxies often use broadband tracers for the SF rate, which must implicitly assume that SF histories (SFHs) have been constant over the timescale of the tracer. In this paper, we compare measures of H I energy to time-resolved SFHs in a number of nearby dwarf galaxies. We find that H I energy surface density is strongly correlated only with SF that occurred 30-40 Myr ago. This timescale corresponds to the approximate lifetime of the lowest mass supernova progenitors ({approx}8 M{sub Sun }). This analysis suggests that the coupling between SF and the neutral interstellar medium is strongest on this timescale, due either to an intrinsic delay between the release of the peak energy from SF or to the coherent effects of many supernova explosions during this interval. At {Sigma}{sub SFR} > 10{sup -3} M{sub Sun} yr{sup -1} kpc{sup -2}, we find a mean coupling efficiency between SF energy and H I energy of {epsilon} = 0.11 {+-} 0.04 using the 30-40 Myr timescale. However, unphysical efficiencies are required in lower {Sigma}{sub SFR} systems, implying that SF is not the primary driver of H I kinematics at {Sigma}{sub SFR} < 10{sup -3} M{sub Sun} yr{sup -1} kpc{sup -2}.

  16. Polony analysis of gene expression in ES cells and blastocysts

    PubMed Central

    Rieger, C.; Poppino, R.; Sheridan, R.; Moley, K.; Mitra, R.; Gottlieb, D.

    2007-01-01

    Expression profiling of stem cells is challenging due to their small numbers and heterogeneity. The PCR colony (polony) approach has theoretical advantages as an assay for stem cells but has not been applied to small numbers of cells. An assay has been developed that is sensitive enough to detect mRNAs from small numbers of ES cells and from fractions of a single mouse blastocyst. Genes assayed include Oct3, Rex1, Nanog, Cdx2 and GLUT-1. The assay is highly sensitive so that multiple mRNAs from a single blastocyst were easily detected in the same assay. In its present version, the assay is an attractive alternative to conventional RT–PCR for profiling small populations of stem cells. The assay is also amenable to improvements that will increase its sensitivity and ability to analyze many cDNAs simultaneously. PMID:18073198

  17. Both the folate cycle and betaine-homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts.

    PubMed

    Zhang, Baohua; Denomme, Michelle M; White, Carlee R; Leung, Kit-Yi; Lee, Martin B; Greene, Nicholas D E; Mann, Mellissa R W; Trasler, Jacquetta M; Baltz, Jay M

    2015-03-01

    The embryonic pattern of global DNA methylation is first established in the inner cell mass (ICM) of the mouse blastocyst. The methyl donor S-adenosylmethionine (SAM) is produced in most cells through the folate cycle, but only a few cell types generate SAM from betaine (N,N,N-trimethylglycine) via betaine-homocysteine methyltransferase (BHMT), which is expressed in the mouse ICM. Here, mean ICM cell numbers decreased from 18-19 in controls to 11-13 when the folate cycle was inhibited by the antifolate methotrexate and to 12-14 when BHMT expression was knocked down by antisense morpholinos. Inhibiting both pathways, however, much more severely affected ICM development (7-8 cells). Total SAM levels in mouse blastocysts decreased significantly only when both pathways were inhibited (from 3.1 to 1.6 pmol/100 blastocysts). DNA methylation, detected as 5-methylcytosine (5-MeC) immunofluorescence in isolated ICMs, was minimally affected by inhibition of either pathway alone but decreased by at least 45-55% when both BHMT and the folate cycle were inhibited simultaneously. Effects on cell numbers and 5-MeC levels in the ICM were completely rescued by methionine (immediate SAM precursor) or SAM. Both the folate cycle and betaine/BHMT appear to contribute to a methyl pool required for normal ICM development and establishing initial embryonic DNA methylation. PMID:25466894

  18. Dihydrolipoic acid induces cytotoxicity in mouse blastocysts through apoptosis processes.

    PubMed

    Houng, Wei-Li; Lin, Cheng-An J; Shen, Ji-Lin; Yeh, Hung-I; Wang, Hsueh-Hsiao; Chang, Walter H; Chan, Wen-Hsiung

    2012-01-01

    α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA). In view of the recent application of DHLA as a hydrophilic nanomaterial preparation, determination of its biosafety profile is essential. In the current study, we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, in vivo implantation by embryo transfer, and early embryonic development in an animal model. Blastocysts treated with 50 μM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover, in vitro treatment with 50 μM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 μM DHLA led to decreased early embryo development, specifically, inhibition of development to the blastocyst stage. However, it appears that concentrations of DHLA lower than 50 μM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 μM DHLA induces apoptosis and retards early pre- and post-implantation development, and support the potential of DHLA to induce embryonic cytotoxicity. PMID:22489194

  19. Factors affecting recovery and quality of oocytes for bovine embryo production in vitro using ovum pick-up technology.

    PubMed

    Ward, F A; Lonergan, P; Enright, B P; Boland, M P

    2000-08-01

    In Experiment 1, different vacuum pressures (30, 50, 70 and 90 mm Hg) were used to aspirate 4156 bovine follicles in vitro, to assess their effect on flow rate and the recovery, morphology and blastocyst formation of the recovered oocytes. Cumulus oocyte complexes (COCs) were classified according to the morphology of the cumulus cells. Data were analyzed using Chi Square analysis. Overall recovery rate declined as the aspiration pressure increased above 50 mm Hg (P<0.05). The recovery rate of Grade 1 oocytes decreased significantly (P<0.05) as the vacuum pressure increased with a corresponding increase in the number of denuded oocytes recovered (P<0.05). The blastocyst yield, expressed as a percentage of recovered COCs decreased significantly as the aspiration pressure increased beyond 50 mm Hg (P<0.05). In Experiment 2, the holding media (hepes- or bicarbonate-buffered TCM 199) and holding time (1 h or 5 h) did not affect the blastocyst formation of the oocytes (P>0.05). In Experiment 3, it was found that individual culture of the oocyte during fertilization or culture had a detrimental effect on the oocytes blastocyst formation (8.8% to 16% blastocyst yield on Day 8) when compared to control (31.3%). In Experiment 4, groups of 5, 10 and 25 oocytes were compared with singly cultured oocytes. There were no significant differences (P<0.05) in the blastocyst formation rate among groups of 5, 10, or 25 oocytes, but there was a significant difference between oocytes processed in groups and those processed individually. In Experiment 5, when groups of 10 oocytes were cultured in different drop sizes, there was no significant difference in cleavage rates between oocytes cultured in 100 microL (85.0%, n = 280) and in 10 microL (86.8%, n = 280) of media, but culture in 50 microL (79.3%, n = 280) resulted in cleavage rates significantly lower (P<0.05) than culture in 10 microL drops. There was no significant difference in the blastocyst formation. However there was a

  20. Effect of α-tocopherol supplementation on in vitro maturation of sheep oocytes and in vitro development of preimplantation sheep embryos to the blastocyst stage

    PubMed Central

    Shankar, Madhira Bhawani; Munuswamy, Deecaraman

    2010-01-01

    Purpose To determine the effects of α-tocopherol supplementation to oocyte maturation media and embryo culture media on the yield of ovine embryos. Methods α-tocopherol, at concentrations of 0, 50, 100, 200, 400 and 500 µM was supplemented to ovine oocyte or embryo culture media and cultured at 5% or 20% O2 levels. Percentages of cleavage, morula and blastocyst, total cell count and comet assay were taken as indicators of developmental competence of embryos. Results 200 µM α-tocopherol in embryo culture medium at 20% O2 level significantly increased the rates of cleavage (P < 0.05), morulae (P < 0.05) and blastocyst (P < 0.01) formation and blastocyst total cell number (P < 0.01) when compared with control. The rates of blastocyst formation were also significantly higher in 100 µM (P < 0.01) and 400 µM (P < 0.05) supplemented groups than control. Conclusion α-tocopherol supplementation may enhance the in vitro developmental competence of ovine embryos by protecting them from oxidative damage. PMID:20454845

  1. Multiple aster formation is frequently observed in bovine oocytes retrieved from 1-day stored ovaries.

    PubMed

    Hara, H; Tagiri, M; Hirabayashi, M; Hochi, S

    2016-02-01

    We have recently reported that multiple aster formation after in vitro fertilization (IVF) was one of the factors that negatively affected the developmental competence of vitrified-warmed bovine matured oocytes, and that short-term culture of the post-warm oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the multiple aster formation and improved the blastocyst yield. The present study was conducted to investigate whether increased multiple aster formation following IVF was involved in impaired developmental competence of stored ovary-derived bovine oocytes. Oocytes retrieved from 1-day stored ovaries had lower developmental potential to day 8 blastocysts when compared with those from fresh ovaries (37 versus 63%). Immunostaining of α-tubulin 10 h post-IVF revealed that a higher incidence of multiple aster formation occurred in oocytes retrieved from stored ovaries than from fresh ovaries (31 versus 15%). Treatment of post-in vitro maturated (post-IVM) oocytes with ROCK inhibitor for 2 h significantly suppressed the incidence of multiple aster formation (10 versus 32% in the control group). However, the suppression effect of ROCK inhibitor on multiple aster formation in IVM/IVF oocytes did not improve blastocyst yield from stored ovary-derived oocytes (41 versus 37% in the control group). These results suggested that the higher incidence of multiple aster formation by bovine ovary storage was not responsible for the decreased developmental competence of IVF oocytes. PMID:25732862

  2. Utility of FT-IR imaging spectroscopy in estimating differences between the quality of bovine blastocysts

    NASA Astrophysics Data System (ADS)

    Wiecheć, A.; Opiela, J.; Lipiec, E.; Kwiatek, W. M.

    2013-10-01

    This study was conducted to verify whether the FT-IR spectroscopy and Focal Plane Array (FPA) imaging can be successfully applied to estimate the quality of bovine blastocysts (on the basis of the concentration of nucleic acids and amides). The FT-IR spectra of inner cell mass from blastocysts of three different culture systems were examined. The spectral changes between blastocysts were analyzed in DNA (spectral range of 1240-950 cm-1) and protein amides (1800-1400 cm-1). Blastocyst 1 (BL1-HA) was developed from the fertilized oocyte cultured with low concentration of hialuronian (HA), Blastocyst 2 and 3 were developed from the oocytes cultured in standard conditions. Cleavage stage blastocyst 2 (BL2-SOF) has been cultured in SOF medium while blastocyst 3 (BL3-VERO) was cultured in co-culture with VERO cells. The multivariate statistical analysis (Hierarchical Cluster Analysis - HCA and Principal Component Analysis - PCA) of single cells spectra showed high similarity of cells forming the inner cell mass within single blastocyst. The main variance between the three examined blastocysts was related to amides bands. Differences in the intensities of the amides' peaks between the bovine blastocysts derived from different culture systems indicated that specific proteins reflecting the appearance of a new phenotype were produced. However, for the three blastocysts, the α-helix typical peak was twice more intensive than the β-sheet typical peak suggesting that the differentiation processes had been started. Taking into account the quantitative and qualitative composition of the protein into examined blastocysts, it can be assumed, that the quality of the BL1-HA turned out much more similar to BL3-VERO than to BL2-SOF. FT-IR spectroscopy can be successfully applied in reproductive biology research for quality estimation of oocytes and embryos at varied stages of their development. Moreover this technique proved to be particularly useful when the quantity of the

  3. Molecular and cellular aspects of blastocyst dormancy and reactivation for implantation.

    PubMed

    Fu, Zheng; Chen, Yongjie; Wu, Weiwei; Wang, Shumin; Wang, Weixiang; Wang, Bingyan; Wang, Haibin

    2013-01-01

    Blastocyst activation, a process for the blastocyst to achieve implantation competency is equally important as attainment of uterine receptivity for the success of embryo implantation. While a wide range of regulatory molecules have been identified as essential players in conferring uterine receptivity in both laboratory animal models and humans, it remains largely unknown how blastocysts achieve implantation competency. This chapter will highlight our current knowledge about the mechanisms governing the process of blastocyst activation. A better understanding of this periimplantation event is hoped to alleviate female infertility and help to develop novel contraceptives and new strategies for accessing embryo quality in clinical practice. PMID:24698984

  4. Comparison of BALB/c and B6-albino mouse strain blastocysts as hosts for the injection of C57BL6/N-derived C2 embryonic stem cells.

    PubMed

    Alcantar, Tuija M; Wiler, Rhonda; Rairdan, Xin Y

    2016-08-01

    Embryonic stem (ES) cells from a C57BL/6N (B6N) background injected into B6(Cg)-Tyrc-2J/J (B6-albino) recipient blastocysts are commonly used for generating genetically modified mouse models. To understand the influence of the recipient blastocyst strain on germline transmission, BALB/cAnNTac and B6-albino germline transmission rates were compared using the C57BL6/N-derived C2 ES cell line. A total of 92 ES cell clones from 27 constructs were injected. We compared blastocyst yield, birth rate, chimera formation rate, and high-percentage (>50 %) male chimera formation rate. For germline transmission, we analyzed 24 clones from 19 constructs, which generated high-percentage male chimeras from both donor strains. B6-albino hosts resulted in higher mean blastocyst yields per donor than did BALB/c ones (3.6 vs. 2.5). However, BALB/c hosts resulted in a higher birth rate than B6-albino ones (36 vs. 27 %), a higher chimera formation rate (50 vs. 42 %), a higher high-percentage male chimera rate (10 vs. 8 %), and a higher germline transmission rate (65 vs. 49 %), respectively. Our data suggest that BALB/c is a suitable blastocyst host strain for C2 ES cells and has an advantage over the B6-albino strain for receiving the injection of C2 ES cells. PMID:26852382

  5. Human Endometrial CD98 Is Essential for Blastocyst Adhesion

    PubMed Central

    Domínguez, Francisco; Simón, Carlos; Quiñonero, Alicia; Ramírez, Miguel Ángel; González-Muñoz, Elena; Burghardt, Hans; Cervero, Ana; Martínez, Sebastián; Pellicer, Antonio; Palacín, Manuel; Sánchez-Madrid, Francisco; Yáñez-Mó, María

    2010-01-01

    Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window. PMID:20976164

  6. Blastocyst recovery and multifactorial gene expression analysis in the wild guinea pig (Cavia aperea).

    PubMed

    Hribal, Romy; Guenther, Anja; Rübensam, Kathrin; Jewgenow, Katarina

    2016-09-15

    The expression of specific developmentally important genes in preimplantation embryos is an accepted marker for unraveling the influence of single factors in studies that are mostly related to artificial reproduction techniques. Such studies, however, often reveal high levels of heterogeneity between single embryos, independently of the influence of factors of interest. A possible explanation for this variation could be the large variety of physiological and environmental factors to which early embryos are exposed and their ability to react to them. Here, we investigated several potentially important parameters of development at the same time, in blastocysts of the wild guinea pig (Cavia aperea) generated in vivo after natural mating. The optimal time for flushing fully developed blastocysts was between 123 and 126 hours after mating. The abundance of POU5F1 (P = 0.042), BAX (P < 0.001), SLC2A1 (P = 0.017), and DNMT3A (P < 0.001) mRNA changed significantly over time after mating. The number of sibling embryos present influenced STAT3 levels significantly (P = 0.02). Levels of BAX and POU5F1 were significantly affected by season (P = 0.03 and 0.04). The temporal pattern of SLC2A1 levels was significantly altered both after feeding a protein-deficient diet (P = 0.04) and temperature treatment (P = 0.04) of the sire. In addition, the identity of the father had a significant influence on POU5F1 (P = 0.049) and STAT3 (P < 0.001) mRNA abundances. These data report that the expression of specific genes in early embryos reflects the entire heterogeneity of their surroundings and that it is a plastic reaction toward a multifactorial environment. PMID:27264741

  7. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst.

    PubMed

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer

    2014-03-01

    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established. PMID:24504341

  8. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

    PubMed Central

    Le Bin, Gloryn Chia; Muñoz-Descalzo, Silvia; Kurowski, Agata; Leitch, Harry; Lou, Xinghua; Mansfield, William; Etienne-Dumeau, Charles; Grabole, Nils; Mulas, Carla; Niwa, Hitoshi; Hadjantonakis, Anna-Katerina; Nichols, Jennifer

    2014-01-01

    The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established. PMID:24504341

  9. Does Powerful Language Training Affect Student Participation, Impression Formation, and Gender Communication in Online Discussions?

    ERIC Educational Resources Information Center

    Thomas, Crystal Ann

    2012-01-01

    The purpose of this dissertation was to investigate whether powerful language training affected student participation, impression formation, and gender communication style in online discussions. Powerful language was defined as a lack of the use of powerless language. Participants in this study were 507 freshmen taking a first-year college…

  10. Colorimetric Method for Identifying Plant Essential Oil Components That Affect Biofilm Formation and Structure

    PubMed Central

    Niu, C.; Gilbert, E. S.

    2004-01-01

    The specific biofilm formation (SBF) assay, a technique based on crystal violet staining, was developed to locate plant essential oils and their components that affect biofilm formation. SBF analysis determined that cinnamon, cassia, and citronella oils differentially affected growth-normalized biofilm formation by Escherichia coli. Examination of the corresponding essential oil principal components by the SBF assay revealed that cinnamaldehyde decreased biofilm formation compared to biofilms grown in Luria-Bertani broth, eugenol did not result in a change, and citronellol increased the SBF. To evaluate these results, two microscopy-based assays were employed. First, confocal laser scanning microscopy (CLSM) was used to examine E. coli biofilms cultivated in flow cells, which were quantitatively analyzed by COMSTAT, an image analysis program. The overall trend for five parameters that characterize biofilm development corroborated the findings of the SBF assay. Second, the results of an assay measuring growth-normalized adhesion by direct microscopy concurred with the results of the SBF assay and CLSM imaging. Viability staining indicated that there was reduced toxicity of the essential oil components to cells in biofilms compared to the toxicity to planktonic cells but revealed morphological damage to E. coli after cinnamaldehyde exposure. Cinnamaldehyde also inhibited the swimming motility of E. coli. SBF analysis of three Pseudomonas species exposed to cinnamaldehyde, eugenol, or citronellol revealed diverse responses. The SBF assay could be useful as an initial step for finding plant essential oils and their components that affect biofilm formation and structure. PMID:15574886

  11. The effect of anxiety on impression formation: affect-congruent or stereotypic biases?

    PubMed

    Curtis, Guy J; Locke, Vance

    2005-03-01

    Two classes of theories propose that anxious individuals will form either more affect-congruent or more stereotypic impressions of others. These theories' predictions are not mutually exclusive. Eighty-one participants were examined to determine if either class of theories was more descriptive of the effect of anxiety on impression formation or whether a theory combining elements of both was more appropriate. Anxious participants read behavioural descriptions about an Australian Aboriginal target that were stereotypic, non-stereotypic, threatening, and non-threatening, and rated the target on traits that corresponded to the behavioural descriptions. Anxious participants formed impressions that were more affect-congruent, but not more stereotypic, than those formed by control participants. This result was replicated in a field study with 61 participants who were waiting to see a dentist. Future studies should examine the cognitive mechanisms that influence and underlie anxious affect-congruent impression formation. PMID:15901392

  12. Assessment, modeling and optimization of parameters affecting the formation of disinfection by-products in water.

    PubMed

    Gougoutsa, Chrysa; Christophoridis, Christophoros; Zacharis, Constantinos K; Fytianos, Konstantinos

    2016-08-01

    This study focused on (a) the development of a screening methodology, in order to determine the main experimental variables affecting chlorinated and brominated disinfection by-product (DBP) formation in water during chlorination experiments and (b) the application of a central composite design (CCD) using response surface methodology (RSM) for the mathematical description and optimization of DBP formation. Chlorine dose and total organic carbon (TOC) were proven to be the main factors affecting the formation of total chlorinated DBPs, while chlorine dose and bromide concentration were the main parameters affecting the total brominated THMs. Longer contact time promoted a rise in chlorinated DBPs' concentration even in the presence of a minimal amount of organic matter. A maximum production of chlorinated DBPs was observed under a medium TOC value and it reduced at high TOC concentrations, possibly due to the competitive production of brominated THMs. The highest concentrations of chlorinated THMs were observed at chlorine dose 10 mg L(-1) and TOC 5.5 mg L(-1). The formation of brominated DBPs is possible even with a minimum amount of NaOCl in the presence of high concentration of bromide ions. Brominated DBPs were observed in maximum concentrations using 8 mg L(-1) of chlorine in the presence of 300 μg L(-1) bromides. PMID:27178297

  13. Transcriptomic Features of Bovine Blastocysts Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Min, Byungkuk; Cho, Sunwha; Park, Jung Sun; Lee, Yun-Gyeong; Kim, Namshin; Kang, Yong-Kook

    2015-01-01

    Reprogramming incompletely occurs in most somatic cell nuclear transfer (SCNT) embryos, which results in misregulation of developmentally important genes and subsequent embryonic malfunction and lethality. Here we examined transcriptome profiles in single bovine blastocysts derived by in vitro fertilization (IVF) and SCNT. Different types of donor cells, cumulus cell and ear-skin fibroblast, were used to derive cSCNT and fSCNT blastocysts, respectively. SCNT blastocysts expressed 13,606 genes on average, similar to IVF (13,542). Correlation analysis found that both cSCNT and fSCNT blastocyst groups had transcriptomic features distinctive from the IVF group, with the cSCNT transcriptomes closer to the IVF ones than the fSCNT. Gene expression analysis identified 56 underrepresented and 78 overrepresented differentially expressed genes in both SCNT groups. A 400-kb locus harboring zinc-finger protein family genes in chromosome 18 were found coordinately down-regulated in fSCNT blastocysts, showing a feature of reprogramming-resistant regions. Probing into different categories of genes important for blastocyst development revealed that genes involved in trophectoderm development frequently were underrepresented, and those encoding epigenetic modifiers tended to be overrepresented in SCNT blastocysts. Our effort to identify reprogramming-resistant, differentially expressed genes can help map reprogramming error-prone loci onto the genome and elucidate how to handle the stochastic events of reprogramming to improve cloning efficiency. PMID:26342001

  14. Transcriptomic Features of Bovine Blastocysts Derived by Somatic Cell Nuclear Transfer.

    PubMed

    Min, Byungkuk; Cho, Sunwha; Park, Jung Sun; Lee, Yun-Gyeong; Kim, Namshin; Kang, Yong-Kook

    2015-12-01

    Reprogramming incompletely occurs in most somatic cell nuclear transfer (SCNT) embryos, which results in misregulation of developmentally important genes and subsequent embryonic malfunction and lethality. Here we examined transcriptome profiles in single bovine blastocysts derived by in vitro fertilization (IVF) and SCNT. Different types of donor cells, cumulus cell and ear-skin fibroblast, were used to derive cSCNT and fSCNT blastocysts, respectively. SCNT blastocysts expressed 13,606 genes on average, similar to IVF (13,542). Correlation analysis found that both cSCNT and fSCNT blastocyst groups had transcriptomic features distinctive from the IVF group, with the cSCNT transcriptomes closer to the IVF ones than the fSCNT. Gene expression analysis identified 56 underrepresented and 78 overrepresented differentially expressed genes in both SCNT groups. A 400-kb locus harboring zinc-finger protein family genes in chromosome 18 were found coordinately down-regulated in fSCNT blastocysts, showing a feature of reprogramming-resistant regions. Probing into different categories of genes important for blastocyst development revealed that genes involved in trophectoderm development frequently were underrepresented, and those encoding epigenetic modifiers tended to be overrepresented in SCNT blastocysts. Our effort to identify reprogramming-resistant, differentially expressed genes can help map reprogramming error-prone loci onto the genome and elucidate how to handle the stochastic events of reprogramming to improve cloning efficiency. PMID:26342001

  15. Affect and Cognition in Attitude Formation toward Familiar and Unfamiliar Attitude Objects

    PubMed Central

    van Giesen, Roxanne I.

    2015-01-01

    At large attitudes are built on earlier experience with the attitude object. If earlier experiences are not available, as is the case for unfamiliar attitude objects such as new technologies, no stored evaluations exist. Yet, people are still somehow able to construct attitudes on the spot. Depending on the familiarity of the attitude object, attitudes may find their basis more in affect or cognition. The current paper investigates differences in reliance on affect or cognition in attitude formation toward familiar and unfamiliar realistic attitude objects. In addition, individual differences in reliance on affect (high faith in intuition) or cognition (high need for cognition) are taken into account. In an experimental survey among Dutch consumers (N = 1870), we show that, for unfamiliar realistic attitude objects, people rely more on affect than cognition. For familiar attitude objects where both affective and cognitive evaluations are available, high need for cognition leads to more reliance on cognition, and high faith in intuition leads to more reliance on affect, reflecting the influence of individually preferred thinking style. For people with high need for cognition, cognition has a higher influence on overall attitude for both familiar and unfamiliar realistic attitude objects. On the other hand, affect is important for people with high faith in intuition for both familiar and unfamiliar attitude objects and for people with low faith in intuition for unfamiliar attitude objects; this shows that preferred thinking style is less influential for unfamiliar objects. By comparing attitude formation for familiar and unfamiliar realistic attitude objects, this research contributes to understanding situations in which affect or cognition is the better predictor of overall attitudes. PMID:26517876

  16. Affect and Cognition in Attitude Formation toward Familiar and Unfamiliar Attitude Objects.

    PubMed

    van Giesen, Roxanne I; Fischer, Arnout R H; van Dijk, Heleen; van Trijp, Hans C M

    2015-01-01

    At large attitudes are built on earlier experience with the attitude object. If earlier experiences are not available, as is the case for unfamiliar attitude objects such as new technologies, no stored evaluations exist. Yet, people are still somehow able to construct attitudes on the spot. Depending on the familiarity of the attitude object, attitudes may find their basis more in affect or cognition. The current paper investigates differences in reliance on affect or cognition in attitude formation toward familiar and unfamiliar realistic attitude objects. In addition, individual differences in reliance on affect (high faith in intuition) or cognition (high need for cognition) are taken into account. In an experimental survey among Dutch consumers (N = 1870), we show that, for unfamiliar realistic attitude objects, people rely more on affect than cognition. For familiar attitude objects where both affective and cognitive evaluations are available, high need for cognition leads to more reliance on cognition, and high faith in intuition leads to more reliance on affect, reflecting the influence of individually preferred thinking style. For people with high need for cognition, cognition has a higher influence on overall attitude for both familiar and unfamiliar realistic attitude objects. On the other hand, affect is important for people with high faith in intuition for both familiar and unfamiliar attitude objects and for people with low faith in intuition for unfamiliar attitude objects; this shows that preferred thinking style is less influential for unfamiliar objects. By comparing attitude formation for familiar and unfamiliar realistic attitude objects, this research contributes to understanding situations in which affect or cognition is the better predictor of overall attitudes. PMID:26517876

  17. Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats.

    PubMed

    Katska-Ksiazkiewicz, L; Opiela, J; Ryńska, B

    2007-09-15

    The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF. PMID:17651793

  18. Mfn2 Affects Embryo Development via Mitochondrial Dysfunction and Apoptosis

    PubMed Central

    Liu, Qun; Xiang, Wenpei

    2015-01-01

    Background Growth factors, energy sources, and mitochondrial function strongly affect embryo growth and development in vitro. The biological role and prospective significance of the mitofusin gene Mfn2 in the development of preimplantation embryos remain poorly understood. Our goal is to profile the role of Mfn2 in mouse embryos and determine the underlying mechanism of Mfn2 function in embryo development. Methods We transfected Mfn2-siRNA into 2-cell fertilized eggs and then examined the expression of Mfn2, the anti-apoptotic protein Bcl-2, and the apoptosis-promoting protein Bax by Western blot. Additionally, we determined the blastocyst formation rate and measured ATP levels, mtDNA levels, mitochondrial membrane potential (ΔΨm), and apoptosis in all of the embryos. Results The results indicate that the Mfn2 and Bcl-2 levels were markedly decreased, whereas Bax levels were increased in the T group (embryos transfected with Mfn2-siRNA) compared with the C group (embryos transfected with control-siRNA). The blastocyst formation rate was significantly decreased in the T group. The ATP content and the relative amounts of mtDNA and cDNA in the T group were significantly reduced compared with the C group. In the T group, ΔΨm and Ca2+ levels were reduced, and the number of apoptotic cells was increased. Conclusion Low in vitro expression of Mfn2 attenuates the blastocyst formation rate and cleavage speed in mouse zygotes and causes mitochondrial dysfunction, as confirmed by the ATP and mtDNA levels and mitochondrial membrane potential. Mfn2 deficiency induced apoptosis through the Bcl-2/Bax and Ca2+ pathways. These findings indicate that Mfn2 could affect preimplantation embryo development through mitochondrial function and cellular apoptosis. PMID:25978725

  19. Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts.

    PubMed

    Milazzotto, Marcella Pecora; Goissis, Marcelo Demarchi; Chitwood, James Lee; Annes, Kelly; Soares, Carlos Alexandre; Ispada, Jéssica; Assumpção, Mayra Elena Ortiz Ávila; Ross, Pablo Juan

    2016-04-01

    Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro-produced early- and late-dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo-produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast- and slow-dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast- and slow-dividing embryo groups, whereas blastocysts from the slow-dividing group were more similar to in vivo-produced blastocysts. Gene ontology enrichment analysis showed that the slow-dividing and in vivo-produced blastocysts shared biological processes related to groups of up- or down-regulated genes when compared to the fast-dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro-produced bovine embryos. Embryos from fast- and slow-dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post-in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage-stage and morulae groups, but was higher in the blastocysts of the slow-dividing group. On the other hand, blastocysts of the fast-dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324-336, 2016. © 2016 Wiley Periodicals, Inc. PMID:26822777

  20. Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos.

    PubMed

    Campelo, Iana S; Pereira, Alexsandra F; Alcântara-Neto, Agostinho S; Canel, Natalia G; Souza-Fabjan, Joanna M G; Teixeira, Dárcio I A; Camargo, Luiz S A; Melo, Luciana M; Rádis-Baptista, Gandhi; Salamone, Daniel F; Freitas, Vicente J F

    2016-02-01

    The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 μM for 6-24 h. PMID:25532535

  1. ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation

    PubMed Central

    Hsueh, Yi-Huang; Ke, Wan-Ju; Hsieh, Chien-Te; Lin, Kuen-Song; Tzou, Dong-Ying; Chiang, Chao-Lung

    2015-01-01

    Zinc oxide nanoparticles (ZnO NPs) are an important antimicrobial additive in many industrial applications. However, mass-produced ZnO NPs are ultimately disposed of in the environment, which can threaten soil-dwelling microorganisms that play important roles in biodegradation, nutrient recycling, plant protection, and ecological balance. This study sought to understand how ZnO NPs affect Bacillus subtilis, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on B. subtilis growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that B. subtilis growth is inhibited by high concentrations of ZnO NPs (≥ 50 ppm), with cells exhibiting a prolonged lag phase and delayed medial FtsZ ring formation. RedoxSensor and Phag-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, B. subtilis reductase activity, membrane stability, and protein expression all decrease. SDS-PAGE Stains-All staining results and FT-IR data further demonstrate that ZnO NPs negatively affect exopolysaccharide production. Moreover, it was found that B. subtilis biofilm surface structures became smooth under ZnO-NP concentrations of only 5–10 ppm, with concentrations ≤ 25 ppm significantly reducing biofilm formation activity. XANES and EXAFS spectra analysis further confirmed the presence of ZnO in co-cultured B. subtilis cells, which suggests penetration of cell membranes by either ZnO NPs or toxic Zn+ ions from ionized ZnO NPs, the latter of which may be deionized to ZnO within bacterial cells. Together, these results demonstrate that ZnO NPs can affect B. subtilis viability through the inhibition of cell growth, cytosolic protein expression, and biofilm formation, and suggest that future ZnO-NP waste management strategies would do well to mitigate the potential environmental impact engendered by the disposal of these nanoparticles. PMID:26039692

  2. Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

    PubMed Central

    SUZUKI, Chie; SAKAGUCHI, Yosuke; HOSHI, Hiroyoshi; YOSHIOKA, Koji

    2015-01-01

    The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin. PMID:26582048

  3. Accumulation of RNA in blastocysts during embryonic diapause and the periimplantation period in the western spotted skunk

    SciTech Connect

    Mead, R.A.; Rourke, A.W.

    1985-07-01

    The in vivo incorporation of TH-uridine into RNA was studied in delayed implanting and activated blastocysts obtained from 33 western spotted skunks. TH-uridine was incorporated into RNA by all blastocysts; however, significantly more label was incorporated as blastocyst diameter increased. Activated blastocysts with diameters of 1.6 mm or greater on average incorporated 65 times more TH-precursor in 5 hr than diapausing blastocysts with diameters of 1.1 mm or less. Polyadenylated RNA was likewise synthesized by delayed implanting and activated skunk blastocysts; however, the proportion of polyadenylated RNA synthesized by the former was greater than in the latter. The data suggest that the transition from embryonic diapause to fully activated blastocysts first occurs gradually for several days before entering a 1-2-day period of rapid development characterized by an abrupt increase in RNA accumulation.

  4. Insertion site and sealing technique affect residual hearing and tissue formation after cochlear implantation.

    PubMed

    Burghard, Alice; Lenarz, Thomas; Kral, Andrej; Paasche, Gerrit

    2014-06-01

    Tissue formation around the electrode array of a cochlear implant has been suggested to influence preservation of residual hearing as well as electrical hearing performance of implanted subjects. Further, inhomogeneity in the electrical properties of the scala tympani shape the electrical field and affect current spread. Intracochlear trauma due to electrode insertion and the insertion site itself are commonly seen as triggers for the tissue formation. The present study investigates whether the insertion site, round window membrane (RWM) vs. cochleostomy (CS), or the sealing material, no seal vs. muscle graft vs. carboxylate cement, have an influence on the amount of fibrous tissue and/or new bone formation after CI implantation in the guinea pig. Hearing thresholds were determined by auditory brainstem response (ABR) measurements prior to implantation and after 28 days. The amount of tissue formation was quantified by evaluation of microscopic images obtained by a grinding/polishing procedure to keep the CI in place during histological processing. An insertion via the round window membrane resulted after 28 days in less tissue formation in the no seal and muscle seal condition compared to the cochleostomy approach. Between these two sealing techniques there was no difference. Sealing the cochlea with carboxylate cement resulted always in a strong new bone formation and almost total loss of residual hearing. The amount of tissue formation and the hearing loss correlated at 1-8 kHz. Consequently, the use of carboxylate cement as a sealing material in cochlear implantation should be avoided even in animal studies, whereas sealing the insertion site with a muscle graft did not induce an additional tissue growth compared to omitting a seal. For hearing preservation the round window approach should be used. PMID:24566091

  5. Bisphenol a influences blastocyst implantation via regulating integrin β3 and trophinin expression levels

    PubMed Central

    Pan, Xiaoyan; Wang, Xiyan; Wang, Zhengchao; Wang, Xuenan; Dou, Zhaohua; Li, Zhixin

    2015-01-01

    Objective: This study is to investigate effects of bisphenol A (BPA) on the blastocyst implantation in endometrium. Methods: Pregnant mice were orally administered with BPA. Implantation sites were examined, and serum estrogen level was assayed with ELISA. Protein expression levels were detected with immunohistochemistry and immunofluorescence staining. Results: High doses (400 and 600 mg/kg/day) of BPA remarkably reduced the implantation sites in the pregnant mice. No significant differences were observed in the serum estrogen level across the groups. Moreover, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression level of endometrial estrogen receptor α (ERα) in the pregnant mice. In addition, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression levels of integrin β3 and trophinin in the endometrium and blastocysts. Conclusion: BPA declines ERα expression in endometrium, and inhibits adhesion protein expression in endometrium and blastocysts, causing the adhesion failure of blastocyst implantation. PMID:26884915

  6. Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency.

    PubMed

    Durruthy-Durruthy, Jens; Wossidlo, Mark; Pai, Sunil; Takahashi, Yusuke; Kang, Gugene; Omberg, Larsson; Chen, Bertha; Nakauchi, Hiromitsu; Reijo Pera, Renee; Sebastiano, Vittorio

    2016-07-11

    Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology. PMID:27404362

  7. Betaine Homocysteine Methyltransferase Is Active in the Mouse Blastocyst and Promotes Inner Cell Mass Development*

    PubMed Central

    Lee, Martin B.; Kooistra, Megan; Zhang, Baohua; Slow, Sandy; Fortier, Amanda L.; Garrow, Timothy A.; Lever, Michael; Trasler, Jacquetta M.; Baltz, Jay M.

    2012-01-01

    Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts. PMID:22847001

  8. Generation of large pig and bovine blastocysts by culturing in human induced pluripotent stem cell medium.

    PubMed

    Gao, Qing-Shan; Jin, Long; Li, Suo; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Jin, Qing-Guo; Kang, Jin-Dan; Yan, Chang-Guo; Yin, Xi-Jun

    2016-04-01

    We investigated the effect of human induced pluripotent stem cell (hiPS) medium on porcine somatic cell nuclear transfer and bovine in vitro fertilized early blastocysts, in comparison with North Carolina State University (NCSU)-37 medium and in vitro culture (IVC)-II medium. After 2 days of culture, the diameter of the portion of the blastocyst that was extruded from the zona pellucid dramatically differed between porcine blastocysts cultured in hiPS medium and those cultured in NCSU-37 medium (221.47 ± 38.94 μm versus 481.87 ± 40.61 μm, P < 0.01). Moreover, the diameter of the portion of the blastocyst significantly differed between bovine blastocysts cultured in hiPS medium and those cultured in IVC-II medium (150.30 ± 29.49 μm versus 195.58 ± 41.59 μm, P < 0.01). Furthermore, the total number of cells per porcine and bovine blastocyst was more than two-fold higher in blastocysts cultured in hiPS medium than in those cultured in NCSU-37 medium (44.33 ± 5.28 and 143.33 ± 16.05, P < 0.01) or IVC-II medium (172.12 ± 45.08 and 604.83 ± 242.64, P < 0.01), respectively. These results indicate that hiPS medium markedly improves the quality of porcine and bovine blastocysts. PMID:25925489

  9. Plant-derived decapeptide OSIP108 interferes with Candida albicans biofilm formation without affecting cell viability.

    PubMed

    Delattin, Nicolas; De Brucker, Katrijn; Craik, David J; Cheneval, Olivier; Fröhlich, Mirjam; Veber, Matija; Girandon, Lenart; Davis, Talya R; Weeks, Anne E; Kumamoto, Carol A; Cos, Paul; Coenye, Tom; De Coninck, Barbara; Cammue, Bruno P A; Thevissen, Karin

    2014-05-01

    We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation. PMID:24566179

  10. Neonatal outcomes after the transfer of vitrified blastocysts: closed versus open vitrification system

    PubMed Central

    2013-01-01

    Background Increasing evidence indicates that closed vitrification has been successfully used in the cryopreservation of human oocytes and embryos. Little information is available regarding the neonatal outcome of closed blastocysts vitrification. The aim of this study was to evaluate the effectiveness and safety of blastocyst vitrification using a high-security closed vitrification system compared with an open vitrification system. Methods A total of 332 vitrified-warmed blastocyst transfer cycles between April 2010 and May 2012 were analyzed retrospectively. The post-thaw survival rate, implantation rate, clinical pregnancy rate, live birth rate, and neonatal outcome were recorded. Results There were no significant differences between the open vitrification group and the close vitrification group regarding the post-thaw survival rate (98% versus 95.8%), clinical pregnancy rate (47.6% versus 42.2%), implantation rate (42.9% versus 35.6%), and live birth rate (39.8% versus 32.1%). In total, 332 warming cycles produced 131 healthy babies. There were no significant differences in the mean gestational age, the birth weight, and the birth length between the two groups. No adverse neonatal outcomes were observed in the children born after the transfer of closed vitrified blastocysts compared with the transfer of open vitrified blastocysts. Conclusions These data suggest that blastocyst vitrification using a closed vitrification device seems safe and effective with results comparable to those obtained through open vitrification. PMID:24256633

  11. The pig blastocyst: its ultrastructure and the uptake of protein macromolecules.

    PubMed

    Stroband, H W; Taverne, N; vd Bogaard, M

    1984-01-01

    Between days 8 and 11 of pregnancy spherical blastocysts from 0.3 to 10 mm in diameter were flushed from the uterine horns of Dutch Landrace pigs. A description of their ultrastructure is given, and the uptake of horseradish peroxidase and ferritin is demonstrated. The ultrastructure of the trophoblast was similar at all ages studied. The trophoblast which has many apical microvilli is able to take up and digest the macromolecules which were offered in the in vitro incubation medium. The hypoblast consists of flattened cells. In blastocysts 2 mm and larger, compact cells bearing microvilli are found below the embryoblast. Cell organelles indicating protein synthesis are found within hypoblast cells of such blastocysts. In the embryoblast, local concentrations of cell organelles are visible, indicating that differentiation has started. After the disappearance of Rauber's layer, which takes place when the blastocyst reaches a diameter of about 2 mm, superficial embryoblast cells develop short microvilli. The cells do not absorb ferritin or peroxidase but are dependent on the trophoblast for their food requirements. All cell layers in the blastocyst contain mitochondria that have characteristics of those found in steroid-producing cells. The significance of the uptake and digestion of macromolecules by trophoblast cells, the synthesis of protein by hypoblast cells and the possible synthesis of steroids is discussed with respect to the relationship between the cell layers of the blastocyst and in the context of concepto-maternal relationships. PMID:6705037

  12. Inhibitory effects of preimplantation exposure to bisphenol-A on blastocyst development and implantation

    PubMed Central

    Pan, Xiaoyan; Wang, Xuenan; Sun, Yanmei; Dou, Zhaohua; Li, Zhixin

    2015-01-01

    The effect of preimplantation exposure to bisphenol-A (BPA) on blastocyst development and implantation is investigated. Mice were orally administered with BPA (200, 400, 600, and 800 mg/kg/day) from Day 0.5 to Day 3.5 of their pregnancy. Blastocyst development was examined on Day 4 of pregnancy. With 400 mg/kg/day BPA, implantation site number and implantation rate significantly reduced. With 600 and 800 mg/kg/day BPA, no implantation site was observed. BPA at 800 mg/kg/day significantly reduced blastocyst development rate and hatching rate. With 400 and 600 mg/kg/day BPA, Blastocyst development rate showed no significant difference whereas hatching rate was lower. With 400, 600, and 800 mg/kg/day BPA, some embryos were detected in the fallopian tube and hatched blastocysts showed greatly increased apoptosis level and endothelial nitric oxide synthase expression. In summary, high concentration BPA delayed the transfer of embryos to the uterus, damaged blastocyst development before implantation, and inhibited embryo implantation. PMID:26309523

  13. How Environment Affects Galaxy Metallicity through Stripping and Formation History: Lessons from the Illustris Simulation

    NASA Astrophysics Data System (ADS)

    Genel, Shy

    2016-05-01

    Recent studies have found higher galaxy metallicities in richer environments. It is not yet clear, however, whether metallicity-environment dependencies are merely an indirect consequence of environmentally dependent formation histories, or of environmentally related processes directly affecting metallicity. Here, we present a first detailed study of metallicity-environment correlations in a cosmological hydrodynamical simulation, in particular, we focus on the Illustris simulation. Illustris galaxies display similar relations to those observed. Utilizing our knowledge of simulated formation histories, and leveraging the large simulation volume, we construct galaxy samples of satellites and centrals with matching formation histories. This allows us to find that ∼ 1/3 of the metallicity-environment correlation is due to different formation histories in different environments. This is a combined effect of satellites (in particular, in denser environments) having on average lower z = 0 star formation rates (SFRs), and of their older stellar ages, even at a given z = 0 SFR. Most of the difference, ∼ 2/3, however, is caused by the higher concentration of star-forming disks of satellite galaxies, as this biases their SFR-weighted metallicities toward their inner, more metal-rich parts. With a newly defined quantity, the “radially averaged” metallicity, which captures the metallicity profile but is independent of the SFR profile, the metallicities of satellites and centrals become environmentally independent once they are matched in formation history. We find that circumgalactic metallicity (defined as rapidly inflowing gas around the virial radius), while sensitive to environment, has no measurable effect on the metallicity of the star-forming gas inside the galaxies.

  14. Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts.

    PubMed

    Ochota, M; Wojtasik, B; Niżański, W

    2016-06-01

    The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre-implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures. PMID:26991408

  15. The effects of blastocyst morphological score and blastocoele re-expansion speed after warming on pregnancy outcomes

    PubMed Central

    Yin, Huiqun; He, Ruibing; Wang, Cunli; Zhu, Jie; Li, Yang

    2016-01-01

    Objective The aim of this study was to investigate associations between the morphology score of blastocysts and blastocoele re-expansion speed after warming with clinical outcomes, which could assist in making correct and cost-effective decisions regarding the appropriate time to vitrify blastocysts and to transfer vitrified-warmed blastocysts. Methods A total of 327 vitrified-warmed two-blastocyst transfer cycles in women 38 years old and younger were included in this retrospective study. Results The clinical pregnancy rate (CPR) and implantation rate (IR) of transfers of two good-morphology grade 4 blastocysts vitrified on day 5 (64.1% and 46.8%, respectively) were significantly higher than the CPR and IR associated with the transfers of two good-morphology grade 3 blastocysts vitrified on day 5 (46.7% and 32.2%, respectively). No significant differences were found in the CPR and IR among the transfers of two good-morphology grade 4 blastocysts regardless of the day of cryopreservation. Logistic regression analysis showed that blastocoele re-expansion speed after warming was associated with the CPR. Conclusion The selection of a good-morphology grade 4 blastocyst to be vitrified could be superior to the choice of a grade 3 blastocyst. Extending the culture of grade 3 blastocysts and freezing grade 4 or higher blastocysts on day 6 could lead to a greater likelihood of pregnancy. Since re-expansion was shown to be a morphological marker of superior blastocyst viability, blastocysts that quickly re-expand after warming should be prioritized for transfer. PMID:27104155

  16. Biofilm Formation by the Fish Pathogen Flavobacterium columnare: Development and Parameters Affecting Surface Attachment

    PubMed Central

    Cai, Wenlong; De La Fuente, Leonardo

    2013-01-01

    Flavobacterium columnare is a bacterial fish pathogen that affects many freshwater species worldwide. The natural reservoir of this pathogen is unknown, but its resilience in closed aquaculture systems posits biofilm as the source of contagion for farmed fish. The objectives of this study were (i) to characterize the dynamics of biofilm formation and morphology under static and flow conditions and (ii) to evaluate the effects of temperature, pH, salinity, hardness, and carbohydrates on biofilm formation. Nineteen F. columnare strains, including representatives of all of the defined genetic groups (genomovars), were compared in this study. The structure of biofilm was characterized by light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. F. columnare was able to attach to and colonize inert surfaces by producing biofilm. Surface colonization started within 6 h postinoculation, and microcolonies were observed within 24 h. Extracellular polysaccharide substances and water channels were observed in mature biofilms (24 to 48 h). A similar time course was observed when F. columnare formed biofilm in microfluidic chambers under flow conditions. The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. Several physicochemical parameters modulate attachment to surfaces, with the largest influence being exerted by hardness, salinity, and the presence of mannose. Maintenance of hardness and salinity values within certain ranges could prevent biofilm formation by F. columnare in aquaculture systems. PMID:23851087

  17. Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes.

    PubMed

    Ochota, Małgorzata; Pasieka, Anna; Niżański, Wojciech

    2016-03-15

    Blastocyst production in vitro seems to be crucial part of assisted reproduction techniques in feline species. However, the results of cats' oocyte maturation and embryo development are still lower than those in other species. The aim of this study was to evaluate whether the supplementation with superoxide dismutase (SOD) and taurine during maturation or culture would improve the blastocyst yield obtained from lower grades of oocytes, that are usually discarded, as not suitable for further in vitro purposes. To investigate the effect of antioxidants' addition, the good- and poor-quality oocytes, were cultured with the addition of 10-mmol taurine and 600 UI/mL SOD. The nuclear maturity, embryo development, and blastocyst quality were subsequently assessed. In control group, without antioxidant supplementation, significantly less poor-quality oocytes matured (42% vs. 62%) and more degenerated (35% vs. 20%), comparing to the experimental group supplemented with SOD and taurine. The amount of obtained blastocyst was much higher, when poor quality oocytes were supplemented with SOD and taurine (supplementation to IVM-4%; supplementation to IVC-5.5%; supplementation to IVM and IVC-5.9% of blastocyst), comparing to not supplemented control group (1.3%). The best blastocysts were obtained when poor oocytes had antioxidants added only during embryo culture (185 ± 13.4 blastomeres vs. 100 ± 1.5 in control). In the present study, we reported that the lower grades of oocytes can better mature and form significantly more blastocysts with better quality, when cultured with addition of SOD and taurine. PMID:26643604

  18. Antioxidants significantly affect the formation of different classes of isoprostanes and neuroprostanes in rat cerebral synaptosomes.

    PubMed

    Montine, Thomas J; Montine, Kathleen S; Reich, Erin E; Terry, Erin S; Porter, Ned A; Morrow, Jason D

    2003-02-15

    Lipid peroxidation has been implicated in the pathogenesis of a number of diseases, including neurodegenerative disorders. Evidence that antioxidants can affect the clinical course of neurodegenerative diseases is limited. In the present study, we examined the ability of five common antioxidants or antioxidant combinations, alpha-tocopherol, gamma-tocopherol, ascorbic acid, GSH ethyl ester, and a combination of ascorbate and alpha-tocopherol, to modulate lipid peroxidation in peroxidizing rat cerebral synaptosomes, a well-characterized model of oxidant injury. In these studies, we quantified isoprostanes (IsoPs) derived from arachidonic acid as an index of whole tissue oxidation and neuroprostanes (NeuroPs) formed from docosahexaenoic acid as a marker of selective neuronal peroxidation. We report that these various antioxidants displayed markedly different capacities to inhibit IsoP and NeuroP formation with the most potent effects on IsoPs observed for ascorbate, GSH ethyl ester, and the alpha-tocopherol-ascorbate combination. alpha-Tocopherol was slightly less potent and gamma-tocopherol significantly less effective. The concentration-response relationships were significantly different for NeuroP formation with the antioxidants being significantly less potent than for IsoP generation. In particular, alpha-tocopherol did not inhibit NeuroP formation at concentrations up to 100 microM. We also determined that tocopherols, in particular alpha-tocopherol, act in vitro as reducing agents to convert IsoP and NeuroP endoperoxides to reduced F-ring compounds, a finding we have observed previously in vivo in brain. These studies are of importance because they have further defined the role of antioxidants to modulate the formation of lipid peroxidation products in peroxidizing brain tissue. In addition, they suggest that alpha-tocopherol may not be a particularly effective agent to inhibit oxidant stress in the terminal compartment of neurons in the central nervous system

  19. Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

    PubMed

    Galeati, Giovanna; Zannoni, Augusta; Spinaci, Marcella; Bucci, Diego; Ostanello, Fabio; Panarese, Serena; Tamanini, Carlo; Sarli, Giuseppe

    2016-04-01

    This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir. PMID:26434667

  20. Good quality blastocyst from non-/mono-pronuclear zygote may be used for transfer during IVF.

    PubMed

    Yin, Bao-Li; Hao, Hao-Ying; Zhang, Ya-Nan; Wei, Duo; Zhang, Cui-Lian

    2016-04-01

    Although healthy infants have developed from non- and mono-pronuclear zygotes, the transfer of embryos from non- and mono-pronuclear zygotes is not recommended because there are no proper selection criteria. In the present study, we discuss how to select non- and mono-pronuclear embryos with the highest developmental potential at 19-20 hours post-insemination. We found that the percentage of blastocysts with normal chromosome constitution in non-pronuclear zygotes was slightly higher than in mono-pronuclear zygotes. Non- and mono-pronuclear embryos that were at the 4-cell stage on D2 and/or at the 6- to 8-cell stage on D3 had higher incidence rates of blastocysts with normal chromosome constitutions. We also found higher incidences of blastocysts with normal chromosome constitution on D6 than on D5. The results suggest that if high quality non- and mono-pronuclear zygotes develop to the 4-cell stage on D2 and the 6-to 8- cell stages on D3, along with high quality D6 blastocysts, the incidence of blastocysts with normal chromosome constitution is higher. PMID:26901373

  1. The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance of Pseudomonas aeruginosa.

    PubMed

    Oglesby-Sherrouse, Amanda G; Djapgne, Louise; Nguyen, Angela T; Vasil, Adriana I; Vasil, Michael L

    2014-04-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment for such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here, we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, nonsiderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by subinhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development. PMID:24436170

  2. Does the tail wag the dog? How the structure of a glycosylphosphatidylinositol anchor affects prion formation.

    PubMed

    Bate, Clive; Nolan, William; Williams, Alun

    2016-03-01

    There is increasing interest in the role of the glycosylphosphatidylinositol (GPI) anchor attached to the cellular prion protein (PrP(C)). Since GPI anchors can alter protein targeting, trafficking and cell signaling, our recent study examined how the structure of the GPI anchor affected prion formation. PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc) in prion-infected neuronal cell lines and in scrapie-infected primary cortical neurons. In uninfected neurons desialylated PrP(C) was associated with greater concentrations of gangliosides and cholesterol than PrP(C). In addition, the targeting of desialylated PrP(C) to lipid rafts showed greater resistance to cholesterol depletion than PrP(C). The presence of desialylated PrP(C) caused the dissociation of cytoplasmic phospholipase A2 (cPLA2) from PrP-containing lipid rafts, reduced the activation of cPLA2 and inhibited PrP(Sc) production. We conclude that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. PMID:26901126

  3. A Pleiotropic Regulator, Frp, Affects Exopolysaccharide Synthesis, Biofilm Formation, and Competence Development in Streptococcus mutans

    PubMed Central

    Wang, Bing; Kuramitsu, Howard K.

    2006-01-01

    Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development. PMID:16861645

  4. Identification of processes affecting excess air formation during natural bank filtration and managed aquifer recharge

    NASA Astrophysics Data System (ADS)

    Massmann, Gudrun; Sültenfuß, Jürgen

    2008-09-01

    SummaryManaged aquifer recharge is gaining importance as a practice to bank and treat surface water for drinking water production. Neon (Ne) concentrations were analysed at four different recharge sites in and near Berlin, where groundwater is recharged directly from surface water courses, either by near-natural bank filtration, induced bank filtration or engineered basin recharge. Neon concentrations in excess of saturation (ΔNe) were used to identify excess air in the infiltrates. Excess air concentrations were around saturation at the near-natural bank filtration site, where river water infiltrates through a permeable river bed into a confined aquifer under completely saturated conditions. At two induced unconfined bank filtration sites, samples generally contained excess air (up to 60% ΔNe). Highest excess air concentrations (up to 81% ΔNe) were encountered at the engineered basin recharge site. The degree of water table fluctuations, the water saturation of the sediments in the infiltration zone and the presence of a confining layer affect the formation of excess air. Excess air can only be used to trace bank filtrate or artificially recharged water in a setting where the ambient groundwater in the near vicinity of production wells is not affected by large water-table fluctuations. Nevertheless, excess air concentrations provide valuable additional information on the type of recharge (saturated or unsaturated, degree of water table fluctuations).

  5. DIETARY ARSENITE AFFECTS DIMETHYLHYDRAZINE (DMH)-INDUCED ABERRANT CRYPT FORMATION IN COLON AND GLOBAL DNA METHYLATION IN LIVER OF RATS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work has shown that arsenic (As) affects methionine metabolism. Alterations in methionine metabolism can affect cancer processes. To determine the effect of dietary As on DMH-induced aberrant crypt formation in colon Fisher-344 male, weanling rats (N=20/group) were fed diets containing 0, 0...

  6. Lactate production by the mammalian blastocyst: Manipulating the microenvironment for uterine implantation and invasion?

    PubMed Central

    Gardner, David K

    2015-01-01

    The mammalian blastocyst exhibits a high capacity for aerobic glycolysis, a metabolic characteristic of tumours. It has been considered that aerobic glycolysis is a means to ensure a high carbon flux to fulfil biosynthetic demands. Here, alternative explanations for this pattern of metabolism are considered. Lactate creates a microenvironment of low pH around the embryo to assist the disaggregation of uterine tissues to facilitate trophoblast invasion. Further it is proposed that lactate acts as a signalling molecule (especially at the reduced oxygen tension present at implantation) to elicit bioactive VEGF recruitment from uterine cells, to promote angiogenesis. Finally it is suggested that the region of high lactate/low pH created by the blastocyst modulates the activity of the local immune response, helping to create immune tolerance. Consequently, the mammalian blastocyst offers a model to study the role of microenvironments, and how metabolites and pH are used in signalling. PMID:25619853

  7. A survey of the effects of proteases and glycosidases on culture of rabbit morulae to blastocysts.

    PubMed

    Kane, M T

    1986-09-01

    The effects of a range of commercially available proteases and glycosidases on blastocyst development and hatching were examined on rabbit embryos cultured from the morula stage in a defined medium supplemented with charcoal-treated bovine serum albumin. The proteases tested were trypsin, alpha-chymotrypsin, thrombin, elastase, plasmin, papain, clostripain, collagenase, Streptomyces griseus protease and cathepsin C. The glycosidases tested were neuraminidase, alpha-mannosidase, beta-galactosidase and hyaluronidase. None of these enzymes appeared to stimulate blastocyst growth. The only enzymes which digested the embryonic investments, the zona and mucin coat, sufficiently to cause complete blastocyst hatching were trypsin and Streptomyces griseus protease at relatively low concentrations (250 ng/ml) and chymotrypsin and elastase at higher concentrations. PMID:3531506

  8. Inositol stimulates DNA and protein synthesis, and expansion by rabbit blastocysts in vitro.

    PubMed

    Fahy, M M; Kane, M T

    1992-04-01

    The effect of different concentrations (0, 0.6, 3, 15, 75 and 375 microM) of myo-inositol on the development of rabbit morulae to expanded blastocysts was investigated in terms of blastocyst expansion and synthesis of DNA and protein, as measured by incorporation of [3H]thymidine and [14C]amino acids into acid-precipitable material. A concentration of 15 microM inositol caused a 2.8-fold increase in blastocyst expansion (P less than 0.01), a 9.9-fold increase in thymidine incorporation into DNA (P less than 0.01) and a 3.6-fold increase in amino acid incorporation into protein (P less than 0.01). There were no significant differences in the range from 15 to 375 microM inositol. PMID:1522201

  9. Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media.

    PubMed

    Malik, Hruda Nanda; Singhal, Dinesh Kumar; Saugandhika, Shrabani; Dubey, Amit; Mukherjee, Ayan; Singhal, Raxita; Kumar, Sudarshan; Kaushik, Jai Kumar; Mohanty, Ashok Kumar; Das, Bikash Chandra; Bag, Sadhan; Bhanja, Subrata Kumar; Malakar, Dhruba

    2015-06-01

    The present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts. PMID:24405529

  10. The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance of Pseudomonas aeruginosa

    PubMed Central

    Oglesby-Sherrouse, Amanda G.; Djapgne, Louise; Nguyen, Angela T.; Vasil, Adriana I.; Vasil, Michael L.

    2014-01-01

    Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment of such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, non-siderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by sub-inhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development. PMID:24436170

  11. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  12. Dynamic changes in leptin distribution in the progression from ovum to blastocyst of the pre-implantation mouse embryo

    PubMed Central

    Schulz, Laura C.; Roberts, R. Michael

    2011-01-01

    The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally-derived leptin from the oocyte becomes differentially distributed among blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased towards a particular fate as early as the 2-cell stage. Here, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 % and 76.9 %, respectively) collected following superovulation, but was reduced, particularly in CF1 embryos (29.8 %; p < 0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division, and was not affected by removal of the zona pellucida. Presence or absence of leptin polarization was not linked to differences in ability of embryos to develop normally to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm but upon blastocoel expansion it was lost from cells. Throughout development leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development. PMID:21444625

  13. Osmotic tolerance of in vitro produced porcine blastocysts assessed by their morphological integrity and cellular actin filament organization.

    PubMed

    Men, Hongsheng; Agca, Yuksel; Mullen, Steven F; Critser, Elizabeth S; Critser, John K

    2005-10-01

    This experiment investigated the osmotic tolerance limits of the morphology and the cellular actin filament organization of porcine blastocysts. In vitro produced Day 6 blastocysts were subjected to osmotic treatments with sucrose solutions of different osmolalities (75, 150, 210, 600, 1200, and 2400 mOsm) and one isotonic solution (NCSU-23, 285 mOsm). Blastocysts were then either fixed immediately, or cultured for 18 h and subsequently fixed with formalin. The morphology of the treated blastocysts was examined under a stereomicroscope and the integrity of the cellular actin filaments of the blastocysts was examined by confocal microscopy after staining with Alexa Fluor 488 phalloidin. The results indicated that there was a significant relationship between the osmotic levels and the probability of blastocysts exhibiting disrupted cellular actin filaments. In addition, blastocysts also collapsed in proportion to the levels of osmotic treatments. The osmotic tolerance limits which would maintain 70% of the blastocysts with their original morphology immediately after the treatment were 90 and 170%, respectively, of isotonicity. After 18 h of culture, the osmotic tolerance limits were 61 and 163%, respectively, of isotonicity. Similarly, the osmotic conditions relative to isotonicity which would maintain the integrity of cellular actin filaments in 70% of treated blastocysts had to be within the range of 87 and 147% immediately after the treatment and 87 and 169% after 18 h of culture. Collectively, these data indicate that in vitro produced porcine blastocysts are very sensitive to osmotic stress. This information can be used to optimize cryopreservation procedures for porcine embryos. PMID:16024011

  14. [Regulation of neurogenesis: factors affecting of new neurons formation in adult mammals brain].

    PubMed

    Respondek, Michalina; Buszman, Ewa

    2015-01-01

    Neurogenesis is a complex and multi-step process of generating completely functional neurons. This process in adult brain is based on pluripotentional neuronal stem cells (NSC), which are able to proliferation and differentiation into mature neurons or glial cells. NSC are located in subgranular zone inside hippocampus and in subventricular zone. The new neurons formation depends on many endo- and exogenous factors which modulate each step of neurogenesis. This article describes the most important regulators of adult neurogenesis, mainly: neurotrophins, growth factors, hormones, neurotransmitters and microenvironment of NSC. Some drugs, especially antipsychotics, antidepressants and normothymics may affect the neurogenic properties of adult brain. Moreover pathological processes such as neuroinflammation, stroke or epilepsy are able to induce proliferation of NSC. The proneurogenic effects of psychotropic drugs and pathological processes are associated with their ability to increase some hormones and neurotrophins level, as well as with rising the expression of antiapoptotic Bcl-2 protein and metalloproteinase MMP-2. Additionaly, some drugs, for example haloperidol, are able to block prolactin and dopaminergic neuroblasts receptors. Down-regulation of adult neurogenesis is associated with alcohol abuse and high stress level. Negative effect of many drugs, such as cytostatics, COX-2 inhibitors and opioides was also observed. The proneurogenic effect of described factors suggest their broad therapeutic potential and gives a new perspective on an effective and modern treatment of many neuropsychiatric disorders. This effect can also help to clarify the pathogenesis of disorders associated with proliferation and degeneration of adult brain cells. PMID:27259217

  15. Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies

    PubMed Central

    Capalbo, Antonio; Ubaldi, Filippo Maria; Cimadomo, Danilo; Maggiulli, Roberta; Patassini, Cristina; Dusi, Ludovica; Sanges, Federica; Buffo, Laura; Venturella, Roberta; Rienzi, Laura

    2016-01-01

    STUDY QUESTION Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners? SUMMARY ANSWER The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment. WHAT IS KNOWN ALREADY Recently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes. STUDY DESIGN, SIZE, DURATION This longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET). PARTICIPANTS/MATERIALS, SETTING, METHODS Seven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical

  16. Blood Clot Formation Does Not Affect Metastasis Formation or Tumor Growth in a Murine Model of Breast Cancer

    PubMed Central

    Rossnagl, Stephanie; von Au, Anja; Vasel, Matthaeus; Cecchini, arco G.; Nakchbandi, Inaam A.

    2014-01-01

    Cancer is associated with increased fracture risk, due either to metastasis or associated osteoporosis. After a fracture, blood clots form. Because proteins of the coagulation cascade and activated platelets promote cancer development, a fracture in patients with cancer often raises the question whether it is a pathologic fracture or whether the fracture itself might promote the formation of metastatic lesions. We therefore examined whether blood clot formation results in increased metastasis in a murine model of experimental breast cancer metastasis. For this purpose, a clot was surgically induced in the bone marrow of the left tibia of immundeficient mice. Either one minute prior to or five minutes after clot induction, human cancer cells were introduced in the circulation by intracardiac injection. The number of cancer cells that homed to the intervention site was determined by quantitative real-time PCR and flow cytometry. Metastasis formation and longitudinal growth were evaluated by bioluminescence imaging. The number of cancer cells that homed to the intervention site after 24 hours was similar to the number of cells in the opposite tibia that did not undergo clot induction. This effect was confirmed using two more cancer cell lines. Furthermore, no difference in the number of macroscopic lesions or their growth could be detected. In the control group 72% developed a lesion in the left tibia. In the experimental groups with clot formation 79% and 65% developed lesions in the left tibia (p = ns when comparing each experimental group with the controls). Survival was similar too. In summary, the growth factors accumulating in a clot/hematoma are neither enough to promote cancer cell homing nor support growth in an experimental model of breast cancer bone metastasis. This suggests that blood clot formation, as occurs in traumatic fractures, surgical interventions, and bruises, does not increase the risk of metastasis formation. PMID:24740307

  17. Oxamflatin Significantly Improves Nuclear Reprogramming, Blastocyst Quality, and In Vitro Development of Bovine SCNT Embryos

    PubMed Central

    Li, Yanyan; Li, Ruizhe; Li, Qian; Wu, Yongyan; Quan, Fusheng; Liu, Jun; Guo, Zekun; Zhang, Yong

    2011-01-01

    Aberrant epigenetic nuclear reprogramming results in low somatic cloning efficiency. Altering epigenetic status by applying histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos. The present study was carried out to examine the effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming and development of bovine SCNT embryos in vitro. We found that Oxamflatin modified the acetylation status on H3K9 and H3K18, increased total and inner cell mass (ICM) cell numbers and the ratio of ICM∶trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts, and significantly enhanced the development of bovine SCNT embryos in vitro. Furthermore, Oxamflatin treatment suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-XL and the pluripotency-related genes OCT4 and SOX2 in SCNT blastocysts. Additionally, the treatment also reduced the DNA methylation level of satellite I in SCNT blastocysts. In conclusion, Oxamflatin modifies epigenetic status and gene expression, increases blastocyst quality, and subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos. PMID:21912607

  18. Ultrastructural characterization of in vivo-produced ovine morulae and blastocysts.

    PubMed

    Bettencourt, E M V; Bettencourt, C M V; Chagas e Silva, J N; Ferreira, P; Oliveira, E; Romão, R; Rocha, A; Sousa, M

    2015-08-01

    The ultrastructure of in vivo-produced ovine embryos, at the morula, early blastocyst and late blastocyst stages, was evaluated using transmission electron microscopy. Embryonic cells were characterized by the presence of intact intercellular junctions, numerous mitochondria, smooth endoplasmic reticulum cisternae and light vesicles. Polyribosomes, rough endoplasmic reticulum cisternae, secondary lysosomes, Golgi complexes and lipid droplets were also observed in the cytoplasm. The nucleus was well defined and organized, with an intact envelope rich in nuclear pore complexes, and one or more reticular nucleoli. Microvilli were present in external blastomeres of morulae and became more abundant in trophectoderm cells of early and late blastocysts. Light vesicles seemed to be associated with small cisternae of Golgi and endoplasmic reticulum origin. These cisternae fused and created light vesicles with engulfed heterogeneous cytosolic structures, small cisternae and vesicles. Their labile membrane enabled them to rapidly coalesce into medium-sized vesicles that began to engulf mitochondria and lipid droplets, forming giant vacuoles mostly filled with fat. Incomplete matured secretory vesicles were observed to exocytose into the perivitelline space of morulae, whereas fully matured secretory vesicles appeared only in trophectoderm cells, being exocytosed into the blastocoelic cavity. These observations suggested that these endoplasmic-/Golgi-derived vesicles behave as active autophagic organelles presenting probably a maturation process from compact morulae to blastocyst. PMID:25076424

  19. Comparative Genomic Hybridization Selection of Blastocysts for Repeated Implantation Failure Treatment: A Pilot Study

    PubMed Central

    Greco, Ermanno; Bono, Sara; Ruberti, Alessandra; Lobascio, Anna Maria; Greco, Pierfrancesco; Biricik, Anil; Spizzichino, Letizia; Greco, Alessia; Tesarik, Jan; Minasi, Maria Giulia; Fiorentino, Francesco

    2014-01-01

    The aim of this study is to determine if the use of preimplantation genetic screening (PGS) by array comparative genomic hybridization (array CGH) and transfer of a single euploid blastocyst in patients with repeated implantation failure (RIF) can improve clinical results. Three patient groups are compared: 43 couples with RIF for whom embryos were selected by array CGH (group RIF-PGS), 33 couples with the same history for whom array CGH was not performed (group RIF NO PGS), and 45 good prognosis infertile couples with array CGH selected embryos (group NO RIF PGS). A single euploid blastocyst was transferred in groups RIF-PGS and NO RIF PGS. Array CGH was not performed in group RIF NO PGS in which 1-2 blastocysts were transferred. One monoembryonic sac with heartbeat was found in 28 patients of group RIF PGS and 31 patients of group NO RIF PGS showing similar clinical pregnancy and implantation rates (68.3% and 70.5%, resp.). In contrast, an embryonic sac with heartbeat was only detected in 7 (21.2%) patients of group RIF NO PGS. In conclusion, PGS by array CGH with single euploid blastocyst transfer appears to be a successful strategy for patients with multiple failed IVF attempts. PMID:24779011

  20. ISOLATION AND CHARACTERIZATION OF PORCINE VISCERAL ENDODERM CELL LINES DERIVED FROM IN VIVO 11-DAY BLASTOCYSTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two porcine cell lines of yolk-sac visceral endoderm, designated PE-1 and PE-2, were derived from in vivo 11-day porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of cell lines was done on STO feeder cells. The PE-1 ...

  1. Cleavage-stage or blastocyst transfer: what are the benefits and harms?

    PubMed

    Glujovsky, Demián; Farquhar, Cynthia

    2016-08-01

    ET is a critical step in an assisted reproduction cycle. Over the past decade there has been an increasing trend to extending culture from cleavage-stage to blastocyst transfer. There has also been a trend to single ET and reporting the success of an assisted reproductive cycle as a cumulative live-birth rate after using both fresh and frozen embryos. There is low evidence that fresh blastocyst transfer is associated with improved live-birth rates compared with fresh cleavage-stage embryos. However, in the few studies that report cumulative pregnancy rates after fresh and frozen transfers, no significant difference was found. Cleavage-stage transfer is associated with greater numbers of embryos available for freezing, and blastocyst transfer is associated with increased number of cycles with no embryos to transfer. Further well-designed studies are warranted to evaluate the outcomes for blastocyst transfer including cumulative live-birth rate after fresh and frozen transfers, time to live birth, costs of the different transfer strategies, and perinatal mortality and severe perinatal morbidity. PMID:27421614

  2. Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells.

    PubMed

    Rayon, Teresa; Menchero, Sergio; Rollán, Isabel; Ors, Inmaculada; Helness, Anne; Crespo, Miguel; Nieto, Andres; Azuara, Véronique; Rossant, Janet; Manzanares, Miguel

    2016-01-01

    The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation. PMID:27256674

  3. Distinct mechanisms regulate Cdx2 expression in the blastocyst and in trophoblast stem cells

    PubMed Central

    Rayon, Teresa; Menchero, Sergio; Rollán, Isabel; Ors, Inmaculada; Helness, Anne; Crespo, Miguel; Nieto, Andres; Azuara, Véronique; Rossant, Janet; Manzanares, Miguel

    2016-01-01

    The first intercellular differences during mammalian embryogenesis arise in the blastocyst, producing the inner cell mass and the trophectoderm. The trophectoderm is the first extraembryonic tissue and does not contribute to the embryo proper, its differentiation instead forming tissues that sustain embryonic development. Crucial roles in extraembryonic differentiation have been identified for certain transcription factors, but a comprehensive picture of the regulation of this early specification is still lacking. Here, we investigated whether the regulatory mechanisms involved in Cdx2 expression in the blastocyst are also utilized in the postimplantation embryo. We analyzed an enhancer that is regulated through Hippo and Notch in the blastocyst trophectoderm, unexpectedly finding that it is inactive in the extraembryonic structures at postimplantation stages. Further analysis identified other Cdx2 regulatory elements including a stem-cell specific regulatory sequence and an element that drives reporter expression in the trophectoderm, a subset of cells in the extraembryonic region of the postimplantation embryo and in trophoblast stem cells. The cross-comparison in this study of cis-regulatory elements employed in the blastocyst, stem cell populations and the postimplantation embryo provides new insights into early mammalian development and suggests a two-step mechanism in Cdx2 regulation. PMID:27256674

  4. Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse.

    PubMed

    Khalifa, E A; Tucker, M J; Hunt, P

    1992-04-01

    Implantation rates remain low following human in-vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). Such techniques must have minimal adverse effects on the embryos, while maximizing the potential for an embryo to hatch fully as a blastocyst. In a mouse model, we compared embryonic development after zona drilling, and cruciate thinning of the zona (CTOZ) intended to simulate the natural thinning of the zona pellucida. Using acidic Tyrode's solution both zona drilling and cruciate-thinning were performed on day 3 morulae. On day 4 the rates of complete hatching of blastocysts were 0/165, 24/172 and 72/175 in control, zona drilled and thinned groups respectively (P less than 0.0001). On day 5 the rates of complete hatching in the same groups were 20/165, 54/172 and 120/175 respectively (P less than 0.00001) and by day 6, 66/165, 74/172 and 130/175 respectively (P less than 0.00001). The rate of arrest at the morula stage was 24/172 versus 8/175 in the zona drilled and thinned groups respectively (P less than 0.005, whilst the rate of arrest at the blastocyst stage was 21/172 versus 14/175 respectively (NS). Hence cruciate thinning of the zona appears less detrimental at the morula stage than zona drilling, but eventual rates of arrest at the blastocyst stage were comparable. Both techniques significantly increased the rate of hatching, but zona drilling did not guarantee complete hatching.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1522198

  5. Identification of sources affecting fog formation using receptor modeling approaches and inventory estimates of sectoral emissions

    NASA Astrophysics Data System (ADS)

    Mehta, Bhavin; Venkataraman, Chandra; Bhushan, Mani; Tripathi, Sachchida Nand

    Positive matrix factorization (PMF) was used to identify factors affecting fog formation in Kanpur during the ISRO-GBP land campaign-II (LC-II) in December 2004. PMF predicted factors were validated by contrasting the emission strength of sources in the foggy and clear periods, using a combination of potential source contribution function (PSCF) analysis and quantitative emission inventory information. A time series aerosol chemical data set of 29 days and 12 species was decomposed to identify 4-factors: Secondary species, Biomass burning, Dust and Sea salt. PMF predicted particle mass with a satisfactory goodness-of-fit (slope of 0.83 ± 0.17 and R2 of 0.8), and strong species within 11-12% relative standard deviation. Mean contributions of anthropogenic factors were significantly higher during the foggy period for secondary species (2.9 ± 0.3) and biomass burning (1.2 ± 0.09) compared to the clear period. Local sources contributing to aerosols that mediated fog events at Kanpur, based on emissions in a 200 km × 200 km area around Kanpur city were thermal power plants and transportation (SO 2) and biofuel combustion (BC and OM). Regional scale sources influencing emissions during the foggy period, in probable source regions identified by PSCF included thermal power plants, transportation, brick kilns and biofuel combustion. While biofuel combustion and transportation are distributed area sources, individual point sources include coal-fired thermal power plants located in Aligarh, Delhi, Ghaziabad, Jhansi, Kanpur, Rae Bareli and Rupnagar and brick kilns located in Allahabad, Agra, Farrukhabad, Ghaziabad, Kanpur, Ludhiana, Lucknow and Rae Bareli. Additionally, in the foggy period, large areas of probable source regions lay outside India, implying the significance of aerosol incursion from outside India.

  6. Verbal versus Numerical Probabilities: Does Format Presentation of Probabilistic Information regarding Breast Cancer Screening Affect Women's Comprehension?

    ERIC Educational Resources Information Center

    Vahabi, Mandana

    2010-01-01

    Objective: To test whether the format in which women receive probabilistic information about breast cancer and mammography affects their comprehension. Methods: A convenience sample of 180 women received pre-assembled randomized packages containing a breast health information brochure, with probabilities presented in either verbal or numeric…

  7. Factors that Affect Science and Mathematics Teachers' Initial Implementation of Technology-Enhanced Formative Assessment Using a Classroom Response System

    ERIC Educational Resources Information Center

    Lee, Hyunju; Feldman, Allan; Beatty, Ian D.

    2012-01-01

    The purpose of this study is to uncover and understand the factors that affect secondary science and mathematics teachers' initial implementation of Technology-Enhanced Formative Assessment (TEFA), a pedagogy developed for teaching with classroom response system (CRS) technology. We sought to identify the most common and strongest factors, and to…

  8. Gene expression of porcine blastocysts from gilts fed organic or inorganic selenium and pyridoxine.

    PubMed

    Dalto, B D; Tsoi, S; Audet, I; Dyck, M K; Foxcroft, G R; Matte, J J

    2015-01-01

    In this study, we determined how maternal dietary supplementation with pyridoxine combined with different sources of selenium (Se) affected global gene expression of porcine expanded blastocysts (PEB) during pregnancy. Eighteen gilts were randomly assigned to one of the three experimental diets (n=6 per treatment): i) basal diet without supplemental Se or pyridoxine (CONT); ii) CONT+0.3 mg/kg of Na-selenite and 10 mg/kg of HCl-pyridoxine (MSeB610); and iii) CONT+0.3 mg/kg of Se-enriched yeast and 10 mg/kg of HCl-pyridoxine (OSeB610). All gilts were inseminated at their fifth post-pubertal estrus and killed 5 days later for embryo harvesting. A porcine embryo-specific microarray was used to detect differentially gene expression between MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610. CONT gilts had lower whole blood Se and erythrocyte pyridoxal-5-P concentrations than supplemented gilts (P<0.05). No treatment effect was observed on blood plasma Se-glutathione peroxidase activity (P=0.57). There were 10, 247, and 96 differentially expressed genes for MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610 respectively. No specific biological process was associated with MSeB610 vs CONT. However, for OSeB610 vs CONT, upregulated genes were related with global protein synthesis but not to selenoproteins. The stimulation of some genes related with monooxygenase and thioredoxin families was confirmed by quantitative real-time RT-PCR. In conclusion, OSeB610 affects PEB metabolism more markedly than MSeB610. Neither Se sources with pyridoxine influenced the Se-glutathione peroxidase metabolic pathway in the PEB, but OSeB610 selectively stimulated genes involved with antioxidant defense. PMID:25326430

  9. Prospection and emotional memory: how expectation affects emotional memory formation following sleep and wake

    PubMed Central

    Cunningham, Tony J.; Chambers, Alexis M.; Payne, Jessica D.

    2014-01-01

    Successful prospective memory is necessarily driven by an expectation that encoded information will be relevant in the future, leading to its preferential placement in memory storage. Like expectation, emotional salience is another type of cue that benefits human memory formation. Although separate lines of research suggest that both emotional information and information explicitly expected to be important in the future benefit memory consolidation, it is unknown how expectation affects the processing of emotional information and whether sleep, which is known to maximize memory consolidation, plays a critical role. The purpose of this study was to investigate how expectation would impact the consolidation of emotionally salient content, and whether this impact would differ across delays of sleep and wake. Participants encoded scenes containing an emotionally charged negative or neutral foreground object placed on a plausible neutral background. After encoding, half of the participants were informed they would later be tested on the scenes (expected condition), while the other half received no information about the test (unexpected condition). At recognition, following a 12-h delay of sleep or wakefulness, the scene components (objects and backgrounds) were presented separately and one at a time, and participants were asked to determine if each component was old or new. Results revealed a greater disparity for memory of negative objects over their paired neutral backgrounds for both the sleep and wake groups when the memory test was expected compared to when it was unexpected, while neutral memory remained unchanged. Analyzing each group separately, the wake group showed a threefold increase in the magnitude of this object/background trade-off for emotional scenes when the memory test was expected compared to when it was unexpected, while those who slept performed similarly across conditions. These results suggest that emotional salience and expectation cues

  10. Factors affecting the efficiency of embryo transfer in the domestic ferret (Mustela putorius furo).

    PubMed

    Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

    2006-07-15

    Embryo transfer (ET) to recipient females is a foundational strategy for a number of assisted reproductive technologies, including cloning by somatic cell nuclear transfer. In an attempt to develop efficient ET in domestic ferrets, factors affecting development of transferred embryo were investigated. Unilateral and bilateral transfer of zygotes or blastocysts in the oviduct or uterus was evaluated in recipient nulliparous or primiparous females. Developing fetuses were collected from recipient animals 21 days post-copulation and examined. The percentage of fetal formation was different (P<0.05) for unilateral and bilateral transfer of zygotes (71%) in nulliparous females with bilateral transfer (56%) in primiparous recipients. The percentage (90%) of fetal formation in nulliparous recipients following unilateral transfer of blastocysts was higher (P<0.05) than that observed in primiparous recipients with bilateral ET (73%). Notably, the percentage of fetal formation was higher (P<0.05) when blastocyts were transferred as compared to zygotes (90% versus 71%). Transuterine migration of embryos occurred following all unilateral transfers and also in approximately 50% of bilateral transfers with different number of embryos in each uterine horn. These data will help to facilitate the development of assisted reproductive strategies in the ferret and could lead to the use of this species for modeling human disease and for conservation of the endangered Mustelidae species such as black-footed ferret and European mink. PMID:16330092

  11. How Interactive Video (ITV) Web-Enhanced Format Affects Instructional Strategy and Instructor Satisfaction

    ERIC Educational Resources Information Center

    Moody, Catrina V.

    2013-01-01

    This qualitative study explored the quality of technology associated with interactive video (ITV) classes in distance education programs and the resulting satisfaction of the instructors teaching this format. The participants were full time instructors of a rural community college that used the ITV format. Community college ITV instructors are…

  12. Understanding the Factors Affecting the Formation of Carbonyl Iron Electrodes in Rechargeable Alkaline Iron Batteries

    SciTech Connect

    Manohar, AK; Yang, CG; Malkhandi, S; Yang, B; Prakash, GKS; Narayanan, SR

    2012-01-01

    Rechargeable iron-based alkaline batteries such as iron - air and nickel - iron batteries are attractive for large-scale electrical energy storage because iron is inexpensive, globally-abundant and environmentally-friendly. Further, the iron electrode is known for its robustness to repeated charge/discharge cycling. During manufacturing these batteries are charged and discharged 20 to 50 times during which the discharge capacity of the iron electrode increases gradually and attains a stable value. This process of achieving stable capacity is called formation. In this study we have focused our efforts on understanding the effect of electrode design on formation. We have investigated the role of wetting agent, pore-former additive, and sulfide additive on the formation of carbonyl iron electrodes. The wetting agent increased the rate of formation while the pore-former additive increased the final capacity. Sodium sulfide added to the electrolyte worked as a de-passivation agent and increased the final discharge capacity. We have proposed a phenomenological model for the formation process that predicts the rate of formation and final discharge capacity given the design parameters for the electrode. The understanding gained here will be useful in reducing the time lost in formation and in maximizing the utilization of the iron electrode. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.021301jes] All rights reserved.

  13. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner.

    PubMed

    Takegahara, Yuki; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. PMID:24720912

  14. The small and the beautiful: how the star formation law affects galactic disc structure

    NASA Astrophysics Data System (ADS)

    Braun, H.; Schmidt, W.

    2015-12-01

    We investigate the influence of different analytical parametrizations and fit functions for the local star formation rate in adaptive mesh refinement simulations of an isolated disc galaxy with the NYX code. Suchparametrizations express the star formation efficiency as function of the local turbulent Mach number and virial parameter. By employing the method of adaptively refined large eddy simulations, we are able to evaluate these physical parameters from the numerically unresolved turbulent energy associated with the grid scale. We consider both single and multi free-fall variants of star formation laws proposed by Padoan & Nordlund, Hennebelle & Chabrier, and Krumholz & McKee, summarized and tested recently with numerical simulations by Federrath & Klessen. We find that the global star formation rate and the relation between the local star formation rate and the gas column density is reproduced in agreement with observational constraints by all multi free-fall models of star formation. Some models with obsolete calibration or a single free-fall time-scale, however, result in an overly clumpy disc that does not resemble the structure of observed spirals.

  15. Effects of lead on the male mouse as investigated by in vitro fertilization and blastocyst culture

    SciTech Connect

    Johansson, L.; Sjoeblom, P.; Wide, M.

    1987-02-01

    Long-term exposure of male mice to inorganic lead (lead chloride, 1 g/liter) in the drinking water reduces their fertility. The cause of this reduction, expressed as a decrease in the number of mated females showing inplantations, was investigated, using an in vivo fertilization method. It was found that spermatozoa from lead-exposed males had a significantly lower ability to fertilize mouse eggs than those from unexposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males. Preimplantation embryos, isolated from uterine horns of mice mated with lead-exposed males, were examined. No morphologically abnormal embryos were found. However, when cultured in vitro over the implantation period, blastocysts of the group mated with lead-exposed males showed an increased frequency of delayed hatching from the zona pellucida or an inability to hatch. Among blastocysts from this group a decreased frequency of inner cell mass development was also found.

  16. Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study

    PubMed Central

    Noli, Laila; Capalbo, Antonio; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

    2015-01-01

    Summary Discordant growth is a common complication of monochorionic/diamniotic pregnancies; in approximately 50% of cases, the cause is unknown. The case presented here suggests that discordant growth of monozygotic twins could start during preimplantation development. Two inner cell masses (ICMs) within the same blastocyst may originate in uneven splitting of a single “parental” ICM, or the two ICMs may be formed independently de novo. We studied the transcriptomes of two morphologically distinct ICMs within a single blastocyst using high-resolution RNA sequencing. The data indicated that the two ICM were at different stages of development; one was in the earliest stages of lineage commitment, while the other had already differentiated into epiblast and primitive endoderm. IGF1-mediated signaling is likely to play a key role in ICM growth and to be the major driver behind these differences. PMID:26584541

  17. Chemokine Receptor Type 4 Regulates Migration and Invasion of Trophectoderm Cell in the Human Blastocyst.

    PubMed

    Bao, Siyu; Li, Tianjie; Long, Xiaoyu; Zhang, Jinjuan; Zhao, Hongcui; Ren, Yun; Zhao, Yue; Li, Rong; Tan, Tao; Yu, Yang; Qiao, Jie

    2016-07-01

    Chemokine receptor type 4 (CXCR4) has been suggested to regulate cell migration and invasion in human somatic cells. However, its role in human oocytes and embryos has not been investigated directly. Here we show that CXCR4 mRNA was initially expressed at the 4-cell stage, and its expression gradually increased until the blastocyst stage, whereas its protein was detectable only after the 8-cell stage. In addition, CXCR4 mRNA and protein were expressed in the inner cell mass (ICM) and trophectoderm (TE) cell of the blastocyst. Furthermore, we collected embryos from women whose embryos had undergone successful implantation (SI) and those whose embryos had failed implantation (FI) in their fresh cycles. TE cells from the FI group had reduced CXCR4 mRNA expression relative to those from the SI group but not in the ICM. Through ICM replacement, we constructed mouse blastocysts in which Cxcr4 was specifically knocked down in TE cells to simulate the CXCR4 expression profile of human blastocysts from the FI group. In this case, we found that the implantation rate significantly decreased after transfer of reconstructed embryos. Bioinformatic analysis indicated that CXCR4 can induce cell apoptosis and migration mediated by Rho signaling. This hypothesis was confirmed by invasion and migration experiments, using a human trophoblast cell line. The present study is the first to explore the characteristics of CXCR4 expression using human oocytes and embryos and suggests that CXCR4 is required upstream of TE cell apoptosis and migration. CXCR4 expression is a potential biomarker to predict implantation competence during assisted reproductive technologies. PMID:27146031

  18. UDP-glucuronosyltransferase 1a enzymes are present and active in the mouse blastocyst.

    PubMed

    Collier, Abby C; Yamauchi, Yasuhiro; Sato, Brittany L M; Rougée, Luc R A; Ward, Monika A

    2014-11-01

    The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development. PMID:25200869

  19. UDP-Glucuronosyltransferase 1a Enzymes Are Present and Active in the Mouse Blastocyst

    PubMed Central

    Yamauchi, Yasuhiro; Sato, Brittany L.M.; Rougée, Luc R.A.; Ward, Monika A.

    2014-01-01

    The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development. PMID:25200869

  20. Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer.

    PubMed

    Du, Yutao; Li, Juan; Kragh, Peter M; Zhang, Yunhai; Schmidt, Mette; Bøgh, Ingrid B; Zhang, Xiuqing; Purup, Stig; Kuwayama, M; Jørgensen, Arne L; Pedersen, Anette M; Villemoes, Klaus; Yang, Huanming; Bolund, Lars; Vajta, Gábor

    2007-01-01

    Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage. PMID:18154508

  1. Karyomapping identifies second polar body DNA persisting to the blastocyst stage: implications for embryo biopsy.

    PubMed

    Ottolini, Christian S; Rogers, Shaun; Sage, Karen; Summers, Michael C; Capalbo, Antonio; Griffin, Darren K; Sarasa, Jonas; Wells, Dagan; Handyside, Alan H

    2015-12-01

    Blastocyst biopsy is now widely used for both preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Although this approach yields good results, variable embryo quality and rates of development remain a challenge. Here, a case is reported in which a blastocyst was biopsied for PGS by array comparative genomic hybridization on day 6 after insemination, having hatched completely. In addition to a small trophectoderm sample, excluded cell fragments from the subzonal space from this embryo were also sampled. Unexpectedly, the array comparative genomic hybridization results from the fragments and trophectoderm sample were non-concordant: 47,XX,+19 and 46,XY, respectively. DNA fingerprinting by short tandem repeat and amelogenin analysis confirmed the sex chromosome difference but seemed to show that the two samples were related but non-identical. Genome-wide single nucleotide polymorphism genotyping and karyomapping identified that the origin of the DNA amplified from the fragments was that of the second polar body corresponding to the oocyte from which the biopsied embryo developed. The fact that polar body DNA can persist to the blastocyst stage provides evidence that excluded cell fragments should not be used for diagnostic purposes and should be avoided when performing embryo biopsies as there is a risk of diagnostic errors. PMID:26380865

  2. Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

    PubMed

    Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu

    2015-01-15

    Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals. PMID:25192056

  3. Factors affecting trihalomethane formation and speciation during chlorination of reclaimed water.

    PubMed

    Ma, Defang; Gao, Baoyu; Wang, Yan; Yue, Qinyan; Li, Qian

    2015-01-01

    A hybrid process with membrane bioreactor (MBR) and powdered activated carbon (PAC), PAC/MBR, was used for real municipal wastewater treatment and reuse. The roles of chlorine dose, contact time, pH and bromide in trihalomethane (THM) formation and speciation during chlorination of the reclaimed water were investigated. Total trihalomethane (TTHM) yield exponentially increased to maximum with increasing chlorine dose (correlation coefficient R2=0.98). Prolonging substrate chlorine contact time significantly promoted TTHM formation. Less than 40% of THMs formed in the first 24 h, indicating that the PAC/MBR effluent organic matters were mostly composed of slow-reacting precursors. Increasing pH and bromide concentration facilitated THM formation. Higher chlorine dose and contact time enhanced chloro-THM formation. The bromo-THM formation was favored at near neutral condition. Despite the variation of chlorine dose, contact time and pH, the yield of THM species in order was usually CHCl3>CHBrCl2>CHBr2Cl>CHBr3. However, THM speciation shifted from chlorinated species to brominated species with increasing bromide concentration. PMID:26247761

  4. Melatonin promotes the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine.

    PubMed

    Wang, Feng; Tian, XiuZhi; Zhang, Lu; Tan, DunXian; Reiter, Russel J; Liu, GuoShi

    2013-10-01

    When a defect occurs in the in vitro development of a pronuclear embryo, the interruption of the subsequent implantation limits the success of assisted conception. This common problem remains to be solved. In this study, we observed that melatonin at its physiological concentration (10(-7)  m) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate, and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0% versus control 67.8%), litter sizes (4.1 pups/litter versus control 2.7 pups/litter), and postnatal survival rates of offspring (96.84% versus control 81.24%) were significantly improved compared with their non-melatonin-treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti-apoptotic factor bcl-2 while downregulating the expression of pro-apoptotic genes p53 and caspase-3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival. PMID:23772689

  5. Cationic Pillararenes Potently Inhibit Biofilm Formation without Affecting Bacterial Growth and Viability.

    PubMed

    Joseph, Roymon; Naugolny, Alissa; Feldman, Mark; Herzog, Ido M; Fridman, Micha; Cohen, Yoram

    2016-01-27

    It is estimated that up to 80% of bacterial infections are accompanied by biofilm formation. Since bacteria in biofilms are less susceptible to antibiotics than are bacteria in the planktonic state, biofilm-associated infections pose a major health threat, and there is a pressing need for antibiofilm agents. Here we report that water-soluble cationic pillararenes differing in the quaternary ammonium groups efficiently inhibited the formation of biofilms by clinically important Gram-positive pathogens. Biofilm inhibition did not result from antimicrobial activity; thus, the compounds should not inhibit growth of natural bacterial flora. Moreover, none of the cationic pillararenes caused detectable membrane damage to red blood cells or toxicity to human cells in culture. The results indicate that cationic pillararenes have potential for use in medical applications in which biofilm formation is a problem. PMID:26745311

  6. In vitro production of multigene transgenic blastocysts via sperm-mediated gene transfer allows rapid screening of constructs to be used in xenotransplantation experiments.

    PubMed

    Vargiolu, A; Manzini, S; de Cecco, M; Bacci, M L; Forni, M; Galeati, G; Cerrito, M G; Busnelli, M; Lavitrano, M; Giovannoni, R

    2010-01-01

    Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic. PMID:20692428

  7. Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis

    PubMed Central

    Khezri, Maryam; Jouzani, Gholamreza Salehi; Ahmadzadeh, Masoud

    2016-01-01

    It is known that there is correlation between biofilm formation and antagonistic activities of Bacillus subtilis strains; but, the mechanism of this correlation is not clear. So, the effect of the plant pathogen (Fusarium culmorum) on the biofilm formation in a B. subtilis strain with high antagonistic and biofilm formation activities was studied. The expression of sinR and tasA genes involved in the biofilm formation was studied in both single culture of bacterium (B) and co-culture with F. culmorum (FB) using real-time PCR. The results revealed that the expression of the sinR gene in both B and FB conditions was continuously decreased during the biofilm formation period and, after 24 h (B4 and FB4), it reached 1% and 0.3% at the planktonic phase (B1), respectively, whereas the expression of the tasA was continuously increased and was 5.27 and 30 times more than that at the planktonic phase (B1) after 24 h, respectively. So, the expression reduction rate for sinR (3 times) and the expression increasing rate for tasA (6 times) were significantly higher in FB conditions than the B ones. The relative expression of sinR in FB1 (planktonic phase), FB2 (8 h), FB3(12 h), and FB4 (24 h) times was 0.65, 0.44, 0.35, and 0.29, whereas the tasA gene expression was 2.98, 3.44, 4.37, and 5.63-fold of the one at coordinate time points in B conditions, respectively. The significant expression reduction of sinR and increase of tasA confirmed that the presence of pathogen could stimulate biofilm formation in the antagonistic bacterium. PMID:26887226

  8. Model systems for evaluating factors affecting acrylamide formation in deep fried foods.

    PubMed

    Lindsay, R C; Jang, S

    2005-01-01

    Simulated food pieces constructed from fiberglass pads (models for French fries and chips) were used as carriers for defined aqueous solutions, dispersions of test substances and ingredients to evaluate acrylamide formation. The pads were loaded with a solution containing asparagine and glucose (10 mM each) plus selected reaction modulators before deep fat frying and analysis for acrylamide. Data from fiberglass models along with companion sliced potato samples were used in developing hypotheses for the mechanisms involved in the suppression of acrylamide formation by polyvalent cations, polyanionic compounds, pH, and altered food polymer states in fried potato products. PMID:16438309

  9. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    SciTech Connect

    Takegahara, Yuki; Yamanouchi, Keitaro Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  10. Submerged Conidiation and Product Formation by Aspergillus niger at Low Specific Growth Rates Are Affected in Aerial Developmental Mutants ▿

    PubMed Central

    Jørgensen, Thomas R.; Nielsen, Kristian F.; Arentshorst, Mark; Park, JooHae; van den Hondel, Cees A.; Frisvad, Jens C.; Ram, Arthur F.

    2011-01-01

    Exposure to an aerial environment or severe nutrient limitation induces asexual differentiation in filamentous fungi. Submerged cultivation of Aspergillus niger in carbon- and energy-limited retentostat cultures both induces and fuels conidiation. Physiological and transcriptomic analyses have revealed that this differentiation strongly affects product formation. Since conidiation is inherent in the aerial environment, we hypothesized that product formation near zero growth can be influenced by affecting differentiation or development of aerial hyphae in general. To investigate this idea, three developmental mutants (ΔfwnA, scl-1, and scl-2 mutants) that have no apparent vegetative growth defects were cultured in maltose-limited retentostat cultures. The secondary-metabolite profile of the wild-type strain defined flavasperone, aurasperone B, tensidol B, and two so far uncharacterized compounds as associated with conidium formation, while fumonisins B2, B4, and B6 were characteristic of early response to nutrient limitation by the vegetative mycelium. The developmental mutants responded differently to the severe substrate limitation, which resulted in distinct profiles of growth and product formation. fwnA encodes the polyketide synthase responsible for melanin biosynthesis during aerial differentiation, and we show that conidial melanin synthesis in submerged retentostat cultures and aurasperone B production are fwnA dependent. The scl-1 and scl-2 strains are two UV mutants generated in the ΔfwnA background that displayed reduced asexual conidiation and formed sclerotium-like structures on agar plates. The reduced conidiation phenotypes of the scl-1 and scl-2 strains are reflected in the retentostat cultivation and are accompanied by elimination or severely reduced accumulation of secondary metabolites and distinctly enhanced accumulation of extracellular protein. This investigation shows that submerged conidiation and product formation of a mitosporic fungus

  11. Indole affects the formation of multicellular aggregate structures in Pantoea agglomerans YS19.

    PubMed

    Yu, Xuemei; Jiang, Jing; Liang, Chen; Zhang, Xiao; Wang, Jieru; Shen, Delong; Feng, Yongjun

    2016-01-01

    Pantoea agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice. As well as having the ability to form a biofilm, as do most bacteria, it is characterized by the formation of a unique multicellular aggregate structure called symplasmata. Indole is traditionally known as a metabolite of the amino acid tryptophan, which, however, has recently been shown to participate in various regulations of bacterial physiological processes, including stress resistance, quorum sensing and biofilm formation. Here, an indole signal was found to promote symplasmata formation, yet inhibit biofilm formation, indicating different regulatory pathways of indole in the construction of the two structures. However, symplasmata showed almost an equivalent stress-resistant capability, as compared with biofilms, for YS19 to confront acids, heavy metals (Cu(2+)), and UV treatments. Moreover, indole was tested to show a promoting effect on exopolysaccharides (EPS) production and an inhibition effect on the expression of an outer membrane protein OmpW. These results provide evidence for understanding the regulatory mechanisms of indole on such multicellular aggregates. PMID:26923129

  12. Polyphenols from olive mill waste affect biofilm formation and motility in Escherichia coli K-12

    PubMed Central

    Carraro, Lisa; Fasolato, Luca; Montemurro, Filomena; Martino, Maria Elena; Balzan, Stefania; Servili, Maurizio; Novelli, Enrico; Cardazzo, Barbara

    2014-01-01

    Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. A potential option for bioremediation to overcome ecological problems is the reutilization of these natural compounds in food production. The aim of this work was to gain a better understanding of the antimicrobial mode of action of a phenols extract from olive vegetation water (PEOVW) at molecular level by studying Escherichia coli as a model microorganism. Genome-wide transcriptional analysis was performed on E. coli K-12 exposed to PEOVW. The repression of genes for flagellar synthesis and the involvement of genes linked to biofilm formation and stress response were observed. Sub-inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility, thus confirming the gene expression data. This study provides interesting insights on the molecular action of PEOVW on E. coli K-12. Given these anti-biofilm properties and considering that biofilm formation is a serious problem for the food industry and human health, PEOVW has proved to be a high-value natural product. Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. Genome-wide transcriptional analysis was performed on E. coli K-12 exposed to phenols extract from olive vegetation water (PEOVW). Sub-inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility. Given these anti-biofilm properties PEOVW has proved to be a high-value natural product. PMID:24628798

  13. Do Content, Format, and Level of Inquiry Affect Scores on Open-Ended Science Tasks?

    ERIC Educational Resources Information Center

    Stecher, Brian M.; Klein, Stephen P.; Solano-Flores, Guillermo; McCaffrey, Dan; Robyn, Abby; Shavelson, Richard J.; Haertel, Edward

    This study investigated three factors that may contribute to the large variation in student performance across open-ended measures. These factors are content domain, format (whether the task required only pencil and paper or involved a hands-on manipulation of equipment), and level of inquiry (whether the task guided the student toward the…

  14. Platelet size and density affect shear-induced thrombus formation in tortuous arterioles

    NASA Astrophysics Data System (ADS)

    Chesnutt, Jennifer K. W.; Han, Hai-Chao

    2013-10-01

    Thrombosis accounts for 80% of deaths in patients with diabetes mellitus. Diabetic patients demonstrate tortuous microvessels and larger than normal platelets. Large platelets are associated with increased platelet activation and thrombosis, but the physical effects of large platelets in the microscale processes of thrombus formation are not clear. Therefore, the objective of this study was to determine the physical effects of mean platelet volume (MPV), mean platelet density (MPD) and vessel tortuosity on platelet activation and thrombus formation in tortuous arterioles. A computational model of the transport, shear-induced activation, collision, adhesion and aggregation of individual platelets was used to simulate platelet interactions and thrombus formation in tortuous arterioles. Our results showed that an increase in MPV resulted in a larger number of activated platelets, though MPD and level of tortuosity made little difference on platelet activation. Platelets with normal MPD yielded the lowest amount of mural thrombus. With platelets of normal MPD, the amount of mural thrombus decreased with increasing level of tortuosity but did not have a simple monotonic relationship with MPV. The physical mechanisms associated with MPV, MPD and arteriole tortuosity play important roles in platelet activation and thrombus formation.

  15. Factors affecting efficiency of introducing foreign DNA and RNA into parthenogenetic or in vitro-fertilized porcine eggs by cytoplasmic microinjection.

    PubMed

    Liu, Shuai; Liu, XiaoQun; Huang, HaiYan; Liu, QingYou; Su, XiaoPing; Zhu, Peng; Li, HongLi; Cui, KuiQing; Xie, BingKun; Shi, DeShun

    2016-08-01

    Cytoplasmic microinjection (CI) of foreign gene into in vivo fertilized zygotes has emerged as a useful tool for transgenic pig production. In the current study, we investigated factors affecting transgenic efficiency and developmental potential of parthenogenetic (PA) and in vitro-fertilized (IVF) porcine embryos produced by CI. These factors included adding of RNase inhibitor, DNA or RNA concentration, injection time, and different structures of plasmids. Our results showed that adding of 1-4 U/μL of RNase inhibitor did not have negative effect on development potential of CI-PA embryos, and RNase inhibitor injection significantly increased EGFP expressing rate of CI-PA embryos. High injection DNA concentration and long injection interval after PA significantly reduced blastocyst formation. Different molecular structures such as DNA or RNA affected CI-PA embryos development, and RNA had little harmful effect on pig's early embryonic development. EGFP expression rate of CI-IVF embryos was improved following the increase of foreign DNA concentration, but blastocyst formation rate was decreased. Injection time after IVF did not show any significant difference on embryonic development, but longer interval resulted in a significantly lower EGFP expressing rate. Cas9 mRNA and myostatin (GDF-8) sgRNA co-injection indicated that the mutation rate of CI-IVF group was significantly higher than that of CI-PA. The CI-IVF-generated embryos were then transferred to six recipient pigs, but no live piglets were obtained. The following pronuclear formation assessment showed more than 76.1% IVF zygotes were polyspermy. These results demonstrate that CI-PA and CI-IVF were effective methods for production of transgenic pig embryos. However, polyspermic fertilization and poor quality of porcine IVF blastocysts are still the main problem of resulting in pregnancy failure. PMID:27130683

  16. Overexpression of AtPTPA in Arabidopsis increases protein phosphatase 2A activity by promoting holoenzyme formation and ABA negatively affects holoenzyme formation

    PubMed Central

    Chen, Jian; Zhu, Xunlu; Shen, Guoxin; Zhang, Hong

    2015-01-01

    AtPTPA is a critical regulator for the holoenzyme assembling of protein phosphatase 2A (PP2A) in Arabidopsis. Characterization of AtPTPA improves our understanding of the function and regulation of PP2A in eukaryotes. Further analysis of AtPTPA-overexpressing plants indicates that AtPTPA increases PP2A activity by promoting PP2A's AC dimer formation, thereby holoenzyme assembling. Plant hormone abscisic acid (ABA) reduces PP2A enzyme activity by negatively affects PP2A's AC dimer formation. Therefore, AtPTPA is a positive factor that promotes PP2A holoenzyme assembly, and ABA is a negative factor that prevents PP2A holoenzyme assembly. PMID:26633567

  17. Cell Signaling and Transcription Factors Regulating Cell Fate During Formation of the Mouse Blastocyst

    PubMed Central

    Frum, Tristan; Ralston, Amy

    2015-01-01

    The first cell fate decisions during mammalian development establish tissues essential for healthy pregnancy. The mouse has served as a valuable model for discovering pathways regulating the first cell fate decisions, because of the ease with which early embryos can be recovered and an arsenal of classical and emerging methods for manipulating gene expression. Here we summarize the major pathways that govern the first cell fate decisions in mouse development. This knowledge serves as a paradigm for exploring how emergent properties of a self-organizing system can dynamically regulate gene expression and cell fate plasticity. Moreover, it brings to light the processes that establish healthy pregnancy and embryonic stem (ES) cells. We also describe unsolved mysteries and new technologies that could help overcome experimental challenges in the field. PMID:25999217

  18. Identity Formation and Affective Spaces in Conflict-Ridden Societies: Inventing Heterotopic Possibilities

    ERIC Educational Resources Information Center

    Zembylas, Michalinos; Ferreira, Ana

    2009-01-01

    In this article, we present vignettes from two projects--one in Cyprus and the other in South Africa--to show how some classrooms enact "heterotopic" affective spaces that oppose normal/ized identities, that is, identities grounded in polarized trauma narratives. The notion of heterotopia is a spatial concept developed by Foucault to emphasize the…

  19. Three-Day-Old Human Unfertilized Oocytes after In Vitro Fertilization/Intracytoplasmic Sperm Injection Can Be Activated by Calcium Ionophore A23187 or Strontium Chloride and Develop to Blastocysts

    PubMed Central

    Han, Xiao-jie; Liu, Ming-hui; Wang, Shu-yu; Jia, Chan-wei; Yu, Lan; Ren, Guoqing; Wang, Li; Li, Wei

    2014-01-01

    Abstract Our objective was to observe the effectiveness of the calcium ionophore A23187 or strontium chloride on the activation and subsequent embryonic development of 3-day-old human unfertilized oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). A total of 279 3-day-old unfertilized oocytes after IVF or ICSI were randomized to be activated by the calcium ionophore A23187 (n=138) or strontium chloride (n=141). The activated oocytes were cultured in vitro for 3–5 days. Activation rate, pronucleus formation, cleavage rate, and developmental potential of parthenotes during culture were evaluated. A total of 170 unfertilized oocytes were activated; 65 developed to cleavage stage, 19 developed to greater than the eight-cell stage, and five blastocysts were obtained. The activation rate of the calcium ionophore A23187 group was higher than that of the strontium chloride group (75.4% and 46.8%, respectively; p<0.05); there was significant difference between two groups (p<0.05). Among the 44 cleaved oocytes in the calcium ionophore A23187 group, eight developed to the two- to four-cell stage, 17 developed to the five- to eight-cell stage, 15 developed to greater than the eight-cell stage, and four blastocysts were obtained. Among the 21 cleaved oocytes in the strontium chloride group, six developed to the two- to four- cell stage, 10 developed to the five- to eight-cell stage, four developed to greater than the eight-cell stage, and one blastocyst was obtained. Three-day-old unfertilized human oocytes after IVF or ICSI could be activated by the calcium ionophore A23187 or strontium chloride, and a small part of parthenogenetic embryos developed into blastocysts. The treatment with the calcium ionophore A23187 was better than that of strontium chloride in respect to the activation rate of 3-day-old unfertilized human oocytes after IVF or ICSI. PMID:24960285

  20. How Environment Affects Star Formation: Tracing Activity in High Redshift Galaxy Clusters

    NASA Astrophysics Data System (ADS)

    Alberts, Stacey; Pope, A.; Brodwin, M.; Atlee, D. W.; Lin, Y.; Chary, R.; Dey, A.; Eisenhardt, P. R.; Gettings, D.; Gonzalez, A. H.; Jannuzi, B.; Mancone, C.; Moustakas, J.; Snyder, G. F.; Stanford, S. A.; Stern, D.; Weiner, B. J.; Zeimann, G.

    2014-01-01

    The emerging picture of the evolution of cluster galaxies indicates that the epoch of z>1 is a crucial period of active star formation and mass assembly in clusters. In this dissertation, I leverage a uniformly-selected cluster sample from the IRAC Shallow Cluster Survey (ISCS) with Herschel imaging to analyse the star formation (SF) activity in cluster galaxies over the past ten billion years. This analysis is two-fold: 1) using 274 clusters across the 9 square degree Bootes field, I perform a stacking analysis of mass-limited samples of cluster and field galaxies using wide-field Herschel observations over a long redshift baseline, z=0.3-1.5. I find that the average SF activity in cluster galaxies is evolving faster than in the field, with field-like SF in the cluster cores and enhanced SF activity in the cluster outskirts at z>1.2. By further breaking down my analysis by galaxy mass and type, I determine which mechanisms are capable of driving this evolution. 2) I use unique, deep Herschel imaging of 11 spectroscopically-confirmed clusters from z=1.1-1.8 to study the properties of individual infrared bright cluster galaxies as a function of redshift and cluster-centric radius. Combined with ancillary data, I determine the star formation, dust, and AGN properties of the most active cluster galaxies and tie the evolution of these properties back to the environment by comparing to field populations. By combining these two approaches, I constrain cluster galaxy properties during a pivotal epoch of dust-obscured star formation activity and mass assembly in some of the most extreme structures in the Universe.

  1. Does student learning style affect performance on different formats of biomechanics examinations?

    PubMed

    Hsieh, Chengtu; Mache, Melissa; Knudson, Duane

    2012-03-01

    Students' learning style preferences have been widely adapted into teaching and learning environments. The purpose of this study was to investigate the relationship between self-reported and assessed learning style preferences (visual, auditory, reading/writing, kinesthetic: VARK) on performance in different types of multiple-choice examinations (T1: text only format and T2: visual format) given in an introductory biomechanics class. Students who enrolled in three biomechanics classes at a state university were recruited to participate in the study. Ninety students (47 males and 43 females) completed a learning style survey and two types of examinations. Results showed that approximately half of the students were assessed and self-reported as kinesthetic for their preferred learning style. There was no significant difference in test performance between students who preferred visual and reading/writing learning styles (self-reported and assessed). These students demonstrated similar learning and comprehension of biomechanical concepts regardless of whether the test material was presented in their preferred sensory mode or not. Interestingly, female students' perceptions of their learning style preference may have a positive effect on the test results when the test is presented in their preferred format. PMID:22518949

  2. Polyphenols from olive mill waste affect biofilm formation and motility in Escherichia coli K-12.

    PubMed

    Carraro, Lisa; Fasolato, Luca; Montemurro, Filomena; Martino, Maria Elena; Balzan, Stefania; Servili, Maurizio; Novelli, Enrico; Cardazzo, Barbara

    2014-05-01

    Olive mill wastes are sources of phenolic compounds with a wide array of biological activities, including antimicrobial effects. A potential option for bioremediation to overcome ecological problems is the reutilization of these natural compounds in food production. The aim of this work was to gain a better understanding of the antimicrobial mode of action of a phenols extract from olive vegetation water (PEOVW) at molecular level by studying Escherichia coli as a model microorganism. Genome-wide transcriptional analysis was performed on E. coli K-12 exposed to PEOVW. The repression of genes for flagellar synthesis and the involvement of genes linked to biofilm formation and stress response were observed. Sub-inhibitory concentrations of PEOVW significantly decreased biofilm formation, swarming and swimming motility, thus confirming the gene expression data. This study provides interesting insights on the molecular action of PEOVW on E. coli K-12. Given these anti-biofilm properties and considering that biofilm formation is a serious problem for the food industry and human health, PEOVW has proved to be a high-value natural product. PMID:24628798

  3. Frequency of Teriparatide Administration Affects the Histological Pattern of Bone Formation in Young Adult Male Mice.

    PubMed

    Yamamoto, Tomomaya; Hasegawa, Tomoka; Sasaki, Muneteru; Hongo, Hiromi; Tsuboi, Kanako; Shimizu, Tomohiro; Ota, Masahiro; Haraguchi, Mai; Takahata, Masahiko; Oda, Kimimitsu; Luiz de Freitas, Paulo Henrique; Takakura, Aya; Takao-Kawabata, Ryoko; Isogai, Yukihiro; Amizuka, Norio

    2016-07-01

    Evidence supports that daily and once-weekly administration of teriparatide, human (h)PTH(1-34), enhance bone mass in osteoporotic patients. However, it is uncertain whether different frequencies of hPTH(1-34) administration would induce bone formation similarly in terms of quantity and quality. To investigate that issue, mice were subjected to different frequencies of PTH administration, and their bones were histologically examined. Frequencies of administration were 1 time/2 days, 1 time a day, and 2 and 4 times a day. Mice were allocated to either to control or to 3 different dosing regimens: 80 μg/kg of hPTH(1-34) per injection (80 μg/kg per dose), 80 μg/kg of hPTH(1-34) per day (80 μg/kg · d), or 20 μg/kg of hPTH(1-34) per day (20 μg/kg · d). With the regimens of 80 μg/kg per dose and 80 μg/kg · d, high-frequency hPTH(1-34) administration increased metaphyseal trabecular number. However, 4 doses per day induced the formation of thin trabeculae, whereas the daily PTH regimen resulted in thicker trabeculae. A similar pattern was observed with the lower daily hPTH(1-34) dose (20 μg/kg · d): more frequent PTH administration led to the formation of thin trabeculae, showing a thick preosteoblastic cell layer, several osteoclasts, and scalloped cement lines that indicated accelerated bone remodeling. On the other hand, low-frequency PTH administration induced new bone with mature osteoblasts lying on mildly convex surfaces representative of arrest lines, which suggests minimodeling-based bone formation. Thus, high-frequency PTH administration seems to increase bone mass rapidly by forming thin trabeculae through accelerated bone remodeling. Alternatively, low-frequency PTH administration leads to the formation of thicker trabeculae through bone remodeling and minimodeling. PMID:27227535

  4. Downregulation of miR-199a-5p Disrupts the Developmental Potential of In Vitro-Fertilized Mouse Blastocysts.

    PubMed

    Tan, Kun; Wang, Xiaodong; Zhang, Zhenni; Miao, Kai; Yu, Yong; An, Lei; Tian, Jianhui

    2016-09-01

    Although in vitro fertilization (IVF), one of the most effective and successful assisted reproductive technologies, is widely used for treating infertility and in animal breeding, increasing evidence indicates that IVF offspring are linked to various short- or long-term consequences. Erroneous epigenetic modifications induced by IVF are suspected of contributing to these consequences. Among these epigenetic modifications, microRNAs may affect embryo implantation and early postimplantation development. Here, we performed comparative microRNA profiling between in vivo-fertilized (IVO group) and in vitro-fertilized (IVF group) mouse embryos at Embryonic Day 3.5 (E3.5) and E7.5. Our dynamic analyses showed that the dysregulated microRNAs were mainly associated with the regulation of genes involved in carcinogenesis, genetic information processing, glucose metabolism, cytoskeleton organization, and neurogenesis. Further analysis showed that miR-199a-5p was consistently downregulated in IVF embryos compared with their IVO counterparts. Through gain- and loss-of-function experiments, we demonstrated that IVF-induced downregulation of miR-199a-5p results in a higher glycolytic rate and lower developmental potential of IVF blastocysts, including cell lineage misallocation and lower fetal survival post implantation. Therefore, preventing downregulation of miR-199a-5p may become an effective strategy for improving the development of IVF peri-implantation embryos in the future. PMID:27488027

  5. Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro.

    PubMed

    Ohboshi, S; Etoh, T; Sakamoto, K; Fujihara, N; Yoshida, T; Tomogane, H

    1997-04-15

    Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar. PMID:16728072

  6. Life cycle stage and water depth affect flooding-induced adventitious root formation in the terrestrial species Solanum dulcamara

    PubMed Central

    Zhang, Qian; Visser, Eric J. W.; de Kroon, Hans; Huber, Heidrun

    2015-01-01

    Background and Aims Flooding can occur at any stage of the life cycle of a plant, but often adaptive responses of plants are only studied at a single developmental stage. It may be anticipated that juvenile plants may respond differently from mature plants, as the amount of stored resources may differ and morphological changes can be constrained. Moreover, different water depths may require different strategies to cope with the flooding stress, the expression of which may also depend on developmental stage. This study investigated whether flooding-induced adventitious root formation and plant growth were affected by flooding depth in Solanum dulcamara plants at different developmental stages. Methods Juvenile plants without pre-formed adventitious root primordia and mature plants with primordia were subjected to shallow flooding or deep flooding for 5 weeks. Plant growth and the timing of adventitious root formation were monitored during the flooding treatments. Key Results Adventitious root formation in response to shallow flooding was significantly constrained in juvenile S. dulcamara plants compared with mature plants, and was delayed by deep flooding compared with shallow flooding. Complete submergence suppressed adventitious root formation until up to 2 weeks after shoots restored contact with the atmosphere. Independent of developmental stage, a strong positive correlation was found between adventitious root formation and total biomass accumulation during shallow flooding. Conclusions The potential to deploy an escape strategy (i.e. adventitious root formation) may change throughout a plant’s life cycle, and is largely dependent on flooding depth. Adaptive responses at a given stage of the life cycle thus do not necessarily predict how the plant responds to flooding in another growth stage. As variation in adventitious root formation also correlates with finally attained biomass, this variation may form the basis for variation in resistance to shallow

  7. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

    PubMed

    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation. PMID:27064112

  8. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

    PubMed Central

    JEON, Yubyeol; NAM, Yeong-Hee; CHEONG, Seung-A; KWAK, Seong-Sung; LEE, Eunsong; HYUN, Sang-Hwan

    2016-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation. PMID:27064112

  9. Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

    PubMed

    O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

    2015-08-01

    The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms. PMID:25912312

  10. Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.

    PubMed

    Ostafin, Magdalena; Pruchniak, Michal Przemyslaw; Ciepiela, Olga; Reznick, Abraham Zeev; Demkow, Urszula

    2016-09-15

    A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample. PMID:27179553

  11. Face format at encoding affects the other-race effect in face memory.

    PubMed

    Zhao, Mintao; Hayward, William G; Bülthoff, Isabelle

    2014-01-01

    Memory of own-race faces is generally better than memory of other-races faces. This other-race effect (ORE) in face memory has been attributed to differences in contact, holistic processing, and motivation to individuate faces. Since most studies demonstrate the ORE with participants learning and recognizing static, single-view faces, it remains unclear whether the ORE can be generalized to different face learning conditions. Using an old/new recognition task, we tested whether face format at encoding modulates the ORE. The results showed a significant ORE when participants learned static, single-view faces (Experiment 1). In contrast, the ORE disappeared when participants learned rigidly moving faces (Experiment 2). Moreover, learning faces displayed from four discrete views produced the same results as learning rigidly moving faces (Experiment 3). Contact with other-race faces was correlated with the magnitude of the ORE. Nonetheless, the absence of the ORE in Experiments 2 and 3 cannot be readily explained by either more frequent contact with other-race faces or stronger motivation to individuate them. These results demonstrate that the ORE is sensitive to face format at encoding, supporting the hypothesis that relative involvement of holistic and featural processing at encoding mediates the ORE observed in face memory. PMID:25104831

  12. Abiotic and Biotic Factors Affecting Resting Spore Formation in the Mite Pathogen Neozygites floridana

    PubMed Central

    da Silveira Duarte, Vanessa; Westrum, Karin; Lopes Ribeiro, Ana Elizabete; Guedes Corrêa Gondim Junior, Manoel; Klingen, Ingeborg; Delalibera Júnior, Italo

    2013-01-01

    Neozygites floridana is an obligate mite pathogenic fungus in the Entomophthoromycota. It has been suggested that resting spores of this fungus are produced as a strategy to survive adverse conditions. In the present study, possible mechanisms involved in the regulation of resting spore formation were investigated in the hosts Tetranychus urticae and Tetranychus evansi. Abiotic and biotic factors mimicking conditions that we, based on earlier field studies, thought might induce resting spores in temperate and tropical regions were tested with isolates from Norway and Brazil. A total of 42 combinations of conditions were tested, but only one induced the formation of a high number of resting spores in only one isolate. The Brazilian isolate ESALQ1420 produced a large number of resting spores (51.5%) in T. urticae at a temperature of 11°C, photoperiod of 10L:14D, and light intensity of 42–46 (μmol m−2 s−1) on nonsenescent plants (nondiapausing females). Resting spores of the Brazilian N. floridana isolate ESALQ1421 were found at very low levels (up to 1.0%). Small percentages of T. urticae with resting spores (0–5.0%) were found for the Norwegian isolate NCRI271/04 under the conditions tested. The percentages of resting spores found for the Norwegian isolate in our laboratory studies are similar to the prevalence reported in earlier field studies. PMID:23878542

  13. Tripeptide SQL Inhibits Platelet Aggregation and Thrombus Formation by Affecting PI3K/Akt Signaling.

    PubMed

    Su, Xing-li; Su, Wen; He, Zhi-long; Ming, Xin; Kong, Yi

    2015-09-01

    Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatelet tripeptide SQL (H-Ser-Gln-Leu-OH) was isolated and characterized from centipede. In this study, we investigated its antithrombotic activities in vivo and underlying mechanism. It was found that SQL inhibited platelet aggregation induced by adenosine diphosphate, thrombin, epinephrine, and collagen and attenuated thrombus formation in both the ferric chloride-induced arterial thrombosis model and arteriovenous shunt thrombosis model in rats. It did not prolong the bleeding time in mice even at the dose of 10 mg/kg that showed potent antithrombosis effects. Molecular docking revealed that SQL binds PI3Kβ with the binding free energy of -24.341 kcal/mol, which is close to that of cocrystallized ligand (-24.220 kcal/mol). Additionally, SQL displayed inhibition on the late (180 seconds) but did not influence the early (60 seconds) Akt Ser473 phosphorylation in the immunoblot assay. These results suggest that SQL inhibits thrombus formation in vivo and that SQL inhibits PI3K-mediated signaling or even the PI3K itself in platelets. This study may help elucidate the mechanism for centipede treating cardiovascular diseases. PMID:25923322

  14. Conditioned water affects pair formation behaviour in the marine polychaete Neanthes (Nereis) acuminata.

    PubMed

    Storey, Ellen J; Reish, Don J; Hardege, Jörg D

    2013-01-01

    Assessing cues from conspecifics is paramount during mate choice decisions. Neanthes acuminata is a marine polychaete with a unique life cycle: pair formation, female death following reproduction, male parental care and male ability to mate again after egg care. Males completing such egg care are 'experienced'. Females have been shown to prefer experienced males over all others, including aggressively dominant males. As the female dies following reproduction, the reproductive success of her offspring depends upon successful parental care by the male. It is therefore vital that the female makes a good mate choice decision. This paper shows that the use of conditioned water from males caring for eggs and newly experienced males caused the female to alter her choice to a previously undesired male. However, conditioned water from males, which had reproduced but were isolated for 2 weeks, did not have the same effect on pairing behaviour. This indicates that the smell of experience is short lived. PMID:22941305

  15. Medicinal plants extracts affect virulence factors expression and biofilm formation by the uropathogenic Escherichia coli.

    PubMed

    Wojnicz, Dorota; Kucharska, Alicja Z; Sokół-Łętowska, Anna; Kicia, Marta; Tichaczek-Goska, Dorota

    2012-12-01

    Medicinal plants are an important source for the therapeutic remedies of various diseases including urinary tract infections. This prompted us to perform research in this area. We decided to focus on medicinal plants species used in urinary tract infections prevention. The aim of our study was to determine the influence of Betula pendula, Equisetum arvense, Herniaria glabra, Galium odoratum, Urtica dioica, and Vaccinium vitis-idaea extracts on bacterial survival and virulence factors involved in tissue colonization and biofilm formation of the uropathogenic Escherichia coli rods. Qualitative and quantitative analysis of plant extracts were performed. Antimicrobial assay relied on the estimation of the colony forming unit number. Hydrophobicity of cells was established by salt aggregation test. Using motility agar, the ability of bacteria to move was examined. The erythrocyte hemagglutination test was used for fimbriae P screening. Curli expression was determined using YESCA agar supplemented with congo red. Quantification of biofilm formation was carried out using a microtiter plate assay and a spectrophotometric method. The results of the study indicate significant differences between investigated extracts in their antimicrobial activities. The extracts of H. glabra and V. vitis-idaea showed the highest growth-inhibitory effects (p < 0.05). Surface hydrophobicity of autoaggregating E. coli strain changed after exposure to all plant extracts, except V. vitis-idaea (p > 0.05). The B. pendula and U. dioica extracts significantly reduced the motility of the E. coli rods (p < 0.05). All the extracts exhibited the anti-biofilm activity. PMID:22915095

  16. Factors affecting treatment efficacy in social phobia: the use of video feedback and individual vs. group formats.

    PubMed

    Aderka, Idan M

    2009-01-01

    This meta-analysis assessed two potential moderators of treatment efficacy in social phobia: video feedback, and treatment format (i.e., individual vs. group). Eighteen recent (2000-2006) trials including a total of 511 participants were sampled. Effect sizes (Cohen's d's) were calculated for each trial while correcting for measurement error. The Q statistic was used to test (a) heterogeneity across trials and (b) potential moderators. Results indicated that use of video feedback was not a moderator of treatment efficacy and did not significantly affect effect sizes. In contrast, treatment format was a moderator of treatment efficacy such that individual treatments reported larger effect sizes and lower attrition rates compared with group treatments. The results suggest that individual treatments in social phobia may be superior to group treatments irrespective of treatment type. PMID:18599263

  17. The impacts of ozonation on oil sands process-affected water biodegradability and biofilm formation characteristics in bioreactors.

    PubMed

    Hwang, Geelsu; Dong, Tao; Islam, Md Sahinoor; Sheng, Zhiya; Pérez-Estrada, Leónidas A; Liu, Yang; Gamal El-Din, Mohamed

    2013-02-01

    To examine the effects of the ozonation process (as an oxidation treatment for water and wastewater treatment applications) on microbial biofilm formation and biodegradability of organic compounds present in oil sands process-affected water (OSPW), biofilm reactors were operated continuously for 6weeks. Two types of biofilm substrate materials: polyethylene (PE) and polyvinylchloride (PVC), and two types of OSPW-fresh and ozonated OSPWs-were tested. Endogenous microorganisms, in OSPW, quickly formed biofilms in the reactors. Without ozonation, the bioreactor (using endogenous microorganisms) removed 13.8% of the total acid-extractable organics (TAO) and 18.5% of the parent naphthenic acids (NAs) from fresh OSPW. The combined ozonation and biodegradation process removed 87.2% of the OSPW TAO and over 99% of the OSPW parent NAs. Further UPLC/HRMS analysis showed that NA biodegradability decreased as the NA cyclization number increased. Microbial biofilm formation was found to depend on the biofilm substrate type. PMID:23313671

  18. Sulforaphane formation and bioaccessibility are more affected by steaming time than meal composition during in vitro digestion of broccoli.

    PubMed

    Sarvan, I; Kramer, E; Bouwmeester, H; Dekker, M; Verkerk, R

    2017-01-01

    Broccoli is a rich source of the glucosinolate glucoraphanin (GR). After hydrolysis of GR by the endogenous enzyme myrosinase, sulforaphane (SF) or sulforaphane nitrile (SFN) are produced, depending on environmental conditions. How the conversion of GR and bioaccessibility of released breakdown products are affected by steaming (raw, 1min, 2min and 3min steamed) and meal composition (protein or lipid addition) was studied with an in vitro digestion model (mouth, stomach, intestine, but not colonic digestion). The main formation of SF and SFN occurred during in vitro chewing. The contents of GR, SF and SFN did not change after further digestion, as the irreversible inactivated myrosinase under gastric conditions caused no further GR hydrolysis. SF concentrations were up to 10 times higher in raw and 1min steamed broccoli samples after digestion compared to longer-steamed broccoli. Protein or lipid addition had no influence on the formation and bioaccessibility of SF or SFN. PMID:27507513

  19. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs

    PubMed Central

    KIM, Jin-Woo; PARK, Hyo-Jin; CHAE, Sung-Kyu; AHN, Jae-Hyun; DO, Geon-Yeop; CHOO, Young-Kug; PARK, Joung Jun; JUNG, Bae Dong; KIM, Sun-Uk; CHANG, Kyu-Tae; KOO, Deog-Bon

    2016-01-01

    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

  20. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs.

    PubMed

    Kim, Jin-Woo; Park, Hyo-Jin; Chae, Sung-Kyu; Ahn, Jae-Hyun; DO, Geon-Yeop; Choo, Young-Kug; Park, Joung Jun; Jung, Bae Dong; Kim, Sun-Uk; Chang, Kyu-Tae; Koo, Deog-Bon

    2016-06-17

    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

  1. Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts.

    PubMed

    Bermejo-Alvarez, P; Rizos, D; Rath, D; Lonergan, P; Gutierrez-Adan, A

    2010-02-23

    Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five (BEX, CAPN6, BEX2, SRPX2, and UBE2A) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage. PMID:20133684

  2. Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts

    PubMed Central

    Bermejo-Alvarez, P.; Rizos, D.; Rath, D.; Lonergan, P.; Gutierrez-Adan, A.

    2010-01-01

    Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five (BEX, CAPN6, BEX2, SRPX2, and UBE2A) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage. PMID:20133684

  3. Stable complex formation between HIV Rev and the nucleosome assembly protein, NAP1, affects Rev function

    SciTech Connect

    Cochrane, Alan; Murley, Laura Lea; Gao Mian; Wong, Raymond; Clayton, Kiera; Brufatto, Nicole; Canadien, Veronica; Mamelak, Daniel; Chen, Tricia; Richards, Dawn; Zeghouf, Mahel; Greenblatt, Jack; Burks, Christian; Frappier, Lori

    2009-05-25

    The Rev protein of HIV-1 is essential for HIV-1 proliferation due to its role in exporting viral RNA from the nucleus. We used a modified version of tandem affinity purification (TAP) tagging to identify proteins interacting with HIV-1 Rev in human cells and discovered a prominent interaction between Rev and nucleosome assembly protein 1 (Nap1). This interaction was also observed by specific retention of Nap1 from human cell lysates on a Rev affinity column. Nap1 was found to bind Rev through the Rev arginine-rich domain and altered the oligomerization state of Rev in vitro. Overexpression of Nap1 stimulated the ability of Rev to export RNA, reduced the nucleolar localization of Rev, and affected Rev nuclear import rates. The results suggest that Nap-1 may influence Rev function by increasing the availability of Rev.

  4. Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification.

    PubMed

    Diaz, Fabian; Bondiolli, Kenneth; Paccamonti, Dale; Gentry, Glen T

    2016-03-15

    Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent

  5. Assessment of cell lineages and cell death in blastocysts by immunostaining.

    PubMed

    Chauveau, Sabine; Chazaud, Claire

    2015-01-01

    During the last decade it has been shown that most mammalian blastocysts consisted of three cell lineages. Immunofluorescence with multiple antibodies enables to identify each cell type allowing an easy detection of eventual defects. It is complementary to RT-PCR experiments as this technique allows to look at cell position and to analyze and count the proportions between the different cell types. Thus after any kind of embryo manipulation such as nuclear transfer (NT), the analysis of the three cell lineages by immunofluorescence will provide criteria for good or poor development. PMID:25287346

  6. Successful pregnancy following single blastocyst transfer in a renal transplant recipient

    PubMed Central

    Muthuvel, V. Arun; Ravindran, Manipriya; Chander, Aravind; Veluswamy, Chandralekha

    2016-01-01

    Numerous spontaneous pregnancies have been reported in renal transplant recipients; however, only a few pregnancies after the use of assisted reproductive techniques. The authors report a case of renal transplant recipient with secondary infertility who delivered a healthy baby without any complications. The report highlights the importance of minimal stimulation protocol during ovarian stimulation, single embryo transfer, and the need for multispecialty care for these patients. To the best of the authors' knowledge, the present report is the first such case from India and also the second in the world to report a blastocyst transfer among renal transplant recipients. PMID:27110079

  7. Multi-level and multi-scale integrative approach to the understanding of human blastocyst implantation.

    PubMed

    Sengupta, Jayasree; Ghosh, Debabrata

    2014-01-01

    Implantation is a complex process which results in fixation of zona pellucida free blastocyst to the maternal uterine endometrium. In the human, it involves progesterone mediated preparation of endometrium, age- and stage-matched development of pre-implantation embryo, and interaction between embryo and endometrium. In the present essay, we present the case to explain why there is a necessity of undertaking multi-level, multi-scale integrative approach to deconstruct the succession process of endometrial development to the climax of implantation. PMID:24342377

  8. Characterization of the Chromosome 4 Genes That Affect Fluconazole-Induced Disomy Formation in Cryptococcus neoformans

    PubMed Central

    Ngamskulrungroj, Popchai; Chang, Yun; Hansen, Bryan; Bugge, Cliff; Fischer, Elizabeth; Kwon-Chung, Kyung J.

    2012-01-01

    Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1Δglo3Δ did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1Δ but without any discernable alteration in gcs2Δ and glo3Δ under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3Δ and gcs2Δ strains. In sey1Δ, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C

  9. Tyrosine Phosphorylation and Dephosphorylation in Burkholderia cenocepacia Affect Biofilm Formation, Growth under Nutritional Deprivation, and Pathogenicity

    PubMed Central

    Andrade, Angel; Tavares-Carreón, Faviola; Khodai-Kalaki, Maryam

    2015-01-01

    Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate p-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated in vivo and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia. PMID:26590274

  10. Sumoylation of eIF4A2 affects stress granule formation

    PubMed Central

    Jongjitwimol, Jirapas; Baldock, Robert A.; Morley, Simon J.

    2016-01-01

    ABSTRACT Regulation of protein synthesis is crucial for cells to maintain viability and to prevent unscheduled proliferation that could lead to tumorigenesis. Exposure to stress results in stalling of translation, with many translation initiation factors, ribosomal subunits and mRNAs being sequestered into stress granules or P bodies. This allows the re-programming of the translation machinery. Many aspects of translation are regulated by post-translational modification. Several proteomic screens have identified translation initiation factors as targets for sumoylation, although in many cases the role of this modification has not been determined. We show here that eIF4A2 is modified by SUMO, with sumoylation occurring on a single residue (K226). We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol. In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules. Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response. PMID:27160682

  11. Physiological variables affecting surface film formation by native lamellar body-like pulmonary surfactant particles.

    PubMed

    Hobi, Nina; Siber, Gerlinde; Bouzas, Virginia; Ravasio, Andrea; Pérez-Gil, Jesus; Haller, Thomas

    2014-07-01

    Pulmonary surfactant (PS) is a surface active complex of lipids and proteins that prevents the alveolar structures from collapsing and reduces the work of breathing by lowering the surface tension at the alveolar air-liquid interface (ALI). Surfactant is synthesized by the alveolar type II (AT II) cells, and it is stored in specialized organelles, the lamellar bodies (LBs), as tightly packed lipid bilayers. Upon secretion into the alveolar lining fluid, a large fraction of these particles retain most of their packed lamellar structure, giving rise to the term lamellar body like-particles (LBPs). Due to their stability in aqueous media, freshly secreted LBPs can be harvested from AT II cell preparations. However, when LBPs get in contact with an ALI, they quickly and spontaneously adsorb into a highly organized surface film. In the present study we investigated the adsorptive capacity of LBPs at an ALI under relevant physiological parameters that characterize the alveolar environment in homeostatic or in pathological conditions. Adsorption of LBPs at an ALI is highly sensitive to pH, temperature and albumin concentration and to a relatively lesser extent to changes in osmolarity or Ca(2+) concentrations in the physiological range. Furthermore, proteolysis of LBPs significantly decreases their adsorptive capacity confirming the important role of surfactant proteins in the formation of surface active films. PMID:24582711

  12. Sumoylation of eIF4A2 affects stress granule formation.

    PubMed

    Jongjitwimol, Jirapas; Baldock, Robert A; Morley, Simon J; Watts, Felicity Z

    2016-06-15

    Regulation of protein synthesis is crucial for cells to maintain viability and to prevent unscheduled proliferation that could lead to tumorigenesis. Exposure to stress results in stalling of translation, with many translation initiation factors, ribosomal subunits and mRNAs being sequestered into stress granules or P bodies. This allows the re-programming of the translation machinery. Many aspects of translation are regulated by post-translational modification. Several proteomic screens have identified translation initiation factors as targets for sumoylation, although in many cases the role of this modification has not been determined. We show here that eIF4A2 is modified by SUMO, with sumoylation occurring on a single residue (K226). We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol. In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules. Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response. PMID:27160682

  13. Hematopoietic Fas Deficiency Does Not Affect Experimental Atherosclerotic Lesion Formation despite Inducing a Proatherogenic State

    PubMed Central

    de Claro, R. Angelo; Zhu, Xiaodong; Tang, Jingjing; Morgan-Stevenson, Vicki; Schwartz, Barbara R.; Iwata, Akiko; Liles, W. Conrad; Raines, Elaine W.; Harlan, John M.

    2011-01-01

    The Fas death receptor (CD95) is expressed on macrophages, smooth muscle cells, and T cells within atherosclerotic lesions. Given the dual roles of Fas in both apoptotic and nonapoptotic signaling, the aim of the present study was to test the effect of hematopoietic Fas deficiency on experimental atherosclerosis in low-density lipoprotein receptor-null mice (Ldlr−/−). Bone marrow from Fas−/− mice was used to reconstitute irradiated Ldlr−/− mice as a model for atherosclerosis. After 16 weeks on an 0.5% cholesterol diet, no differences were noted in brachiocephalic artery lesion size, cellularity, or vessel wall apoptosis. However, Ldlr−/− mice reconstituted with Fas−/− hematopoietic cells had elevated hyperlipidemia [80% increase, relative to wild-type (WT) controls; P < 0.001] and showed marked elevation of plasma levels of CXCL1/KC, CCL2/MCP-1, IL-6, IL-10, IL-12 subunit p70, and soluble Fas ligand (P < 0.01), as well as systemic microvascular inflammation. It was not possible to assess later stages of atherosclerosis because of increased mortality in Fas−/− bone marrow recipients. Our data indicate that hematopoietic Fas deficiency does not affect early atherosclerotic lesion development in Ldlr−/− mice. PMID:21550016

  14. Biological Filtration Limits Carbon Availability and Affects Downstream Biofilm Formation and Community Structure†

    PubMed Central

    Pang, Chee Meng; Liu, Wen-Tso

    2006-01-01

    Carbon removal strategies have gained popularity in the mitigation of biofouling in water reuse processes, but current biofilm-monitoring practices based on organic-carbon concentrations may not provide an accurate representation of the in situ biofilm problem. This study evaluated a submerged microtiter plate assay for direct and rapid monitoring of biofilm formation by subjecting the plates to a continuous flow of either secondary effluent (SE) or biofilter-treated secondary effluent (BF). This method was very robust, based on a high correlation (R2 = 0.92) between the biomass (given by the A600 in the microtiter plate assay) and the biovolume (determined from independent biofilms developed on glass slides under identical conditions) measurements, and revealed that the biomasses in BF biofilms were consistently lower than those in SE biofilms. The influence of the organic-carbon content on the biofilm community composition and succession was further evaluated using molecular tools. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed a group of pioneer colonizers, possibly represented by Sphingomonadaceae and Caulobacter organisms, to be common in both SE and BF biofilms. However, differences in organic-carbon availabilities in the two water samples eventually led to the selection of distinct biofilm communities. Alphaproteobacterial populations were confirmed by fluorescence in situ hybridization to be enriched in SE biofilms, while Betaproteobacteria were dominant in BF biofilms. Cloning analyses further demonstrated that microorganisms adapted for survival under low-substrate conditions (e.g., Aquabacterium, Caulobacter, and Legionella) were preferentially selected in the BF biofilm, suggesting that carbon limitation strategies may not achieve adequate biofouling control in the long run. PMID:16957184

  15. Osteoprotegerin exposure at different stages of osteoclastogenesis differentially affects osteoclast formation and function.

    PubMed

    Zhao, Hongyan; Gu, Jianhong; Dai, Nannan; Gao, Qian; Wang, Dong; Song, Ruilong; Liu, Wei; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Liu, Zongping

    2016-08-01

    This study aimed to investigate the effects of osteoprotegerin (OPG), a decoy receptor for receptor activator for nuclear factor κB ligand (RANKL), during the various stages of osteoclast differentiation, and additionally investigate its effects on osteoclast adhesion and activity. RAW264.7 murine monocytic cells were incubated with macrophage colony-stimulating factor and RANKL for 1, 3, 5, or 7 days, followed by an additional 24-h incubation in the presence or absence of OPG (80 ng/mL). We examined osteoclast differentiation and adhesion capacity using the tartrate-resistant acid phosphatase (TRAP) assay and immunofluorescence microscopy, and additionally examined cell growth in real time using the xCELLigence system. Furthermore, the expression levels of TRAP, RANK, integrin β3, matrix metalloproteinase 9, cathepsin K, carbonic anhydrase II, and vesicular-type H(+)-ATPase A1 were examined using western blotting. OPG exposure on day 1 enhanced the osteoclast growth curve as well as adhesion, and increased RANK and integrin β3 expression. In contrast, exposure to OPG at later time points (days 3-7) inhibited osteoclast differentiation, adhesion structure formation, and protease expression. In conclusion, the biological effects of OPG exposure at the various stages of osteoclast differentiation were varied, and included the enhanced adhesion and survival of preosteoclasts, the block of differentiation from the early to the terminal stages of osteoclastogenesis, and suppression of mature osteoclast activation following OPG exposure during the terminal differentiation stage, suggesting that the effects of OPG exposure differ based on the stage of differentiation. PMID:26044733

  16. Launch conditions might affect the formation of blood vessels in the quail chorioallantoic membrane.

    PubMed

    Henry, M K; Unsworth, B R; Sychev, V; Guryeva, T S; Dadasheva, O A; Piert, S J; Lagel, K E; Dubrovin, L C; Jahns, G C; Boda, K; Sabo, V; Samet, M M; Lelkes, P I

    1998-01-01

    As a part of the first joint USA-Russian MIR/Shuttle program, fertilized quail eggs were flown on the MIR 18 mission. Post-flight examination indicated impaired survival of both the embryos in space and also of control embryo exposed to vibrational and g-forces simulating the condition experienced during the launch of Progress 227. We hypothesized that excess mechanical forces and/or other conditions during the launch might cause abnormal development or the blood supply in the chorioallantoic membrane (CAM) leading to the impaired survival of the embryos. The CAM, a highly vascularized extraembryonic organ, provides for the oxygen exchange across the egg shell and is thus pivotal for proper embryonic development. To test our hypothesis, we compared angiogenesis in CAMs of eggs which were either exposed to the vibration and g-force profile simulating the conditions at launch of Progress 227 (synchronous controls), or kept under routine conditions in a laboratory incubator (laboratory controls). At various time points during incubation, the eggs were fixed in paraformaldehyde for subsequent dissection. At the time of dissection, the CAM was carefully lifted from the egg shell and examined as whole mounts by bright-field and fluorescent microscopy. The development of the vasculature (angiogenesis) was assessed from the density of blood vessels per viewing field and evaluated by computer aided image analysis. We observed a significant decrease in blood-vessel density in the synchronous controls versus "normal" laboratory controls beginning from day 10 of incubation. The decrease in vascular density was restricted to the smallest vessels only, suggesting that conditions during the launch and/or during the subsequent incubation of the eggs may affect the normal progress of angiogenesis in the CAM. Abnormal angiogensis in the CAM might contribute to the impaired survival of the embryos observed in synchronous controls as well as in space. PMID:11543304

  17. Launch Conditions Might Affect the Formation of Blood Vessel in the Quail Chorioallantoic Membrane

    NASA Technical Reports Server (NTRS)

    Henry, M. K.; Unsworth, B. R.; Sychev, B. R.; Guryeva, T. S.; Dadasheva, O. A.; Piert, S. J.; Lagel, K. E.; Dubrovin, L. C.; Jahns, G. C.; Boda, K.; Sabo, V.; Samet, M. M.; Lelkes, P. I.

    1998-01-01

    AS 2 part of the first joint USA-Russian MIR/Shuttle program, fertilized quail eggs were flown on the MIR 18 mission. Post-flight examination indicated impaired survival of both the embryos in space and also of control embryos exposed to vibrational and g-forces simulating the conditions experienced during the launch of Progress 227. We hypothesized that excess mechanical forces and/or other conditions during the launch might cause abnormal development of the blood supply in the chorioallantoic membrane (CAM) leading to the impaired survival of the embryos. The CAM, a highly vascularized extraembryonic organ, provides for the oxygen exchange across the egg shell and is thus pivotal for proper embryonic development. To test our hypothesis, we compared angiogenesis In CAMS of eggs which were either exposed to the vibration and g-force profile simulating the conditions at launch of Progress 227 (synchronous controls), or kept under routine conditions in a laboratory Incubator (laboratory controls). At various time points during Incubation, the eggs were fixed in paraformaldehyde for subsequent dissection. At the time of dissection, the CAM was carefully lifted from the egg shell and examined as whole mounts by bright-field and fluorescent microscopy. The development or the vasculature (angiogenesis) was assessed from the density of blood vessels per viewing field and evaluated by computer aided image analysis. We observed a significant decrease In blood-vessel density in the synchronous controls versus "normal" laboratory controls beginning from day 10 of Incubation. The decrease in vascular density was restricted to the smallest vessels only, suggesting that conditions during the launch and/or during the subsequent Incubation of the eggs may affect the normal progress of angiogenesis in the CAM. Abnormal angiogenesis In the CAM might contribute to the impaired survival of the embryos observed in synchronous controls as well as in space.

  18. N-acetyl-L-cysteine affects growth, extracellular polysaccharide production, and bacterial biofilm formation on solid surfaces.

    PubMed

    Olofsson, Ann-Cathrin; Hermansson, Malte; Elwing, Hans

    2003-08-01

    N-Acetyl-L-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces. PMID:12902275

  19. Isolation and characterization of mutants with lesions affecting pellicle formation and erythrocyte agglutination by type 1 piliated Escherichia coli.

    PubMed Central

    Harris, S L; Elliott, D A; Blake, M C; Must, L M; Messenger, M; Orndorff, P E

    1990-01-01

    The product of the pilE (also called fimH) gene is a minor component of type 1 pili in Escherichia coli. Mutants that have insertions in the pilE gene are fully piliated but unable to bind to and agglutinate guinea pig erythrocytes, a characteristic of wild-type type 1 piliated E. coli. In this paper we describe the isolation of 48 mutants with point lesions that map to the pilE gene. Such mutants were isolated by using mutT mutagenesis and an enrichment procedure devised to favor the growth of individuals that could form a pellicle in static broth containing alpha-methylmannoside, an inhibitor of erythrocyte binding and pellicle formation. Results indicated that the enrichment favored mutants expressing pilE gene products that were defective in mediating erythrocyte binding. Characterization of 12 of the mutants in greater detail revealed that certain lesions affected pilus number and length. In addition, a mutant that was temperature sensitive for erythrocyte binding was isolated and used to provide evidence that pellicle formation relies on the intercellular interaction of pilE gene products. Our results suggest a molecular explanation for the old and paradoxical observations connecting pellicle formation and erythrocyte agglutination by type 1 piliated E. coli. Images PMID:1977736

  20. Pharmacologic regulation of AMPK in breast cancer affects cytoskeletal properties involved with microtentacle formation and re-attachment

    PubMed Central

    Chakrabarti, Kristi R.; Whipple, Rebecca A.; Boggs, Amanda E.; Hessler, Lindsay K.; Bhandary, Lekhana; Vitolo, Michele I.; Thompson, Keyata; Martin, Stuart S.

    2015-01-01

    The presence of tumor cells in the circulation is associated with a higher risk of metastasis in patients with breast cancer. Circulating breast tumor cells use tubulin-based structures known as microtentacles (McTNs) to re-attach to endothelial cells and arrest in distant organs. McTN formation is dependent on the opposing cytoskeletal forces of stable microtubules and the actin network. AMP-activated protein kinase (AMPK) is a cellular metabolic regulator that can alter actin and microtubule organization in epithelial cells. We report that AMPK can regulate the cytoskeleton of breast cancer cells in both attached and suspended conditions. We tested the effects of AMPK on microtubule stability and the actin-severing protein, cofilin. AMPK inhibition with compound c increased both microtubule stability and cofilin activation, which also resulted in higher McTN formation and re-attachment. Conversely, AMPK activation with A-769662 decreased microtubule stability and cofilin activation with concurrent decreases in McTN formation and cell re-attachment. This data shows for the first time that AMPK shifts the balance of cytoskeletal forces in suspended breast cancer cells, which affect their ability to form McTNs and re-attach. These results support a model where AMPK activators may be used therapeutically to reduce the metastatic efficiency of breast tumor cells. PMID:26431377

  1. Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts.

    PubMed

    Jiang, Zongliang; Harrington, Patrick; Zhang, Ming; Marjani, Sadie L; Park, Joonghoon; Kuo, Lynn; Pribenszky, Csaba; Tian, Xiuchun Cindy

    2016-01-01

    High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes. PMID:26883277

  2. Transcriptome Profiling of Rabbit Parthenogenetic Blastocysts Developed under In Vivo Conditions

    PubMed Central

    Naturil-Alfonso, Carmen; Saenz-de-Juano, María dels Desamparats; Peñaranda, David S.

    2012-01-01

    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. PMID:23251477

  3. Effects of High Hydrostatic Pressure on Expression Profiles of In Vitro Produced Vitrified Bovine Blastocysts

    PubMed Central

    Jiang, Zongliang; Harrington, Patrick; Zhang, Ming; Marjani, Sadie L.; Park, Joonghoon; Kuo, Lynn; Pribenszky, Csaba; Tian, Xiuchun (Cindy)

    2016-01-01

    High hydrostatic pressure (HHP) has been used to pre-condition embryos before essential, yet potentially detrimental procedures such as cryopreservation. However, the mechanisms for HHP are poorly understood. We treated bovine blastocysts with three different HHP (40, 60 and 80 MPa) in combination with three recovery periods (0, 1 h, 2 h post HHP). Re-expansion rates were significantly higher at 40 and 60 but lower at 80 MPa after vitrification-warming in the treated groups than controls. Microarray analysis revealed 399 differentially expressed transcripts, representing 254 unique genes, among different groups. Gene ontology analysis indicated that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes in cell death and apoptosis, and up-regulation of genes in RNA processing, cellular growth and proliferation. In contrast, 80 MPa up-regulated genes in apoptosis, and down-regulated protein folding and cell cycle-related genes. Moreover, gene expression was also influenced by the length of the recovery time after HHP. The significantly over-represented categories were apoptosis and cell death in the 1 h group, and protein folding, response to unfolded protein and cell cycle in the 2 h group compared to 0 h. Taken together, HHP promotes competence of vitrified bovine blastocysts through modest transcriptional changes. PMID:26883277

  4. Analysis of marker expression in porcine cell lines derived from blastocysts produced in vitro and in vivo.

    PubMed

    Vackova, Irena; Novakova, Zora; Krylov, Vladimir; Okada, Konosuke; Kott, Tomas; Fulka, Helena; Motlik, Jan

    2011-10-01

    The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ES-like morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (cytokeratin 18, lamins A/C, transferrin, α-fetoprotein and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies. PMID:21685711

  5. Melt front propagation in dielectrics upon femtosecond laser irradiation: Formation dynamics of a heat-affected layer

    NASA Astrophysics Data System (ADS)

    Garcia-Lechuga, Mario; Solis, Javier; Siegel, Jan

    2016-04-01

    Several studies in dielectrics have reported the presence of a thin heat-affected layer underneath the ablation crater produced by femtosecond laser irradiation. In this work, we present a time-resolved microscopy technique that is capable of monitoring the formation dynamics of this layer and apply it to the study of a phosphate glass exposed to single pulses below the ablation threshold. A few nanoseconds after laser excitation, a melt front interface can be detected, which propagates into the bulk, gradually slowing down its speed. By means of image analysis combined with optical modeling, we are able to determine the temporal evolution of the layer thickness and its refractive index. Initially, a strong transient decrease in the refractive index is observed, which partially recovers afterwards. The layer resolidifies after approximately 1 μs after excitation, featuring a maximum thickness of several hundreds of nanometers.

  6. Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic Sperm Injection in Caprine Oocytes.

    PubMed

    Kharche, S D; Pathak, J; Agarwal, S; Kushwah, B; Sikarwar, Aks

    2016-08-01

    The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes. PMID:27170442

  7. Ultrastructure of IVM-IVF bovine blastocysts vitrified after equilibration in glycerol 1,2-propanediol using 2-step and 16-step procedures.

    PubMed

    Kuwayama, M; Fujikawa, S; Nagai, T

    1994-10-01

    Two experiments were conducted to investigate the relationship between survival rates and ultrastructural appearance, using the freeze-replica technique, of bovine blastocysts after vitrification. In experiment 1, blastocysts obtained from in vitro-matured and in vitro-fertilized (IVM-IVF) bovine oocytes were either equilibrated in a stepwise manner in a vitrification solution (VS; 22.5% glycerol + 22.5% 1,2-propanediol) using 16 steps for 18 min in total (the 16-step method) or equilibrated with 10% glycerol + 20% 1,2-propanediol for 10 min and then exposed to VS (the 2-step method). The blastocysts were then vitrified by plunging them into liquid nitrogen. All samples were subsequently thawed in a water bath at 37 degrees C and cultured in vitro with a monolayer of cumulus cells. The survival rate obtained for blastocysts equilibrated by the 16-step method was 83.3% (25/30). In contrast, no blastocysts survived by the 2-step method (0/30). In experiment 2, freeze-replica observations were carried out on blastocysts vitrified by the 16-step method and the 2-step method. In all the blastocysts, no ice crystals were observed in the cytoplasm, blastocoelic cavity, or extracellular areas, which confirmed the occurrence of complete vitrification. Little ultrastructural change was observed in the plasma membrane of the blastocysts equilibrated by the 16-step method. In contrast, small vesicles and distinct intramembrane particle (IMP) aggregation were frequently observed in the plasma membranes of blastocysts equilibrated by the 2-step method. These results indicate that the successful cryopreservation of blastocysts following the 16-step equilibration requires not only complete vitrification, but also minimization of ultrastructural damage to the plasma membrane. PMID:7988150

  8. Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism, acid tolerance response and biofilm formation

    PubMed Central

    Liao, S.; Bitoun, J.P.; Nguyen, A.H.; Bozner, D.; Yao, X.; Wen, Z.T.

    2015-01-01

    Summary Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by realtime polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyl-transferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6. Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation. PMID:25421565

  9. Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism, acid tolerance response and biofilm formation.

    PubMed

    Liao, S; Bitoun, J P; Nguyen, A H; Bozner, D; Yao, X; Wen, Z T

    2015-08-01

    Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real-time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6 . Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation. PMID:25421565

  10. Factors that Affect Science and Mathematics Teachers' Initial Implementation of Technology-Enhanced Formative Assessment Using a Classroom Response System

    NASA Astrophysics Data System (ADS)

    Lee, Hyunju; Feldman, Allan; Beatty, Ian D.

    2012-10-01

    The purpose of this study is to uncover and understand the factors that affect secondary science and mathematics teachers' initial implementation of Technology-Enhanced Formative Assessment (TEFA), a pedagogy developed for teaching with classroom response system (CRS) technology. We sought to identify the most common and strongest factors, and to understand the general process of how teachers adopt TEFA. We identified ten main hindering factors reported by teachers, and found that time limitations and question development difficulties are reported as the most problematic. In this paper we provide five vignettes of teachers' initial implementation experiences, illustrating different courses that TEFA adoption can follow. We classify our ten factors into four groups: contextual factors that directly hinder teachers' attempts to implement TEFA (extrinsic type I); circumstances that affect teachers' teaching in general (extrinsic type 0); gaps that teachers have in the knowledge and skills they need to adopt TEFA (intrinsic type I); and ways of being a teacher that describe teachers' deeper perspectives and beliefs, which may be consonant or dissonant with TEFA (intrinsic type II). Finally, we identify four general categories that describe the teachers' initial TEFA implementation.

  11. Affects, trauma, and mechanisms of symptom formation: a tribute to John C. Nemiah, MD (1918-2009).

    PubMed

    Taylor, Graeme J

    2010-01-01

    John Nemiah was interested in the impact of emotionally traumatic events on mental and bodily processes and in conceptualizing the psychological defenses and deficits that contribute to the development of psychological and somatic symptoms. He viewed dissociation as the central psychological mechanism in the formation of a spectrum of symptoms, and conceptualized alexithymia as a deficit in the cognitive processing of emotion such that stress-induced arousal could bypass the psyche and produce somatic symptoms. This article briefly reviews some of Nemiah's conceptual ideas and relates them to several new theories and concepts and findings from empirical research. His concept of the 'psychic elaboration' of emotion is consistent with contemporary theories of the cognitive processing of emotions that emphasize the importance of imagery and linguistic symbolizations. Alexithymia is inversely related to mentalization and is associated with insecure attachment styles and emotional trauma, which influence the capacity to regulate affects induced by stressful events. A multiple code theory of emotional information processing links psychological and somatic symptoms to various degrees of dissociation within and between the elements comprising emotion schemas and to compensatory attempts at repair. Recent studies support Nemiah's view that alexithymia and intrapsychic conflicts may both contribute to the pathogenesis of panic attacks. There is also substantial evidence of an association between childhood trauma and the development of somatic disease in adult life. Secure attachments and well-developed capacities for symbolization and affect regulation seem to render individuals more resilient to the traumas and stressful events of everyday life. PMID:20733344

  12. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos.

    PubMed

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  13. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers. PMID:25592083

  14. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

    PubMed Central

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  15. GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst.

    PubMed

    Schrode, Nadine; Saiz, Néstor; Di Talia, Stefano; Hadjantonakis, Anna-Katerina

    2014-05-27

    Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell-resolution image analysis pipeline to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling and propose a model where interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification. PMID:24835466

  16. Mouse and human blastocyst-derived stem cells: vive les differences.

    PubMed

    Rossant, Janet

    2015-01-01

    Lessons learned from conserved vertebrate developmental pathways have catalyzed rapid advances in pluripotent stem cell differentiation towards therapeutically relevant cell types. The most highly conserved phases of development are associated with the early patterning of the body plan - the so-called phylotypic stage. Both prior to and after this stage there is much more divergence across species. Developmental differences between human and mouse at the blastocyst and early post-implantation stages might help explain the differences among the different stem cell lines derived from these embryos. A better understanding of these early stages of human development will aid our ability to generate and manipulate human stem cells and their derivatives. PMID:25516964

  17. TallyHO obese female mice experience poor reproductive outcomes and abnormal blastocyst metabolism which is reversed by metformin

    PubMed Central

    Louden, Erica D.; Luzzo, Kerri M.; Jimenez, Patricia T.; Chi, Tiffany; Chi, Maggie; Moley, Kelle H.

    2015-01-01

    Objective Obese women experience worse reproductive outcomes compared to normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay. The objective of this study was to use a genetic mouse model of obesity in order to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. Methods New inbred, polygenic Type 2 diabetic TallyHO mice and age matched control C57BL/6 mice were superovulated to obtain morulae or blastocysts stage embryos which were cultured in human tubal fluid media. Deoxyglucose uptake was performed on insulin-stimulated individual blastocysts. Apoptosis was detected by confocal microscopy using TUNEL assay and Topro-3 nuclear dye. Embryos were scored for %TUNEL positive/total nuclei. AMPK activation, TNFα expression, and adiponectin expression were analyzed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by Bodipy. Finally all measured parameters were compared between TallyHO mice in morulaes cultured to blastocyst embryos in either human tubal fluid (HTF) media or HTF with 25ug/ml metformin added. Results TallyHo mice developed whole body abnormal insulin tolerance, decreased litter number and increased NEFA. Blastocysts demonstrated increased apoptosis, decreased insulin sensitivity, and decreased activation of AMP activated protein-kinase (AMPK). As a possible cause of the insulin resistance/abnormal P-AMPK, we found that Tumor necrosis Factor (TNFα) expression and lipid accumulation as detected by BODIPY were increased in TallyHO blastocysts and adiponectin was decreased. Culturing TallyHO morulae with the AMPK activator, metformin lead to a reversal of all abnormal findings, including increased p-AMPK, improved insulin-stimulated glucose uptake and normalization of lipid accumulation. Conclusions Women with obesity and insulin resistance experience poor pregnancy outcomes. Previously we have shown in mouse

  18. Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

    PubMed Central

    van Leeuwen, Jessica; Berg, Debra K.; Pfeffer, Peter L.

    2015-01-01

    A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology. PMID:26076128

  19. The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation.

    PubMed

    Ivanchenko, Maria G; Zhu, Jinsheng; Wang, Bangjun; Medvecká, Eva; Du, Yunlong; Azzarello, Elisa; Mancuso, Stefano; Megraw, Molly; Filichkin, Sergei; Dubrovsky, Joseph G; Friml, Jiří; Geisler, Markus

    2015-02-15

    Cyclophilin A is a conserved peptidyl-prolyl cis-trans isomerase (PPIase) best known as the cellular receptor of the immunosuppressant cyclosporine A. Despite significant effort, evidence of developmental functions of cyclophilin A in non-plant systems has remained obscure. Mutations in a tomato (Solanum lycopersicum) cyclophilin A ortholog, DIAGEOTROPICA (DGT), have been shown to abolish the organogenesis of lateral roots; however, a mechanistic explanation of the phenotype is lacking. Here, we show that the dgt mutant lacks auxin maxima relevant to priming and specification of lateral root founder cells. DGT is expressed in shoot and root, and localizes to both the nucleus and cytoplasm during lateral root organogenesis. Mutation of ENTIRE/IAA9, a member of the auxin-responsive Aux/IAA protein family of transcriptional repressors, partially restores the inability of dgt to initiate lateral root primordia but not the primordia outgrowth. By comparison, grafting of a wild-type scion restores the process of lateral root formation, consistent with participation of a mobile signal. Antibodies do not detect movement of the DGT protein into the dgt rootstock; however, experiments with radiolabeled auxin and an auxin-specific microelectrode demonstrate abnormal auxin fluxes. Functional studies of DGT in heterologous yeast and tobacco-leaf auxin-transport systems demonstrate that DGT negatively regulates PIN-FORMED (PIN) auxin efflux transporters by affecting their plasma membrane localization. Studies in tomato support complex effects of the dgt mutation on PIN expression level, expression domain and plasma membrane localization. Our data demonstrate that DGT regulates auxin transport in lateral root formation. PMID:25617431

  20. Factors Affecting the Formation, Severity and Location of White Spot Lesions during Orthodontic Treatment with Fixed Appliances

    PubMed Central

    2014-01-01

    ABSTRACT Objectives The purpose of the present study was to investigate factors affecting the formation, severity and location of white spot lesions in patients completing fixed appliance therapy. Material and Methods A total of 45 patients (19 males and 26 females, mean age 15.81 years, standard deviation 2.89 years) attending consecutively Aberdeen Dental Hospital (ADH) between January and June 2013 to have their fixed appliances removed were given a questionnaire to elicit information regarding their dental care and diet. They were then examined clinically as well as their pre-treatment photographs to record treatment data and white spot lesion (WSL) location and severity using a modified version of Universal Visual Scale for Smooth Surfaces (UniViSS Smooth). Absolute risk (AR) and risk ratios (RR) were also calculated. Results The incidence of at least one WSL observed in patients was 42%, with males displaying a higher incidence than females. The highest incidence of WSLs was recorded on the maxillary canines and lateral incisors, and on the maxillary and mandibular premolars and first molars. The gingival areas of the maxillary and mandibular teeth were the most affected surfaces. Significant (P < 0.05) relationships were found between the presence of WSLs and the following factors: poor oral hygiene (OH), males, increased treatment length, lack of use of fluoride supplements, use of carbonated soft drinks and/or fruit juices and the use of sugary foods. Poor OH posed the highest risk of developing WSL (RR = 8.55). Conclusions 42% of patients have developed white spot lesions during fixed appliance therapy. Various contributing risk factors were identified with the greatest risk posed by a poor oral hygiene. PMID:24800054

  1. Precursors and factors affecting formation of haloacetonitriles and chloropicrin during chlor(am)ination of nitrogenous organic compounds in drinking water.

    PubMed

    Jia, Aiyin; Wu, Chunde; Duan, Yan

    2016-05-01

    This study investigated the precursors and factors affecting formation of haloacetonitriles (HANs) and chloropicrin (TCNM) during chlorination/chloramination of eight amino acids in the effluent water of V-type clarifying filtration from a drinking water treatment plant. The yields of trichloroacetonitrile (TCAN), dichloroacetonitrile (DCAN) and TCNM were higher during chlorination than during chloramination. Tyrosine and tryptophan produced the greatest amount of DCAN and also generated a small amount of TCAN during chlorination process. Besides, the yields of DCAN were higher than TCNM during chlorination/chloramination. Contact time, Cl2:org-N molar ratios, pH, temperature and bromide ion affected nitrogenous disinfection by-products (N-DBPs) formation during chlorination of tryptophan in different degrees. TCAN, DCAN and TCNM formation showed the increasing and then decreasing with prolonged contact time. Higher Cl2:org-N molar ratios improved N-DBPs formation within a certain range. The pH affected N-DBPs formation differently. HANs increased with increasing pH from 5 to 6 and decreased with increasing pH from 6 to 9, while TCNM increased with increasing pH from 5 to 9. Higher temperatures enhanced TCNM formation, but reduced the formation of TCAN and DCAN. The presence of bromide ions improved the yields of HANs and TCNM and shifted N-DBPs to more brominated ones. PMID:26859617

  2. Deregulated expression of Cdc6 in the skin facilitates papilloma formation and affects the hair growth cycle.

    PubMed

    Búa, Sabela; Sotiropoulou, Peggy; Sgarlata, Cecilia; Borlado, Luis R; Eguren, Manuel; Domínguez, Orlando; Ortega, Sagrario; Malumbres, Marcos; Blanpain, Cedric; Méndez, Juan

    2015-01-01

    Cdc6 encodes a key protein for DNA replication, responsible for the recruitment of the MCM helicase to replication origins during the G1 phase of the cell division cycle. The oncogenic potential of deregulated Cdc6 expression has been inferred from cellular studies, but no mouse models have been described to study its effects in mammalian tissues. Here we report the generation of K5-Cdc6, a transgenic mouse strain in which Cdc6 expression is deregulated in tissues with stratified epithelia. Higher levels of CDC6 protein enhanced the loading of MCM complexes to DNA in epidermal keratinocytes, without affecting their proliferation rate or inducing DNA damage. While Cdc6 overexpression did not promote skin tumors, it facilitated the formation of papillomas in cooperation with mutagenic agents such as DMBA. In addition, the elevated levels of CDC6 protein in the skin extended the resting stage of the hair growth cycle, leading to better fur preservation in older mice. PMID:26697840

  3. Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection.

    PubMed

    Oikawa, T; Takada, N; Kikuchi, T; Numabe, T; Takenaka, M; Horiuchi, T

    2005-04-01

    The objective of this study was to compare the effectiveness of different methods of bovine oocyte activation following intracytoplasmic sperm injection (ICSI) in terms of oocyte cleavage and blastocyst rates, and calf production. Oocytes were harvested, post mortem, from the ovaries of Japanese Black heifers or cows. ICSI was carried out using a piezo-electric actuator. The injected or sham-injected oocytes that were assigned to three activation treatments, each replicated three times, were studied: (1) exposure to 5 microM ionomycin for 5 min (ionomycin); (2) exposure to 5 microM ionomycin for 5 min followed by culture in TCM199 for 3 h and a further 3h culture in 1.9 mM 6-dimethylaminopurine (DMAP-ionomycin+DMAP); (3) exposure to 7% ethanol in TCM199 for 5 min, 4 h after ICSI (ethanol). One or two blastocysts from the ionomycin+DMAP (8 recipients) and ethanol (17 recipients) oocyte activation treatments were non-surgically transferred into Holsteins for the study of calf production. The highest cleavage and blastocyst production rates were observed in the ionomycin+DMAP treatment (83.9% and 40.1%) by the ICSI. These rates were significantly (P<0.05) higher than those for the ionomycin oocyte activation treatment (57.6% and 18.2%) but did not differ from the ethanol treatment (75.6% and 29.4%). In the sham-injected, the highest blastocyst production rates were observed for the ionomycin+DMAP and ethanol treatments (10.7% and 11.3%). Pregnancy and birth rates for blastocysts derived from the ethanol oocyte activation treatment (58.8% and 47.4%) were significantly higher (P<0.05) than those of the ionomycin+DMAP treatment (12.5% and 9.2%). The results showed that post-ICSI oocyte activation with ethanol is more effective than activation with ionomycin alone or with ionomycin+DMAP for the production of viable blastocysts and calves. PMID:15766799

  4. Null mutation of chloride channel 7 (Clcn7) impairs dental root formation but does not affect enamel mineralization.

    PubMed

    Guo, Jing; Bervoets, Theodore J M; Henriksen, Kim; Everts, Vincent; Bronckers, Antonius L J J

    2016-02-01

    ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization. PMID:26346547

  5. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

    PubMed Central

    2014-01-01

    Background Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3 days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. Results T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25–100% of the embryos were knockout embryos and 7–57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. Conclusions The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos. PMID:25042988

  6. Introduction: Examining the many potential reasons why euploid blastocysts do not always result in viable pregnancies: part 1.

    PubMed

    Meldrum, David R

    2016-03-01

    The environment of the oocyte and embryo includes the metabolic effects of aging, lifestyle issues, psychological stress, nutritional supplements/replacements, cellular energy levels, endocrine and paracrine factors, the cumulus, exogenous gonadotropins and adjunctive medications, culture media, and the in vitro fertilization laboratory's conditions and manipulations. These complex factors, which will be examined in the following series of reviews, explain in part why not all euploid blastocysts result in viable pregnancies. PMID:26730497

  7. Quantifying the Biomechanics of Conception: L-Selectin-Mediated Blastocyst Implantation Mechanics with Engineered “Trophospheres”

    PubMed Central

    Jost, Monika; Rothstein, Dianne; Robertson, Noreen; Marcolongo, Michele S.

    2014-01-01

    An estimated 12% of women in the United States suffer from some form of infertility. In vitro fertilization (IVF) is the most common treatment for infertility encompassing over 99% of all assisted reproductive technologies. However, IVF has a low success rate. Live birth rates using IVF can range from 40% in women younger than 35 years to 4% in women older than 42 years. Costs for a successful IVF outcome can be upward of $61,000. The low success rate of IVF has been attributed to the inability of the blastocyst to implant to the uterus. Blastocyst implantation is initiated by L-selectin expressing cells, trophoblasts, binding to L-selectin ligands, primarily sialyl Lewis X (sLeX), on the uterine surface endometrium. Legal and ethical considerations have limited the research on human subjects and tissues, whereas animal models are costly or do not properly mimic human implantation biochemistry. In this work, we describe a cellular model system for quantifying L-selectin adhesion mechanics. L-selectin expression was confirmed in Jeg-3, JAR, and BeWo cell lines, with only Jeg-3 cells exhibiting surface expression. Jeg-3 cells were cultured into three-dimensional spheres, termed “trophospheres,” as a mimic to human blastocysts. Detachment assays using a custom-built parallel plate flow chamber show that trophospheres detach from sLeX functionalized slides with 2.75×10−3 dyn of force and 7.5×10−5 dyn-cm of torque. This work marks the first time a three-dimensional cell model has been utilized for quantifying L-selectin binding mechanics related to blastocyst implantation. PMID:23927766

  8. Melatonin Improves the Quality of In Vitro Produced (IVP) Bovine Embryos: Implications for Blastocyst Development, Cryotolerance, and Modifications of Relevant Gene Expression

    PubMed Central

    Wang, Feng; Tian, XiuZhi; Zhou, YanHua; Tan, DunXian; Zhu, ShiEn; Dai, YunPing; Liu, GuoShi

    2014-01-01

    To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus–oocyte complexes (COCs) were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10−7 M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10−7 M) significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes. PMID:24695534

  9. Effect of the bovine oviductal fluid on in vitro fertilization, development and gene expression of in vitro-produced bovine blastocysts.

    PubMed

    Cebrian-Serrano, A; Salvador, I; García-Roselló, E; Pericuesta, E; Pérez-Cerezales, S; Gutierrez-Adán, A; Coy, P; Silvestre, M A

    2013-04-01

    Oviductal microenvironment generally provides better conditions for early embryo development than the conventional in vitro system. In an attempt to simulate the oviduct conditions or the main potentially influencing factors, the effect was studied of a bovine oviductal fluid (bOF) treatment applied prior to IVF on (i) IVF parameters, (ii) cleavage rate, (iii) blastocyst yield and (iv) blastocyst quality. Embryo quality was assessed by morphological embryo quality and relative transcript abundance of several developmental genes in bovine blastocysts. Furthermore, to study the effect of bOF without the male effect and zona-sperm interaction, artificially activated metaphase II oocytes were also treated with bOF. In vitro-matured bovine oocytes from abattoir ovaries were treated or untreated with bOF for 30 min and then washed prior to IVF or activation. Subsequently, in vitro-fertilized and parthenogenetic embryos were in vitro cultured for 7 to 8 days. The bOF treatment had no effect on fertilization parameters, cleavage, blastocyst rates both on parthenogenetic and IVF bovine embryos and neither on morphological quality of IVF blastocysts. G6PD and SOD2 genes from IVF blastocysts showed significant changes in their expression after a bOF treatment. Significant differences were found for the expression of SCL2A1, GPX1, BAX, AKR1B1 and PLAC8 genes between excellent or good blastocysts (Grade 1) and fair blastocysts (Grade 2). To our knowledge, this is the first study that evaluates the effect of bOF oocyte treatment on fertilization parameters, development and quality of bovine embryos. PMID:22908847

  10. Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

    PubMed

    Scavizzi, Ferdinando; Ryder, Edward; Newman, Stuart; Raspa, Marcello; Gleeson, Diane; Wardle-Jones, Hannah; Montoliu, Lluis; Fernandez, Almudena; Dessain, Marie-Laure; Larrigaldie, Vanessa; Khorshidi, Zuzana; Vuolteenaho, Reetta; Soininen, Raija; André, Philippe; Jacquot, Sylvie; Hong, Yi; de Angelis, Martin Hrabe; Ramirez-Solis, Ramiro; Doe, Brendan

    2015-10-01

    With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects. PMID:26178246

  11. Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

    PubMed Central

    Qi, Qian-Rong; Xie, Qing-Zhen; Liu, Xue-Li; Zhou, Yun

    2014-01-01

    Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion. PMID:25133541

  12. Freeze-all: enhanced outcomes with cryopreservation at the blastocyst stage versus pronuclear stage using slow-freeze techniques.

    PubMed

    Surrey, Eric; Keller, Jennifer; Stevens, John; Gustofson, Robert; Minjarez, Debra; Schoolcraft, William

    2010-09-01

    This retrospective cohort study compared outcomes from transfer of embryos cryopreserved at the pronuclear versus blastocyst stage following 'freeze-all' IVF cycles without fresh transfer for 87 consecutive IVF patients <40 years, who underwent cryopreservation of all viable embryos followed by at least one subsequent frozen embryo transfer (FET) between January 2003 and July 2007. Cryopreservation of all embryos from one oocyte retrieval was performed at either the pronuclear (1.5 mol/l propanediol and 0.1 mol/l sucrose) (group A) or blastocyst (10% glycerol) (group B) stage. Main outcome measures included survival, live birth and implantation rates. A total of 110 FET cycles were analysed. Live birth and implantation rates observed after the first FET were significantly higher (P=0.025 and P=0.002) in group B (67.7% and 40.8%) than in group A (41.1% and 21.5%) despite a higher survival rate in group A. After two FET cycles, 32.1% of group A had not conceived despite thaw of all available embryos, compared with 6.5% of group B. When freeze-all is necessary, blastocyst cryopreservation leads to higher implantation and live birth rates compared with pronuclear-stage cryopreservation despite lower survival rates. Prolonged embryo culture may allow for more optimal embryo selection. PMID:20638905

  13. Transfer of blastocysts with deviant morphological and morphokinetic parameters at early stages of in-vitro development: a case series.

    PubMed

    Stecher, Astrid; Vanderzwalmen, Pierre; Zintz, Martin; Wirleitner, Barbara; Schuff, Maximilian; Spitzer, Dietmar; Zech, Nicolas H

    2014-04-01

    Time-lapse imaging is increasingly applied as an adjunct to reproductive medicine. The gained information of the morphological and morphokinetic variables before the onset of transcription are supposed to be good predictors for the selection of the best embryo for transfer and are often seen in line with clinical outcomes. This retrospective case series investigated the outcome of transferred blastocysts that did not fulfil the proposed embryo scores at early cleavage or at later stages of development. The observations were made by time-lapse imaging. This study reports the birth of 16 healthy children after day-5 blastocyst transfer, of which at least one of the transferred embryos originated from deviant morphology and/or kinetic cleavage patterns. This case series suggests that some blastocysts derived from embryos with poor conventional morphological score and/or suboptimal morphokinetics can be successfully transferred and might result in live births. Such results might raise awareness that discarding embryos based only on early events is not a suitable approach to give patients the chance to conceive. In conclusion, to date only the transfer of viable embryos after culturing them until day 5 guarantees optimal embryo selection and helps to prevent embryo wastage. PMID:24581992

  14. Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: can supplementary progesterone rescue small embryos?

    PubMed

    O'Hara, Lydia; Forde, Niamh; Kelly, Alan K; Lonergan, Patrick

    2014-05-01

    Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro-produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)-releasing intravaginal device (PRID) with the administration of a prostaglandin F2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro-produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%-55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group

  15. Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos

    PubMed Central

    2011-01-01

    Background The in vitro culture of presumed zygotes derived from single cow ovum pick-up (OPU) is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality. Methods Cumulus oocyte complexes (COCs) were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured in vitro, fertilized in vitro with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1) control OPU (controlOPU) with single cow OPU-derived presumed zygotes (2~8); (2) agar chip-embedded slaughterhouse helper embryo coculture (agarOPU) with ten presumed zygotes including all presumed zygotes from a cow (2~8) and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2); and (3) slaughterhouse in vitro embryo production (sIVP) with ten slaughterhouse ovary-derived presumed zygotes, each in 50 μL droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR. Results The coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P < 0.01). Genes predicted to be involved in implantation failure and/or embryo resorption were down-regulated (P < 0.05) in control OPU zygotes (CD9, 0.4-fold; AKRAB1, 0.3-fold) and in cocultured zygotes (CD9, 0.3-fold; AKRAB1, 0.3-fold) compared to sIVP blastocysts (1.0-fold). Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P < 0.05 to P < 0.01) in control OPU zygotes (PGSH2, 5.0-fold; TXN, 4.3-fold; PLAU, 1.7-fold) and cocultured zygotes (PGSH2, 14.5-fold; TXN, 3.2-fold; PLAU, 6.8-fold) compared to sIVP (1.0-fold) blastocysts. However

  16. A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently.

    PubMed

    Jensen, Pia Rosgaard; Andersen, Thomas Levin; Pennypacker, Brenda L; Duong, Le T; Engelholm, Lars H; Delaissé, Jean-Marie

    2014-01-10

    The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is

  17. Flavor of oranges as impacted by abscission zone formation for trees affected by huanglongbing disease and Lasiodiploida infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trees affected by Huanglongbing (HLB) exhibit excessive fruit drop, which is exacerbated by secondary infection of the abscission zone by the fungus Lasiodiplodia. ‘Hamlin’ orange trees, both healthy and affected by HLB, Candidatus Liberibacter asiaticus (CLas, determined by Polymerase chain reactio...

  18. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

    PubMed Central

    Gerwig, Jan; Kiley, Taryn B.; Gunka, Katrin; Stanley-Wall, Nicola

    2014-01-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  19. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis.

    PubMed

    Gerwig, Jan; Kiley, Taryn B; Gunka, Katrin; Stanley-Wall, Nicola; Stülke, Jörg

    2014-04-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  20. Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer.

    PubMed

    Rodríguez-Alvarez, Lleretny; Manriquez, Jose; Velasquez, Alejandra; Castro, Fidel Ovidio

    2013-10-01

    Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r = 0.92; p = 0.02), number of grade I blastocysts (r = 0.96; p = 0.01), and total cell number (r = 0.98; p = 0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p = 0.03; p = 0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines. PMID:23846396

  1. Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer.

    PubMed

    Lee, Joon-Hee; Campbell, Keith H S

    2008-09-01

    Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are key regulators of both meiotic and mitotic cycles. Oocytes arrested at metaphase of the second meiotic division (MII) contain high levels of both kinases; however, these activities decline with age. Caffeine (an inhibitor of Myt1/Wee1 activity) can increase MPF and MAPK activities in ovine oocytes; however, the effects of caffeine treatment on the activation, nuclear configuration and developmental potential of ovine SC nuclear transfer (SCNT) embryos were unknown. We examined the effects of aging and caffeine treatment on MPF and MAPK activities, activation, development, and nuclear remodeling of SCNT embryos. Both kinases reached maximum activities at 24-h postonset of maturation (hpm) and then decreased with time. The decline in MPF activity occurred rapidly, whereas MAPK activity declined more slowly. Caffeine treatment (10.0 mM) of aging oocytes prevented the decline in activities associated with both kinases and prevented the acquisition of activation competence by a single activation stimulus. However, treatment of aged oocytes with caffeine could not increase kinase activities or reverse the acquisition of activation competence. Enucleation did not affect kinase activities, but caffeine treatment significantly increased both. Caffeine treatment did not affect the decline in MPF or MAPK activities following activation or significantly affect development of parthenogenetically activated oocytes. When SCNT reconstructed embryos were treated with caffeine following fusion, no increase in the frequency of development to blastocyst was observed; however, a significant increase in the occurrence of nuclear envelope break-down (NEBD) and an increase in total cell numbers occurred. PMID:18673075

  2. Succinimidyl residue formation in hen egg-white lysozyme favors the formation of intermolecular covalent bonds without affecting its tertiary structure.

    PubMed

    Desfougères, Yann; Jardin, Julien; Lechevalier, Valérie; Pezennec, Stéphane; Nau, Françoise

    2011-01-10

    Protein chemical degradations occur naturally into living cells as soon as proteins have been synthesized. Among these modifications, deamidation of asparagine or glutamine residues has been extensively studied, whereas the intermediate state, a succinimide derivative, was poorly investigated because of the difficulty of isolating those transient species. We used an indirect method, a limited thermal treatment in the dry state at acidic pH, to produce stable cyclic imide residues in hen lysozyme molecules, enabling us to examine the structural and functional properties of so modified proteins. Five cyclic imide rings have been located at sites directly accessible to solvent and did not lead to any changes in secondary or tertiary structures. However, they altered the catalytic properties of lysozyme and significantly decreased the intrinsic stability of the molecules. Moreover, dimerization occurred during the treatment, and this phenomenon was proportional to the extent of chemical degradation. We propose that succinimide formation could be responsible for covalent bond formation under specific physicochemical conditions that could be found in vivo. PMID:21166442

  3. Antisense Inhibition of Expression of Cysteine Proteinases Affects Entamoeba histolytica-Induced Formation of Liver Abscess in Hamsters

    PubMed Central

    Ankri, Serge; Stolarsky, Tamara; Bracha, Rivka; Padilla-Vaca, Felipe; Mirelman, David

    1999-01-01

    Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters. PMID:9864246

  4. Do radial oxygen loss and external aeration affect iron plaque formation and arsenic accumulation and speciation in rice?

    PubMed Central

    Wu, Chuan; Ye, Zhihong; Li, Hui; Wu, Shengchun; Deng, Dan; Zhu, Yongguan; Wong, Minghung

    2012-01-01

    Hydroponic experiments were conducted to investigate the effect of radial oxygen loss (ROL) and external aeration on iron (Fe) plaque formation, and arsenic (As) accumulation and speciation in rice (Oryza sativa L.). The data showed that there were significant correlations between ROL and Fe concentrations in Fe plaque produced on different genotypes of rice. There were also significant differences in the amounts of Fe plaque formed between different genotypes in different positions of roots and under different aeration conditions (aerated, normal, and stagnant treatments). In aerated treatments, rice tended to have a higher Fe plaque formation than in a stagnant solution, with the greatest formation at the root tip decreasing with increasing distances away, in accordance with a trend of spatial ROL. Genotypes with higher rates of ROL induced higher degrees of Fe plaque formation. Plaques sequestered As on rice roots, with arsenate almost double that with arsenite, leading to decreased As accumulation in both roots and shoots. The major As species detected in roots and shoots was arsenite, ranging from 34 to 78% of the total As in the different treatments and genotypes. These results contribute to our understanding of genotypic differences in As uptake by rice and the mechanisms causing rice genotypes with higher ROL to show lower overall As accumulation. PMID:22345639

  5. Estradiol-15 alpha-hydroxylation: a new avenue of estrogen metabolism in peri-implantation pig blastocysts.

    PubMed

    Chakraborty, C; Davis, D L; Dey, S K

    1990-02-01

    Pig blastocysts have the capacity to convert estradiol into catechol estrogens. Our present study shows that they also have the capacity to hydroxylate estradiol in cycloaliphatic C-atom 15, and this aliphatic hydroxylation reaction is more predominate than the aromatic hydroxylations. The conversion of [4-14C]estradiol to [4-14C]15 alpha-hydroxyestradiol by mitochondrial-rich/microsomal fractions was examined by isolation of this product using reversed phase high-performance liquid chromatography (HPLC) attached to a radiometric flow detector, and its identification by gas chromatography-mass spectrometry. The enzyme kinetics for estrogen 15 alpha-hydroxylase were performed in the pig blastocyst obtained on Day 13 of pregnancy (Day 0 = first acceptance of the male). The enzyme follows classical Michaelis-Menten kinetics. The apparent Kms for estradiol were 2.47 and 1.85 microM, and the apparent Vmaxs were 0.25 and 0.197 nmol/mg/min in the mitochondrial-rich and microsomal fractions, respectively. The enzyme activity was inhibited by different steroidal compounds and non-steroidal estrogens, as well as by CO, SKF-525A, piperonyl butoxide and antibody to cytochrome P450 reductase. Ontogenesis of the blastocyst's estrogen 15 alpha-hydroxylase follows a similar pattern to that of estrogen-2/4-hydroxylase. Thus, highest activity was observed on Days 12 and 13 and lowest was on Day 15 of pregnancy. Furthermore, the enzyme is abundant primarily in the extraembryonic tissues rather than in the embryo proper. The abundance of the enzyme in the extraembryonic tissues, and its surge at a critical time of pregnancy recognition and just prior to implantation suggest that 15 alpha-hydroxylated estradiol could be involved in these processes. PMID:2155354

  6. Efficacy of hyaluronan-rich transfer medium on implantation and pregnancy rates in fresh and frozen-thawed blastocyst transfers in Korean women with previous implantation failure

    PubMed Central

    Chun, Sungwook; Seo, Jung Eun; Rim, Yun Jeung; Joo, Jae Hong; Lee, Yong Chan

    2016-01-01

    Objective To evaluate the effect of hyaluronan-rich transfer medium on pregnancy and implantation rates in fresh and frozen-thawed embryo transfers in Korean women with previous implantation failure. Methods This retrospective study included 283 blastocyst transfers in patients with previous embryo transfer failure at a private fertility clinic. In the study group (n=88), blastocyst transfers were performed using an hyaluronan-rich transfer medium prior to transfer, whereas blastocyst transfers without any treatment served as controls (n=195). According to the type of transfer (fresh elective or frozen-thawed), all the blastocyst transfers were divided into two study and two control groups. Results The patient's mean age, serum anti-Müllerian hormone level, causes of infertility, embryo quality, and the number of transferred embryos were comparable between the study and control groups. There were no significant differences in clinical pregnancy rate (45.5% vs. 43.1%), implantation rate (28.9% vs. 28.8%), and clinical abortion rate (10.0% vs. 8.3%) between the two groups, and these findings were not changed after subgroup analysis according to the type of transfer. Conclusion The use of hyaluronan-rich transfer medium in the blastocyst transfer does not appear to have any significant effect on the implantation and pregnancy rates in patients with previous implantation failure. PMID:27200310

  7. Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression.

    PubMed

    Mori, Miyuki; Kasa, Shojiro; Isozaki, Yoshihiro; Kamori, Tsugumitsu; Yamaguchi, Shoichiro; Ueda, Shuji; Kuwano, Toshio; Eguchi, Minako; Isayama, Keishiro; Nishimura, Shotaro; Tabata, Shoji; Yamauchi, Nobuhiko; Hattori, Masa-aki

    2013-01-01

    The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression. PMID:23164983

  8. Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number.

    PubMed

    Reader, Karen L; Cox, Neil R; Stanton, Jo-Ann L; Juengel, Jennifer L

    2015-06-01

    Viable lambs can be produced after transfer of in vitro-derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for β-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine-treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0

  9. Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel (Camelus dromedarius).

    PubMed

    Herrid, M; Billah, M; Malo, C; Skidmore, J A

    2016-03-01

    The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me2SO) for equilibration, and cooling in 16% EG + 16% Me2SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ∼24 °C/15 min and body 37 °C/3 min), and the replacement of Me2SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and re-expansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37 °C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me2SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (P < 0.05). The modified protocol of loading EG at 37 °C for 3 minutes has increased the embryo survival of the original protocol from 67% to 91% and the developmental rate from 57% to 83% at 5-day culture. These results were comparable to or better than those reported in human or other

  10. Does receiving an earnings supplement affect union formation? Estimating effects for program participants using propensity score matching.

    PubMed

    Harknett, Kristen

    2006-12-01

    This article demonstrates a novel application of propensity score matching techniques: to estimate nonexperimental impacts on program participants within the context of an experimental research design. The author examines the relationship between program participation, defined as qualifying for an earnings supplement by working full-time, and marital union formation among low-income mothers in two Canadian provinces. The author finds that receipt of an earnings supplement substantially increased union formation in one province but not the other. A subgroup analysis based on propensities of program participation revealed that the positive effect on unions was concentrated among relatively disadvantaged participants. The techniques demonstrated in the article are broadly applicable to studies in which take-up is less than 100% among those randomly assigned to a program group. PMID:17093107

  11. Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-{beta}1

    SciTech Connect

    Schmid, Stephan A. . E-mail: leoni.kunz-schughart@oncoray.de; Gaumann, Andreas; Wondrak, Marit; Eckermann, Christoph; Schulte, Stephanie; Mueller-Klieser, Wolfgang; Wheatley, Denys N.; Kunz-Schughart, Leoni A.

    2007-07-15

    When lactate accumulation in a tumor microenvironment reaches an average concentration of 10-20 mM, it tends to reflect a high degree of malignancy. However, the hypothesis that tumor-derived lactate has a number of partially adverse biological effects on malignant and tumor-associated host cells requires further evidence. The present study attempted to evaluate the impact of lactate on the process of angiogenesis, in particular on the formation of tubular structures. The endothelial cell (EC) network in desmoplastic breast tumors is primarily located in areas of reactive fibroblastic stroma. We employed a fibroblast-endothelial cell co-culture model as in vitro angiogenesis system normally producing florid in vitro tubule formation to analyze this situation. In contrast to previous studies, we found that lactate significantly reduces EC network formation in a dose-dependent manner as quantified by semi-automated morphometric analyses following immunohistochemical staining. The decrease in CD31-positive tubular structures and the number of intersections was independent of VEGF supplementation and became more pronounced in the presence of protons. The number of cells, primarily of the fibroblast population, was reduced but cell loss could not be attributed to a decrease in proliferative activity or pronounced apoptotic cell death. Treatment with 10 mM lactate was accompanied by enhanced mRNA expression and release of TGF-{beta}1, which also shows anti-angiogenic activity in the model. Both TGF-{beta}1 and lactate induced myofibroblastic differentiation adjacent to the EC tubular structures. The lactate response on the EC network was diminished by TGF-{beta}1 neutralization, indicating a causal relationship between lactate and TGF-{beta}1 in the finely tuned processes of vessel formation and maturation which may also occur in vivo within tumor tissue.

  12. NMDA-R inhibition affects cellular process formation in Tilapia melanocytes; a model for pigmented adrenergic neurons in process formation and retraction.

    PubMed

    Ogundele, Olalekan Michael; Okunnuga, Adetokunbo Adedotun; Fabiyi, Temitope Deborah; Olajide, Olayemi Joseph; Akinrinade, Ibukun Dorcas; Adeniyi, Philip Adeyemi; Ojo, Abiodun Ayodele

    2014-06-01

    Parkinson's disease has long been described to be a product of dopamine and (or) melanin loss in the substanstia nigra (SN). Although most studies have focused on dopaminergic neurons, it is important to consider the role of pigment cells in the etiology of the disease and to create an in vitro live cell model for studies involving pigmented adrenergic cells of the SN in Parkinsonism. The Melanocytes share specific features with the pigmented adrenergic neurons as both cells are pigmented, contain adrenergic receptors and have cellular processes. Although the melanocyte cellular processes are relatively short and observable only when stimulated appropriately by epinephrine and other factors or molecules. This study employs the manipulation of N-Methyl-D-Aspartate Receptor (NMDA-R), a major receptor in neuronal development, in the process formation pattern of the melanocyte in order to create a suitable model to depict cellular process elongation and shortening in pigmented adrenergic cells. NMDA-R is an important glutamate receptor implicated in neurogenesis, neuronal migration, maturation and cell death, thus we investigated the role of NMDA-R potentiation by glutamate/KCN and its inhibition by ketamine in the behavior of fish scale melanocytes in vitro. This is aimed at establishing the regulatory role of NMDA-R in this cell type (melanocytes isolated form Tilapia) in a similar manner to what is observable in the mammalian neurons. In vitro live cell culture was prepared in modified Ringer's solution following which the cells were treated as follows; Control, Glutamate, Ketamine, Glutamate + Ketamine, KCN + Ketamine and KCN. The culture was maintained for 10 min and the changes were captured in 3D-Time frame at 0, 5 and 10 min for the control and 5, 7 and 10 min for each of the treatment category. Glutamate treatment caused formation of short cellular processes localized directly on the cell body while ketamine treatment (inhibition of NMDA-R) facilitated

  13. Nutrient limitation leads to penetrative growth into agar and affects aroma formation in Pichia fabianii, P. kudriavzevii and Saccharomyces cerevisiae.

    PubMed

    van Rijswijck, Irma M H; Dijksterhuis, Jan; Wolkers-Rooijackers, Judith C M; Abee, Tjakko; Smid, Eddy J

    2015-01-01

    Among fermentative yeast species, Saccharomyces cerevisiae is most frequently used as a model organism, although other yeast species may have special features that make them interesting candidates to apply in food-fermentation processes. In this study, we used three yeast species isolated from fermented masau (Ziziphus mauritiana) fruit, S. cerevisiae 131, Pichia fabianii 65 and Pichia kudriavzevii 129, and determined the impact of nitrogen and/or glucose limitation on surface growth mode and the production of volatile organic compounds (VOCs). All three species displayed significant changes in growth mode in all nutrient-limited conditions, signified by the formation of metafilaments or pseudohyphae. The timing of the transition was found to be species-specific. Transition in growth mode is suggested to be linked to the production of certain fusel alcohols, such as phenylethyl alcohol, which serve as quorum-sensing molecules. Interestingly, we did not observe concomitant increased production of phenylethyl alcohol and filamentous growth. Notably, a broader range of esters was found only for the Pichia spp. grown on nitrogen-limited agar for 21 days compared to nutrient-rich agar, and when grown on glucose- and glucose- plus nitrogen-limited agar. Our data suggest that for the Pichia spp., the formation of esters may play an important role in the switch in growth mode upon nitrogen limitation. Further biological or ecological implications of ester formation are discussed. PMID:25308873

  14. Silencing of the gibberellin receptor homolog, CsGID1a, affects locule formation in cucumber (Cucumis sativus) fruit.

    PubMed

    Liu, Bin; Liu, Xingwang; Yang, Sen; Chen, Chunhua; Xue, Shudan; Cai, Yanling; Wang, Dandan; Yin, Shuai; Gai, Xinshuang; Ren, Huazhong

    2016-04-01

    Gibberellins are phytohormones with many roles, including the regulation of fruit development. However, little is known about the relationship between GA perception and fleshy fruit ontogeny, and particularly locule formation. We characterized the expression of cucumber (Cucumis sativus) GA receptor gene (CsGID1a) using quantitative real-time PCR, in situ hybridization and a promoter::β-glucuronidase (GUS) assay. CsGID1a-RNAi cucumber fruits were observed by dissecting microscope, scanning electron microscopy and transmission electron microscopy. Finally, genome-wide gene expression in young fruits from a control and the RNAi line was compared using a digital gene expression (DGE) analysis approach. The expression pattern of CsGID1a was found to be closely correlated with fruit locule formation, and silencing CsGID1a in cucumber resulted in fruits with abnormal carpels and locules. Overexpression of CsGID1a in the Arabidopsis thaliana double mutant (gid1a gid1c) resulted in 'cucumber locule-like' fruits. The DGE analysis suggested that expression of genes related to auxin synthesis and transport, as well as the cell cycle, was altered in CsGID1a-RNAi fruits, a result that was supported by comparing the auxin content and cellular structures of the control and transgenic fruits. This study demonstrates a previously uncharacterized GA signaling pathway that is essential for cucumber fruit locule formation. PMID:26701170

  15. Linking automatic evaluation to mood and information processing style: consequences for experienced affect, impression formation, and stereotyping.

    PubMed

    Chartrand, Tanya L; van Baaren, Rick B; Bargh, John A

    2006-02-01

    According to the feelings-as-information account, a person's mood state signals to him or her the valence of the current environment (N. Schwarz & G. Clore, 1983). However, the ways in which the environment automatically influences mood in the first place remain to be explored. The authors propose that one mechanism by which the environment influences affect is automatic evaluation, the nonconscious evaluation of environmental stimuli as good or bad. A first experiment demonstrated that repeated brief exposure to positive or negative stimuli (which leads to automatic evaluation) induces a corresponding mood in participants. In 3 additional studies, the authors showed that automatic evaluation affects information processing style. Experiment 4 showed that participants' mood mediates the effect of valenced brief primes on information processing. PMID:16478316

  16. The effect of different cardiovascular risk presentation formats on intentions, understanding and emotional affect: a randomised controlled trial using a web-based risk formatter (protocol)

    PubMed Central

    2010-01-01

    Background The future risk of heart disease can be predicted with increasing precision. However, more research is needed into how this risk is conveyed and presented. The aim of this study is to compare the effects of presenting cardiovascular risk in different formats on individuals' intention to change behaviour to reduce risk, understanding of risk information and emotional affect. Methods/design A randomised controlled trial comprising four arms, with a between subjects design will be performed. There will be two intervention groups and two control groups. The first control comprises a pre-intervention questionnaire and presents risk in a bar graph format. The second control presents risk in a bar graph format without pre-intervention questionnaire. These two control groups are to account for the potential Hawthorne effect of thinking about cardiovascular risk before viewing actual risk. The two intervention groups comprise presenting risk in either a pictogram or metonym format (image depicting seriousness of having a myocardial infarction). 800 individuals' aged between 45 and 64 years, who have not been previously diagnosed with heart disease and have access to a computer with internet, will be given a link to a website comprising a risk calculator and electronic questionnaires. 10-year risk of having a coronary heart disease event will be assessed and presented in one of the three formats. A post-intervention questionnaire will be completed after viewing the risk format. Main outcome measures are (i) intention to change behaviour, (ii) understanding of risk information, (iii) emotional affect and (iv) worry about future heart disease. Secondary outcomes are the sub-components of the theory of planned behaviour: attitudes, perceived behavioural control and subjective norms. Discussion Having reviewed the literature, we are not aware of any other studies which have used the assessment of actual risk, in a trial to compare different graphical cardiovascular

  17. Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

    PubMed

    Sakharova, N Yu; Mezhevikina, L M; Smirnov, A A; Vikhlyantseva, E F

    2014-05-01

    We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos. PMID:24913583

  18. SUBTASK 1.7 EVALUATION OF KEY FACTORS AFFECTING SUCCESSFUL OIL PRODUCTION IN THE BAKKEN FORMATION, NORTH DAKOTA PHASE II

    SciTech Connect

    Darren D. Schmidt; Steven A. Smith; James A. Sorensen; Damion J. Knudsen; John A. Harju; Edward N. Steadman

    2011-10-31

    Production from the Bakken and Three Forks Formations continues to trend upward as forecasts predict significant production of oil from unconventional resources nationwide. As the U.S. Geological Survey reevaluates the 3.65 billion bbl technically recoverable estimate of 2008, technological advancements continue to unlock greater unconventional oil resources, and new discoveries continue within North Dakota. It is expected that the play will continue to expand to the southwest, newly develop in the northeastern and northwestern corners of the basin in North Dakota, and fully develop in between. Although not all wells are economical, the economic success rate has been near 75% with more than 90% of wells finding oil. Currently, only about 15% of the play has been drilled, and recovery rates are less than 5%, providing a significant future of wells to be drilled and untouched hydrocarbons to be pursued through improved stimulation practices or enhanced oil recovery. This study provides the technical characterizations that are necessary to improve knowledge, provide characterization, validate generalizations, and provide insight relative to hydrocarbon recovery in the Bakken and Three Forks Formations. Oil-saturated rock charged from the Bakken shales and prospective Three Forks can be produced given appropriate stimulation treatments. Highly concentrated fracture stimulations with ceramic- and sand-based proppants appear to be providing the best success for areas outside the Parshall and Sanish Fields. Targeting of specific lithologies can influence production from both natural and induced fracture conductivity. Porosity and permeability are low, but various lithofacies units within the formation are highly saturated and, when targeted with appropriate technology, release highly economical quantities of hydrocarbons.

  19. Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development.

    PubMed

    Spinaci, M; De Ambrogi, M; Volpe, S; Galeati, G; Tamanini, C; Seren, E

    2005-07-01

    The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos. PMID:15935852

  20. SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.

    PubMed

    Northrop, L E; Treff, N R; Levy, B; Scott, R T

    2010-08-01

    Although selection of chromosomally normal embryos has the potential to improve outcomes for patients undergoing IVF, the clinical impact of aneuploidy screening by fluorescence in situ hybridization (FISH) has been controversial. There are many putative explanations including sampling error due to mosaicism, negative impact of biopsy, a lack of comprehensive chromosome screening, the possibility of embryo self-correction and poor predictive value of the technology itself. Direct analysis of the negative predictive value of FISH-based aneuploidy screening for an embryo's reproductive potential has not been performed. Although previous studies have found that cleavage-stage FISH is poorly predictive of aneuploidy in morphologically normal blastocysts, putative explanations have not been investigated. The present study used a single nucleotide polymorphism (SNP) microarray-based 24 chromosome aneuploidy screening technology to re-evaluate morphologically normal blastocysts that were diagnosed as aneuploid by FISH at the cleavage stage. Mosaicism and preferential segregation of aneuploidy to the trophectoderm (TE) were evaluated by characterization of multiple sections of the blastocyst. SNP microarray technology also provided the first opportunity to evaluate self-correction mechanisms involving extrusion or duplication of aneuploid chromosomes resulting in uniparental disomy (UPD). Of all blastocysts evaluated (n = 50), 58% were euploid in all sections despite an aneuploid FISH result. Aneuploid blastocysts displayed no evidence of preferential segregation of abnormalities to the TE. In addition, extrusion or duplication of aneuploid chromosomes resulting in UPD did not occur. These findings support the conclusion that cleavage-stage FISH technology is poorly predictive of aneuploidy in morphologically normal blastocysts. PMID:20479065

  1. Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence

    PubMed Central

    Sadaie, Mahito; Salama, Rafik; Carroll, Thomas; Tomimatsu, Kosuke; Chandra, Tamir; Young, Andrew R.J.; Narita, Masako; Pérez-Mancera, Pedro A.; Bennett, Dorothy C.; Chong, Heung; Kimura, Hiroshi; Narita, Masashi

    2013-01-01

    Senescence is a stress-responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for Lys9 trimethylation on histone H3 (H3K9me3). LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3-positive heterochromatin, thus promoting SAHF formation, which could be inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genome-wide redistribution: first, through the spatial reorganization of chromatin and, second, through gene repression. PMID:23964094

  2. Knockout of Extracytoplasmic Function Sigma Factor ECF-10 Affects Stress Resistance and Biofilm Formation in Pseudomonas putida KT2440

    PubMed Central

    Tettmann, Beatrix; Dötsch, Andreas; Armant, Olivier; Fjell, Christopher D.

    2014-01-01

    Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA, ttgB, and ttgC, which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440. PMID:24907323

  3. Cell motility and biofilm formation in Bacillus subtilis are affected by the ribosomal proteins, S11 and S21.

    PubMed

    Takada, Hiraku; Morita, Masato; Shiwa, Yuh; Sugimoto, Ryoma; Suzuki, Shota; Kawamura, Fujio; Yoshikawa, Hirofumi

    2014-01-01

    Bacillus subtilis differentiates into various cellular states in response to environmental changes. It exists in two states during the exponential growth phase: motile cells and connected chains of sessile cells. Here, we identified new regulators of cell motility and chaining, the ribosomal proteins S21 (rpsU) and S11 (rpsK). Their mutants showed impaired cell motility (observed in a laboratory strain) and robust biofilm formation (observed in an undomesticated strain). The two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were strongly expressed in the rpsU mutant, whereas the flagellin-encoding hag gene and other SigD-dependent motility regulons were not. Genetic analysis revealed that the mutation of remA, the transcriptional activator of the eps operon, is epistatic to that of rpsU, whereas the mutation of antagonistic regulators of SinR is not. Our studies demonstrate that S11 and S21 participate in the regulation of bistability via the RemA/RemB pathway. PMID:25035996

  4. RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3.

    PubMed

    Liu, Xiaoguang; Wu, Yan; Chen, Yuanyuan; Xu, Fang; Halliday, Nigel; Gao, Kexiang; Chan, Kok Gan; Cámara, Miguel

    2016-04-01

    The σ(S) subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses. PMID:26671319

  5. Ectopic expression a tomato KNOX Gene Tkn4 affects the formation and the differentiation of meristems and vasculature.

    PubMed

    Yan, Fang; Hu, Guojian; Ren, Zhenxin; Deng, Wei; Li, Zhengguo

    2015-12-01

    The KNOTTED-LIKE HOMEODOMAIN genes are involved in maintenance of the shoot apical meristem which produces the whole above-ground body of vascular plants. In this report, a tomato homolog gene, named as Tkn4 (a nucleus targeted transcription factor) was identified and characterized. By performing RT-PCR, the transcript level of Tkn4 was separately found in stem, root, stamen, stigma, fruit and sepal but hardly visible in the leaf. Besides, Tkn4 was induced by a series of plant hormones. Overexpression of Tkn4 gene in tomato resulted in dwarf phenotype and strongly repressed the formation of shoot apical meristem, lateral meristem and cambiums in transgenic lines. The transgenic lines had wrinkled leaves and anatomic analysis showed that there was no obvious palisade tissues in the leaves and the layer of cells changed in vascular tissue (xylem and phloem). To explore the regulation network of Tkn4, RNA-sequencing was performed in overexpression lines and wild type plants, by which many genes related to the synthesis and the signal transduction of cytokinin, auxin, gibberellin, ethylene, abscisic acid, and tracheary element differentiation or extracellular matrix synthesis were significantly regulated. Taken together, our results demonstrate that Tkn4 plays important roles in regulating the biosynthesis and signal transduction of diverse plant hormones, and the formation and differentiation of meristems and vasculature in tomato. PMID:26456092

  6. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study

    PubMed Central

    2012-01-01

    Background Single embryo transfer (SET) remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH) to provide this advantage and improve pregnancy rate from SET. Methods First-time IVF patients with a good prognosis (age <35, no prior miscarriage) and normal karyotype seeking elective SET were prospectively randomized into two groups: In Group A, embryos were selected on the basis of morphology and comprehensive chromosomal screening via aCGH (from d5 trophectoderm biopsy) while Group B embryos were assessed by morphology only. All patients had a single fresh blastocyst transferred on d6. Laboratory parameters and clinical pregnancy rates were compared between the two groups. Results For patients in Group A (n = 55), 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient). Aneuploidy was detected in 191/425 (44.9%) of blastocysts in this group. For patients in Group B (n = 48), 389 blastocysts were microscopically examined (8.1 blastocysts/patient). Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017); ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009). There were no twin pregnancies. Conclusion Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss), this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9%) among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used

  7. Intragenic sex-chromosomal crossovers of Xmrk oncogene alleles affect pigment pattern formation and the severity of melanoma in Xiphophorus.

    PubMed Central

    Gutbrod, H; Schartl, M

    1999-01-01

    The X and Y chromosomes of the platyfish (Xiphophorus maculatus) contain a region that encodes several important traits, including the determination of sex, pigment pattern formation, and predisposition to develop malignant melanoma. Several sex-chromosomal crossovers were identified in this region. As the melanoma-inducing oncogene Xmrk is the only molecularly identified constituent, its genomic organization on both sex chromosomes was analyzed in detail. Using X and Y allele-specific sequence differences a high proportion of the crossovers was found to be intragenic in the oncogene Xmrk, concentrating in the extracellular domain-encoding region. The genetic and molecular data allowed establishment of an order of loci over approximately 0.6 cM. It further revealed a sequence located within several kilobases of the extracellular domain-encoding region of Xmrk that regulates overexpression of the oncogene. PMID:9927468

  8. Inactivation of Cyclic Di-GMP Binding Protein TDE0214 Affects the Motility, Biofilm Formation, and Virulence of Treponema denticola

    PubMed Central

    Bian, Jiang; Liu, Xiangyang; Cheng, Yi-Qiang

    2013-01-01

    As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 μM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola. PMID:23794624

  9. Elevated growth temperature can enhance photosystem I trimer formation and affects xanthophyll biosynthesis in Cyanobacterium Synechocystis sp. PCC6803 cells.

    PubMed

    Kłodawska, Kinga; Kovács, László; Várkonyi, Zsuzsanna; Kis, Mihály; Sozer, Özge; Laczkó-Dobos, Hajnalka; Kóbori, Ottilia; Domonkos, Ildikó; Strzałka, Kazimierz; Gombos, Zoltán; Malec, Przemysław

    2015-03-01

    In the thylakoid membranes of the mesophilic cyanobacterium Synechocystis PCC6803, PSI reaction centers (RCs) are organized as monomers and trimers. PsaL, a 16 kDa hydrophobic protein, a subunit of the PSI RC, was previously identified as crucial for the formation of PSI trimers. In this work, the physiological effects accompanied by PSI oligomerization were studied using a PsaL-deficient mutant (ΔpsaL), not able to form PSI trimers, grown at various temperatures. We demonstrate that in wild-type Synechocystis, the monomer to trimer ratio depends on the growth temperature. The inactivation of the psaL gene in Synechocystis grown phototropically at 30°C induces profound morphological changes, including the accumulation of glycogen granules localized in the cytoplasm, resulting in the separation of particular thylakoid layers. The carotenoid composition in ΔpsaL shows that PSI monomerization leads to an increased accumulation of myxoxantophyll, zeaxanthin and echinenone irrespective of the temperature conditions. These xanthophylls are formed at the expense of β-carotene. The measured H2O→CO2 oxygen evolution rates in the ΔpsaL mutant are higher than those observed in the wild type, irrespective of the growth temperature. Moreover, circular dichroism spectroscopy in the visible range reveals that a peak attributable to long-wavelength-absorbing carotenoids is apparently enhanced in the trimer-accumulating wild-type cells. These results suggest that specific carotenoids are accompanied by the accumulation of PSI oligomers and play a role in the formation of PSI oligomer structure. PMID:25520404

  10. The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation.

    PubMed

    Bitterlich, Michael; Krügel, Undine; Boldt-Burisch, Katja; Franken, Philipp; Kühn, Christina

    2014-06-01

    Mycorrhizal plants benefit from the fungal partners by getting better access to soil nutrients. In exchange, the plant supplies carbohydrates to the fungus. The additional carbohydrate demand in mycorrhizal plants was shown to be balanced partially by higher CO2 assimilation and increased C metabolism in shoots and roots. In order to test the role of sucrose transport for fungal development in arbuscular mycorrhizal (AM) tomato, transgenic plants with down-regulated expression of three sucrose transporter genes were analysed. Plants that carried an antisense construct of SlSUT2 (SlSUT2as) repeatedly exhibited increased mycorrhizal colonization and the positive effect of plants to mycorrhiza was abolished. Grafting experiments between transgenic and wild-type rootstocks and scions indicated that mainly the root-specific function of SlSUT2 has an impact on colonization of tomato roots with the AM fungus. Localization of SISUT2 to the periarbuscular membrane indicates a role in back transport of sucrose from the periarbuscular matrix into the plant cell thereby affecting hyphal development. Screening of an expression library for SlSUT2-interacting proteins revealed interactions with candidates involved in brassinosteroid (BR) signaling or biosynthesis. Interaction of these candidates with SlSUT2 was confirmed by bimolecular fluorescence complementation. Tomato mutants defective in BR biosynthesis were analysed with respect to mycorrhizal symbiosis and showed indeed decreased mycorrhization. This finding suggests that BRs affect mycorrhizal infection and colonization. If the inhibitory effect of SlSUT2 on mycorrhizal growth involves components of BR synthesis and of the BR signaling pathway is discussed. PMID:24654931

  11. COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation.

    PubMed

    Pérez-Pérez, Rafael; Lobo-Jarne, Teresa; Milenkovic, Dusanka; Mourier, Arnaud; Bratic, Ana; García-Bartolomé, Alberto; Fernández-Vizarra, Erika; Cadenas, Susana; Delmiro, Aitor; García-Consuegra, Inés; Arenas, Joaquín; Martín, Miguel A; Larsson, Nils-Göran; Ugalde, Cristina

    2016-08-30

    Mitochondrial respiratory chain (MRC) complexes I, III, and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here, we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and, to a minor extent, to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization, and function of the individual oxidative phosphorylation complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist. PMID:27545886

  12. Growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of Candida glabrata are affected by different glucose concentrations.

    PubMed

    Ng, Tzu Shan; Desa, Mohd Nasir Mohd; Sandai, Doblin; Chong, Pei Pei; Than, Leslie Thian Lung

    2016-06-01

    Glucose is an important fuel source to support many living organisms. Its importance in the physiological fitness and pathogenicity of Candida glabrata, an emerging human fungal pathogen has not been extensively studied. The present study aimed to investigate the effects of glucose on the growth, biofilm formation, antifungal susceptibility and oxidative stress resistance of C. glabrata. In addition, its effect on the expression of a putative high affinity glucose sensor gene, SNF3 was also investigated. Glucose concentrations were found to exert effects on the physiological responses of C. glabrata. The growth rate of the species correlated positively to the amount of glucose. In addition, low glucose environments were found to induce C. glabrata to form biofilm and resist amphotericin B. Conversely, high glucose environments promoted oxidative stress resistance of C. glabrata. The expression of CgSNF3 was found to be significantly up-regulated in low glucose environments. The expression of SNF3 gene in clinical isolates was found to be higher compared to ATCC laboratory strains in low glucose concentrations, which may explain the better survivability of clinical isolates in the low glucose environment. These observations demonstrated the impact of glucose in directing the physiology and virulence fitness of C. glabrata through the possible modulation by SNF3 as a glucose sensor, which in turn aids the species to adapt, survive and thrive in hostile host environment. PMID:26358577

  13. An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster.

    PubMed Central

    Luschnig, Stefan; Moussian, Bernard; Krauss, Jana; Desjeux, Isabelle; Perkovic, Josip; Nüsslein-Volhard, Christiane

    2004-01-01

    Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning. PMID:15166158

  14. Soy protein isolate does not affect ellagitannin bioavailability and urolithin formation when mixed with pomegranate juice in humans.

    PubMed

    Yang, Jieping; Lee, Rupo; Henning, Susanne M; Thames, Gail; Hsu, Mark; ManLam, Hei; Heber, David; Li, Zhaoping

    2016-03-01

    We investigated the effect of mixing soy protein isolate and pomegranate juice (PJ) on the bioavailability and metabolism of ellagitannins (ETs) in healthy volunteers. Eighteen healthy volunteers consumed PJ alone or PJ premixed with soy protein isolate (PJSP). The concentration of plasma ellagic acid (EA) and urine urolithins was measured. There was no significant difference in plasma EA over a 6-h period between the two interventions. While the maximum concentration of plasma EA after PJSP consumption was slightly but significantly lower than after PJ consumption, EA remained in the plasma longer with an elimination half-life t1/2E at 1.36±0.59 versus 1.06±0.47h for PJSP and PJ consumption, respectively. Urinary urolithin A, B and C was not significantly different between the two interventions. In conclusion, premixing soy protein isolate and PJ did not affect the bioavailability or the metabolism of pomegranate ETs in healthy volunteers. PMID:26471685

  15. Deletion of the Scl +19 enhancer increases the blood stem cell compartment without affecting the formation of mature blood lineages.

    PubMed

    Spensberger, Dominik; Kotsopoulou, Ekaterini; Ferreira, Rita; Broccardo, Cyril; Scott, Linda M; Fourouclas, Nasios; Ottersbach, Katrin; Green, Anthony R; Göttgens, Berthold

    2012-07-01

    The stem cell leukemia (Scl)/Tal1 gene is essential for normal blood and endothelial development, and is expressed in hematopoietic stem cells (HSCs), progenitors, erythroid, megakaryocytic, and mast cells. The Scl +19 enhancer is active in HSCs and progenitor cells, megakaryocytes, and mast cells, but not mature erythroid cells. Here we demonstrate that in vivo deletion of the Scl +19 enhancer (Scl(Δ19/Δ19)) results in viable mice with normal Scl expression in mature hematopoietic lineages. By contrast, Scl expression is reduced in the stem/progenitor compartment and flow cytometry analysis revealed that the HSC and megakaryocyte-erythroid progenitor populations are enlarged in Scl(Δ19/Δ19) mice. The increase in HSC numbers contributed to enhanced expansion in bone marrow transplantation assays, but did not affect multilineage repopulation or stress responses. These results affirm that the Scl +19 enhancer plays a key role in the development of hematopoietic stem/progenitor cells, but is not necessary for mature hematopoietic lineages. Moreover, active histone marks across the Scl locus were significantly reduced in Scl(Δ19/Δ19) fetal liver cells without major changes in steady-state messenger RNA levels, suggesting post-transcriptional compensation for loss of a regulatory element, a result that might be widely relevant given the frequent observation of mild phenotypes after deletion of regulatory elements. PMID:22401818

  16. Glucose concentration alters dissolved oxygen levels in liquid cultures of Beauveria bassiana and affects formation and bioefficacy of blastospores.

    PubMed

    Mascarin, Gabriel Moura; Jackson, Mark A; Kobori, Nilce Naomi; Behle, Robert W; Dunlap, Christopher A; Delalibera Júnior, Ítalo

    2015-08-01

    The filamentous fungus Beauveria bassiana is an economically important pathogen of numerous arthropod pests and is able to grow in submerged culture as filaments (mycelia) or as budding yeast-like blastospores. In this study, we evaluated the effect of dissolved oxygen and high glucose concentrations on blastospore production by submerged cultures of two isolates of B. bassiana, ESALQ1432 and GHA. Results showed that maintaining adequate dissolved oxygen levels coupled with high glucose concentrations enhanced blastospore yields by both isolates. High glucose concentrations increased the osmotic pressure of the media and coincided with higher dissolved oxygen levels and increased production of significantly smaller blastospores compared with blastospores produced in media with lower concentrations of glucose. The desiccation tolerance of blastospores dried to less than 2.6 % moisture was not affected by the glucose concentration of the medium but was isolate dependent. Blastospores of isolate ESALQ1432 produced in media containing 140 g glucose L(-1) showed greater virulence toward whitefly nymphs (Bemisia tabaci) as compared with blastospores produced in media containing 40 g glucose L(-1). These results suggest a synergistic effect between glucose concentration and oxygen availability on changing morphology and enhancing the yield and efficacy of blastospores of B. bassiana, thereby facilitating the development of a cost-effective production method for this blastospore-based bioinsecticide. PMID:25947245

  17. Social Memory Formation Rapidly and Differentially Affects the Motivation and Performance of Vocal Communication Signals in the Bengalese Finch (Lonchura striata var. domestica).

    PubMed

    Toccalino, Danielle C; Sun, Herie; Sakata, Jon T

    2016-01-01

    Cognitive processes like the formation of social memories can shape the nature of social interactions between conspecifics. Male songbirds use vocal signals during courtship interactions with females, but the degree to which social memory and familiarity influences the likelihood and structure of male courtship song remains largely unknown. Using a habituation-dishabituation paradigm, we found that a single, brief (<30 s) exposure to a female led to the formation of a short-term memory for that female: adult male Bengalese finches were significantly less likely to produce courtship song to an individual female when re-exposed to her 5 min later (i.e., habituation). Familiarity also rapidly decreased the duration of courtship songs but did not affect other measures of song performance (e.g., song tempo and the stereotypy of syllable structure and sequencing). Consistent with a contribution of social memory to the decrease in courtship song with repeated exposures to the same female, the likelihood that male Bengalese finches produced courtship song increased when they were exposed to a different female (i.e., dishabituation). Three consecutive exposures to individual females also led to the formation of a longer-term memory that persisted over days. Specifically, when courtship song production was assessed 2 days after initial exposures to females, males produced fewer and shorter courtship songs to familiar females than to unfamiliar females. Measures of song performance, however, were not different between courtship songs produced to familiar and unfamiliar females. The formation of a longer-term memory for individual females seemed to require at least three exposures because males did not differentially produce courtship song to unfamiliar females and females that they had been exposed to only once or twice. Taken together, these data indicate that brief exposures to individual females led to the rapid formation and persistence of social memories and support the

  18. Social Memory Formation Rapidly and Differentially Affects the Motivation and Performance of Vocal Communication Signals in the Bengalese Finch (Lonchura striata var. domestica)

    PubMed Central

    Toccalino, Danielle C.; Sun, Herie; Sakata, Jon T.

    2016-01-01

    Cognitive processes like the formation of social memories can shape the nature of social interactions between conspecifics. Male songbirds use vocal signals during courtship interactions with females, but the degree to which social memory and familiarity influences the likelihood and structure of male courtship song remains largely unknown. Using a habituation-dishabituation paradigm, we found that a single, brief (<30 s) exposure to a female led to the formation of a short-term memory for that female: adult male Bengalese finches were significantly less likely to produce courtship song to an individual female when re-exposed to her 5 min later (i.e., habituation). Familiarity also rapidly decreased the duration of courtship songs but did not affect other measures of song performance (e.g., song tempo and the stereotypy of syllable structure and sequencing). Consistent with a contribution of social memory to the decrease in courtship song with repeated exposures to the same female, the likelihood that male Bengalese finches produced courtship song increased when they were exposed to a different female (i.e., dishabituation). Three consecutive exposures to individual females also led to the formation of a longer-term memory that persisted over days. Specifically, when courtship song production was assessed 2 days after initial exposures to females, males produced fewer and shorter courtship songs to familiar females than to unfamiliar females. Measures of song performance, however, were not different between courtship songs produced to familiar and unfamiliar females. The formation of a longer-term memory for individual females seemed to require at least three exposures because males did not differentially produce courtship song to unfamiliar females and females that they had been exposed to only once or twice. Taken together, these data indicate that brief exposures to individual females led to the rapid formation and persistence of social memories and support the

  19. NADPH oxidase 4 deficiency leads to impaired wound repair and reduced dityrosine-crosslinking, but does not affect myofibroblast formation.

    PubMed

    Lévigne, Dominik; Modarressi, Ali; Krause, Karl-Heinz; Pittet-Cuénod, Brigitte

    2016-07-01

    NADPH oxidases (NOX) mediate redox signaling by generating superoxide and/or hydrogen peroxide, which are involved in biosynthetic pathways, e.g. thyroid hormone generation, dityrosine crosslinking, as well as bacterial killing. Data investigating the role of NOX enzymes in cutaneous wound repair is limited and specifically their function in skin myofibroblast expression is unknown. The isoform NOX4 was recently shown to be a pre-requisite for the differentiation of cardiac and pulmonary myofibroblasts. In this study we investigate the role of NOX4 in wound repair using a wound model in NOX4 knockout mice (n=16) and wildtype mice (n=16). Wounds were photographed daily until complete wound closure. Mice were sacrificed at day 3, 7, 14; wound tissue was harvested. NOX4-deficient mice healed significantly slower (22 days, SD=1.9) than wild-type mice (17 days, SD=1.4, p<0.005). However, there was no difference in myofibroblast expression. Strong dityrosine formation was observed, but was significantly weaker in NOX4-/- mice (p<0.05). NOX2, HIF1α and CD31 expression was significantly weaker in NOX4-/- mice (p<0.05). In this study we show for the first time that NOX4 plays a role in cutaneous wound repair. Our data suggests that NOX4 mediates HIF1α expression and neoangiogenesis during wound repair. NOX4 deletion led to a decreased expression of NOX2, implying a role of NOX4 in phagocytic cell recruitment. NOX4 was required for effective wound contraction but not myofibroblast expression. We suggest that myofibroblast contraction in NOX4-deficient mice is less effective in contracting the wound because of insufficient dityrosine-crosslinking of the ECM, providing the first indication for a physiological function of dityrosine crosslinking in higher animals. PMID:27140231

  20. The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

    PubMed Central

    Sordelli, Micaela S.; Beltrame, Jimena S.; Burdet, Juliana; Zotta, Elsa; Pardo, Romina; Cella, Maximiliano; Franchi, Ana M.; Ribeiro, Maria Laura

    2011-01-01

    Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data

  1. Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

    PubMed

    Eyestone, W H; First, N L

    1989-03-01

    In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2704004

  2. Establishment of bovine trophoblast stem-like cells from in vitro-produced blastocyst-stage embryos using two inhibitors.

    PubMed

    Huang, Xianghua; Han, Xuejie; Uyunbilig, Borjigin; Zhang, Manling; Duo, Shuguang; Zuo, Yongchun; Zhao, Yuhang; Yun, Ting; Tai, Dapeng; Wang, Chen; Li, Jinhua; Li, Xueling; Li, Rongfeng

    2014-07-01

    The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-τ, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90%, NANOG 74.40%, and SOX2 8.50%) compared with blastocysts (OCT4 8.90%, NANOG 34.4%, and SOX2 3.80%). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80% and 3.90%) compared with blastocysts (18.80% and 9.10%). The TSC lines that were established in this study may be used either for basic

  3. Null Mutations of NT-3 and Bax Affect Trigeminal Ganglion Cell Number but Not Brainstem Barrelette Pattern Formation

    PubMed Central

    Mosconi, Tony; Arends, J.J.; Jacquin, Mark F.

    2014-01-01

    Trigeminal ganglion (TG) neurons innervate the grid-like array of whisker follicles on the face of the mouse. Central TG axons project to the trigeminal (V) brainstem nuclear complex, including the nucleus principalis (PrV), and the spinal subnucleus interpolaris (SpVi), where they innervate barrelettes that are organized in a pattern that recapitulates the whisker pattern on the face. Neurotrophin-3 (NT-3) supports a population of TG cells that supply slowly adapting mechanoreceptors in the whisker pad. We examined mice at embryonic day 17 (E17) and on the day of birth (P0) with null mutations of NT-3, Bax, a proapoptotic gene associated with naturally occurring cell death, and Bax/NT-3 double knockout mutants to determine if: 1) the number of TG cells would be reduced; 2) eliminating the Bax gene would rescue the NT-3 dependent neurons; and 3) the central projections of the rescued axons in the Bax/NT-3 double knockout mice would fail to develop the barrelette patterns in the PrV and SpVi subnuclei. In mice at E17, NT-3−/− mutants had 65% fewer TG neurons than found in age matched wild-type (WT) mice, and at P0, the number was reduced by 55% (p < 0.001 for both). Bax null mutant mice at E17 had 132% of the WT number of TG cells (p < 0.001), although the numbers returned to WT levels by P0. Bax/NT-3 double knockout mice at E17 had TG cell numbers equal to those seen in WT, but the double knockout failed to retain WT TG neuron numbers in P0 mice (39% fewer cells; p < 0.001). In all cases of reduced experimental neuron numbers, and in the E17 Bax−/− mice with supernumerary cells, the barrelette patterns in the PrV and SpVi were normal. Only a slight qualitative reduction in overall barrelette field area and clarity of barrelettes were seen. These results suggest that NT-3 is not necessary for barrelette pattern formation in the brainstem. PMID:23614607

  4. Apatite formation on bioactive calcium-silicate cements for dentistry affects surface topography and human marrow stromal cells proliferation.

    PubMed

    Gandolfi, Maria Giovanna; Ciapetti, Gabriela; Taddei, Paola; Perut, Francesca; Tinti, Anna; Cardoso, Marcio Vivan; Van Meerbeek, Bart; Prati, Carlo

    2010-10-01

    The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cementsformation of apatite nano-spherulites; (2) the alpha-TCP doped cement aged for 28 days displayed the highest bioactivity and cell proliferation; (3) the deleterious effect of bismuth on cell

  5. Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves.

    PubMed

    Fang, Likui; Zhao, Fangming; Cong, Yunfei; Sang, Xianchun; Du, Qing; Wang, Dezhong; Li, Yunfeng; Ling, Yinghua; Yang, Zhenglin; He, Guanghua

    2012-06-01

    As an important agronomic trait, leaf rolling in rice (Oryza sativa L.) has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the amount of photosynthesis in cultivars and hence raises grain yield. Here, we describe the map-based cloning of the gene RL14, which was found to encode a 2OG-Fe (II) oxygenase of unknown function. rl14 mutant plants had incurved leaves because of the shrinkage of bulliform cells on the adaxial side. In addition, rl14 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, as compared with the wild type. Defective development could be rescued functionally by the expression of wild-type RL14. RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature. Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, and cellulose and lignin content were altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results in water deficiency, which is the major reason for the abnormal shape of the bulliform cells. PMID:22329407

  6. Comparison between Cleavage Stage versus Blastocyst Stage Embryo Transfer in an Egyptian Cohort Undergoing in vitro Fertilization: A Possible Role for Laser Assisted Hatching

    PubMed Central

    Hendawy, Sherif F.; Raafat, TA

    2011-01-01

    Background Extended in vitro embryo culture and blastocyst transfer have emerged as essential components of the advanced reproductive technology armamentarium, permitting selection of more advanced embryos considered best suited for transfer. Aim of study The aim of this study was to compare between cleavage stage and blastocyst stage embryo transfer in patients undergoing intracytoplasmic sperm injection, and to assess the role of assisted hatching technique in patients undergoing blastocyst transfer. Patients and methods This study was carried out on two groups. Group I: 110 patients who underwent 120 cycles of intracytoplasmic sperm injection with day 2–3 embryo transfer—for unexplained infertility or male factor within the previous 3 years. Their data obtained retrospectively from medical records. Group II: 46 age matched infertile female patients undergoing 51 intracytoplasmic sperm injection cycles for similar causes. Patients in Group II were further subdivided into 2 equal subgroups; Group IIa (23 patients), which had laser assisted hatching and Group IIb (23 patients), which did not have assisted hatching. All patients had an infertility workup including basal hormonal profile, pelvic ultrasound, hysterosalpingogram and/or laparoscope and semen analysis of the patient’s partner. All patients underwent controlled ovarian hyperstimulation: Using long protocol of ovulation induction. Laser assisted hatching was done for blastocysts of 23 patients. Results Comparison between both groups as regards the reproductive outcome showed a significant difference in pregnancy and implantation rates, both being higher in group II (P < 0.05) Comparison between both subgroups as regards the reproductive outcome showed a highly significant difference in pregnancy and implantation rates, both being higher in Group IIa (P < 0.01). There was also a significantly higher rate of multiple pregnancies among Group IIa (P < 0.05). Conclusion Blastocyst transfer is a successful

  7. The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

    PubMed Central

    Hou, Dongxia; Su, Min; Li, Xiawei; Li, Zhiying; Yun, Ting; Zhao, Yuhang; Zhang, Manling; Zhao, Lihua; Li, Rongfeng; Yu, Haiquan; Li, Xueling

    2015-01-01

    Trophoblasts (TR) are specialized cells of the placenta and play an important role in embryo implantation. The in vitro culture of trophoblasts provided an important tool to investigate the mechanisms of implantation. In the present study, porcine trophoblast cells were derived from pig in vitro fertilized (IVF) and parthenogenetically activated (PA) blastocysts via culturing in medium supplemented with KnockOut serum replacement (KOSR) and basic fibroblast growth factor (bFGF) on STO feeder layers, and the effect of ROCK (Rho-associated coiled-coil protein kinases) inhibiter Y-27632 on the cell lines culture was tested. 5 PA blastocyst derived cell lines and 2 IVF blastocyst derived cell lines have been cultured more than 20 passages; one PA cell lines reached 110 passages without obvious morphological alteration. The derived trophoblast cells exhibited epithelium-like morphology, rich in lipid droplets, and had obvious defined boundaries with the feeder cells. The cells were histochemically stained positive for alkaline phosphatase. The expression of TR lineage markers, such as CDX2, KRT7, KRT18, TEAD4, ELF5 and HAND1, imprinted genes such as IGF2, PEG1 and PEG10, and telomerase activity related genes TERC and TERF2 were detected by immunofluorescence staining, reverse transcription PCR and quantitative real-time PCR analyses. Both PA and IVF blastocysts derived trophoblast cells possessed the ability to differentiate into mature trophoblast cells in vitro. The addition of Y-27632 improved the growth of both PA and IVF blastocyst derived cell lines and increased the expression of trophoblast genes. This study has provided an alternative highly efficient method to establish trophoblast for research focused on peri-implantation and placenta development in IVF and PA embryos. PMID:26555939

  8. Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro.

    PubMed

    Pampfer, S; Vanderheyden, I; McCracken, J E; Vesela, J; De Hertogh, R

    1997-12-01

    The morphogenetic function of the transient phase of cell death that occurs during blastocyst maturation is not known but it is thought that its regulation results from a delicate balance between survival and lethal signals in the uterine milieu. In this paper, we show that blastocysts from diabetic rats have a higher incidence of dead cells than control embryos. Differential lineage staining indicated that increased nuclear fragmentation occurred mainly in the inner cell mass. In addition, terminal transferase-mediated dUTP nick end labeling (TUNEL) demonstrated an increase in the incidence of non-fragmented DNA-damaged nuclei in these blastocysts. Analysis of the expression of clusterin, a gene associated with apoptosis, by quantitative reverse transcription-polymerase chain reaction detected an increase in the steady-state level of its transcripts in blastocysts from diabetic rats. In situ hybridization revealed that about half the cells identified as expressing clusterin mRNA exhibited signs of nuclear fragmentation. In vitro experiments demonstrated that high D-glucose increased nuclear fragmentation, TUNEL labeling and clusterin transcription. Tumor necrosis factor-alpha (TNF-alpha), a cytokine whose synthesis is up-regulated in the diabetic uterus, did not induce nuclear fragmentation nor clusterin expression but increased the incidence of TUNEL-positive nuclei. The data suggest that excessive cell death in the blastocyst, most probably resulting from the overstimulation of a basal suicidal program by such inducers as glucose and TNF-alpha, may be a contributing factor of the early embryopathy associated with maternal diabetes. PMID:9428419

  9. Immobilized enzymes affect biofilm formation.

    PubMed

    Cordeiro, Ana L; Hippius, Catharina; Werner, Carsten

    2011-09-01

    The effect of the activity of immobilized enzymes on the initial attachment of pathogenic bacteria commonly associated with nosocomial infections (Pseudomonas aeruginosa and Staphylococcus epidermidis) was investigated. The proteolytic enzymes, subtilisin A and the glycoside hydrolase cellulose, were covalently attached onto poly(ethylene-alt-maleic) anhydride copolymer films. A comparison between active and heat-inactivated surfaces showed that while the activity of immobilized cellulase reduced the attachment of S. epidermidis by 67%, it had no effect on the attachment of P. aeruginosa. Immobilized subtilisin A had opposite effects: the active enzyme had no effect on the attachment of S. epidermidis but reduced the attachment of P. aeruginosa by 44%. The results suggest that different biomolecules are involved in the initial steps of attachment of different bacteria, and that the development of broad-spectrum antifouling enzymatic coatings will need to involve the co-immobilization of enzymes. PMID:21618024

  10. Switch of Voltage-Gated K+ Channel Expression in the Plasma Membrane of Chondrogenic Cells Affects Cytosolic Ca2+-Oscillations and Cartilage Formation

    PubMed Central

    Matta, Csaba; Fodor, János; Katona, Éva; Bartok, Adam; Oláh, Tamás; Sebe, Attila; Csernoch, László; Panyi, Gyorgy; Zákány, Róza

    2011-01-01

    Background Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca2+-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation. Methodology/Principal Findings Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated NaV1.4, KV1.1, KV1.3 and KV4.1 channels, although KV1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of NaV1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K+ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced KV currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca2+-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive KV1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive KV4.1 did not affect cartilage formation. Conclusions/Significance These data demonstrate that the differentiation and proliferation of chondrogenic cells depend

  11. Spatial pattern formation of microbes at the soil microscale affect soil C and N turnover in an individual-based microbial community model

    NASA Astrophysics Data System (ADS)

    Kaiser, Christina; Evans, Sarah; Dieckmann, Ulf; Widder, Stefanie

    2016-04-01

    At the μm-scale, soil is a highly structured and complex environment, both in physical as well as in biological terms, characterized by non-linear interactions between microbes, substrates and minerals. As known from mathematics and theoretical ecology, spatial structure significantly affects the system's behaviour by enabling synergistic dynamics, facilitating diversity, and leading to emergent phenomena such as self-organisation and self-regulation. Such phenomena, however, are rarely considered when investigating mechanisms of microbial soil organic matter turnover. Soil organic matter is the largest terrestrial reservoir for organic carbon (C) and nitrogen (N) and plays a pivotal role in global biogeochemical cycles. Still, the underlying mechanisms of microbial soil organic matter buildup and turnover remain elusive. We explored mechanisms of microbial soil organic matter turnover using an individual-based, stoichiometrically and spatially explicit computer model, which simulates the microbial de-composer system at the soil microscale (i.e. on a grid of 100 x 100 soil microsites). Soil organic matter dynamics in our model emerge as the result of interactions among individual microbes with certain functional traits (f.e. enzyme production rates, growth rates, cell stoichiometry) at the microscale. By degrading complex substrates, and releasing labile substances microbes in our model continusly shape their environment, which in turn feeds back to spatiotemporal dynamics of the microbial community. In order to test the effect of microbial functional traits and organic matter input rate on soil organic matter turnover and C and N storage, we ran the model into steady state using continuous inputs of fresh organic material. Surprisingly, certain parameter settings that induce resource limitation of microbes lead to regular spatial pattern formation (f.e. moving spiral waves) of microbes and substrate at the μm-scale at steady-state. The occurrence of these

  12. Antiestrogenic and Anti-Inflammatory Potential of n-Hexane Fraction of Vitex negundo Linn Leaf Extract: A Probable Mechanism for Blastocyst Implantation Failure in Mus musculus

    PubMed Central

    Jivrajani, Mehul; Ravat, Nirav; Anandjiwala, Sheetal; Nivsarkar, Manish

    2014-01-01

    The anti-implantation potential of different fractions of Vitex negundo Linn leaf extract was evaluated in female Swiss Albino mice. Animals from different groups were dosed orally either with 0.2% agar (vehicle) or with fractions of V. negundo leaf extract (n-hexane, chloroform, n-butanol, and remnant fractions) at 10:00 a.m., from day 1 to day 6 of pregnancy. The pregnant females from each group were sacrificed on different days of pregnancy (n = 6), and uterus was excised and used for estimation of lipid peroxidation and assay of superoxide dismutase activity as a marker for blastocyst implantation. Animals treated with n-hexane fraction showed altered level of superoxide anion radical and superoxide dismutase activity as compared to control animals. The probable mechanism by which this extract exhibits inhibition of blastocyst implantation is through the anti-inflammatory and antiestrogenic potential. PMID:27351007

  13. Concept Formation.

    ERIC Educational Resources Information Center

    Vaidya, Narendera

    This document, published in India by the Regional College of Education, deals with 13 subjects: the tough context (thinking), definitions of concept, functions of concept, the process of concept formation, discriminant learning, mediation process, second signalling system, factors affecting concept formation, studies in concept formation, the…

  14. GmEXPB2, a Cell Wall β-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development1[OPEN

    PubMed Central

    Li, Xinxin; Zhao, Jing; Tan, Zhiyuan; Liao, Hong

    2015-01-01

    Nodulation is an essential process for biological nitrogen (N2) fixation in legumes, but its regulation remains poorly understood. Here, a β-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. β-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N2 fixation and growth of soybean. PMID:26432877

  15. Soil-restoration rate and initial soil formation trends on example of anthropogenically affected soils of opencast mine in Kursk region, Russian Federation

    NASA Astrophysics Data System (ADS)

    Pigareva, Tatiana

    2015-04-01

    The mining industry is one of the main factors which anthropogenically change the environment. Mining process results in removing of the rocks and mechanical changes of considerable amounts of ground. One of the main results of mining arising of antropic ecosystems as well as increasing of the new created soils total area is technosols. The main factor controlling the soil formation in postmining environment is the quality of spoiled materials. Initial soil formation has been investigated on spoils of the largest iron ore extraction complex in Russia - Mikhailovsky mining and concentration complex which is situated in Kursk region, Russia. Investigated soils are presented by monogenetic weak developed soils of different age (10-15-20 years). Young soils are formed on the loess parent materials (20 year-old soil), or on a mix of sand and clay overburdens (15 and 10-year-old soils). Anthropogenically affected soils are characterized by well-developed humus horizon which is gradually replaced by weakly changed soil-building rocks (profile type A-C for 10-, 15-years old soils, and A-AC-C for 20 years old soils). Gray-humus soils are characterized by presence of diagnostic humus horizon gradually replaced by soil-building rock. The maximum intensity of humus accumulation has been determined in a semi-hydromorphic 10-year-old soil developed on the mixed heaps which is connected with features of water-air conditions complicating mineralization of plant remnants. 20-year-old soil on loess is characterized by rather high rate of organic substances accumulation between all the automorphous soils. It was shown that one of the most effective restoration ways for anthropogenically affected soils is a biological reclamation. Since overburdens once appeared on a day surface are overgrown badly in the first years, they are subject to influence of water and wind erosion. Our researchers have found out that permanent grasses are able to grow quickly; they accumulate a considerable

  16. A UV-to-MIR Monitoring of DR Tau: Exploring How Water Vapor in the Planet Formation Region is Affected by Stellar Accretion Variability

    NASA Astrophysics Data System (ADS)

    Banzatti, A.; Meyer, M. R.; Manara, C. F.; Pontoppidan, K. M.; Testi, L.

    2014-01-01

    Young stars are known to show variability due to non-steady mass accretion rate from their circumstellar disks. Accretion flares can produce strong energetic irradiation and heating that may affect the disk in the planet formation region, close to the central star. During an extreme accretion outburst in the young star EX Lupi, the prototype of EXor variables, remarkable changes in molecular gas emission from ~1 AU in the disk have recently been observed. Here, we focus on water vapor and explore how it is affected by variable accretion luminosity in T Tauri stars. We monitored a young highly variable solar-mass star, DR Tau, using simultaneously two high/medium-resolution spectrographs at the European Southern Observatory Very Large Telescope: VISIR at 12.4 μm to observe water lines from the disk and X-shooter covering from 0.3 to 2.5 μm to constrain the stellar accretion. Three epochs spanning timescales from several days to several weeks were obtained. The accretion luminosity was estimated to change within a factor of ~2 and no change in water emission was detected at a significant level. In comparison with EX Lupi and EXor outbursts, DR Tau suggests that the less long-lived and weaker variability phenomena typical of T Tauri stars may leave water at planet-forming radii in the disk mostly unaffected. We propose that these systems may provide evidence for two processes that act over different timescales: ultraviolet photochemistry in the disk atmosphere (faster) and heating of the deeper disk layers (slower).

  17. A UV-to-MIR monitoring of DR Tau: Exploring how water vapor in the planet formation region is affected by stellar accretion variability

    SciTech Connect

    Banzatti, A.; Meyer, M. R.; Manara, C. F.; Testi, L.; Pontoppidan, K. M.

    2014-01-01

    Young stars are known to show variability due to non-steady mass accretion rate from their circumstellar disks. Accretion flares can produce strong energetic irradiation and heating that may affect the disk in the planet formation region, close to the central star. During an extreme accretion outburst in the young star EX Lupi, the prototype of EXor variables, remarkable changes in molecular gas emission from ∼1 AU in the disk have recently been observed. Here, we focus on water vapor and explore how it is affected by variable accretion luminosity in T Tauri stars. We monitored a young highly variable solar-mass star, DR Tau, using simultaneously two high/medium-resolution spectrographs at the European Southern Observatory Very Large Telescope: VISIR at 12.4 μm to observe water lines from the disk and X-shooter covering from 0.3 to 2.5 μm to constrain the stellar accretion. Three epochs spanning timescales from several days to several weeks were obtained. The accretion luminosity was estimated to change within a factor of ∼2 and no change in water emission was detected at a significant level. In comparison with EX Lupi and EXor outbursts, DR Tau suggests that the less long-lived and weaker variability phenomena typical of T Tauri stars may leave water at planet-forming radii in the disk mostly unaffected. We propose that these systems may provide evidence for two processes that act over different timescales: ultraviolet photochemistry in the disk atmosphere (faster) and heating of the deeper disk layers (slower).

  18. Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation.

    PubMed

    Talbot, Neil C; Powell, Anne M; Camp, Mary; Ealy, Alan D

    2007-02-01

    Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming. PMID:17570020

  19. The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage

    PubMed Central

    2013-01-01

    Background The Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220°C/min is far lower than that reported with open vitrification systems such as the cryoloop (−15,000°C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an “open” vitrification system routinely used in our laboratory. Methods Human embryos were vitrified at either the 8–10 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (2–3 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of < 0.05 was considered significant. Results A total of 486 vitrified-warmed embryos were assessed and 92% of them were transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going. Conclusions The Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 8–10 cell

  20. Effects of grade of oocyte-cumulus complex and the interactions between grades on the production of blastocysts in the cow, ewe and lamb.

    PubMed

    Kelly, J M; Kleemann, D O; Rudiger, S R; Walker, S K

    2007-12-01

    There is an association between the size of the cumulus investment and the in vitro developmental ability of the oocyte-cumulus complex (OCC) that provides a basis for the selection of OCCs. However, the value of selection is confounded by humoral interactions between OCCs that influence the development of OCCs of other grade(s). This study examined the effect of size of the cumulus investment (OCC grade) and the interactions between grades on the developmental ability of oocytes collected from the cow, ewe and lamb. OCCs were classified into A, B and C grades on visual assessment of the number of cumulus cell layers or left unselected (Unselected). In the cow, there were 669 +/- 228 to 4763 +/- 228 cells per OCC whereas comparable figures in the ewe and lamb were 593 +/- 252 to 3716 +/- 252 and 366 +/- 228 to 3263 +/- 228 respectively (A > Unselected > B > C; Experiment 1). In Experiment 2, OCCs were made to mature within grade and the efficiency of blastocyst production and blastocyst quality was compared with that obtained in the Unselected group. Grade was associated with significant (p < 0.05) differences in cleavage rate, blastocyst production rate and the mean number of nuclei per embryo (generally A > B > C across animal types). However, the performance of A grade OCCs in the cow and lamb did not differ significantly from that obtained in the Unselected group whereas in the ewe, A grade OCCs were significantly (p < 0.05) better. Furthermore, the performance of the Unselected group was significantly (p < 0.05) better than that of the combined grades (A + B + C) in the cow but there were no differences in either the ewe or lamb. It is concluded that (i) interactions between OCCs of different grade influence the developmental ability of OCCs in the cow and, to a lesser extent, the lamb, (ii) selection of OCCs in the cow and lamb would lead to the exclusion of many OCCs that have the ability to develop into blastocysts and (iii) selection in the ewe would

  1. Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure.

    PubMed

    Egert, Markus; Friedrich, Michael W

    2003-05-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate

  2. How the presence of a gas giant affects the formation of mean-motion resonances between two low-mass planets in a locally isothermal gaseous disc

    NASA Astrophysics Data System (ADS)

    Podlewska-Gaca, E.; Szuszkiewicz, E.

    2014-03-01

    In this paper we investigate the possibility of a migration-induced resonance locking in systems containing three planets, namely an Earth analogue (1 M⊕), a super-Earth (4 M⊕) and a gas giant (one Jupiter mass). The planets have been listed in order of increasing orbital periods. All three bodies are embedded in a locally isothermal gaseous disc and orbit around a solar mass star. We are interested in answering the following questions: will the low-mass planets form the same resonant structures with each other in the vicinity of the gas giant as in the case when the gas giant is absent? More in general, how will the presence of the gas giant affect the evolution of the two low-mass planets? When there is no gas giant in the system, it has been already shown that if the two low-mass planets undergo a convergent differential migration, they will capture each other in a mean-motion resonance. For the choices of disc parameters and planet masses made in this paper, the formation of the 5:4 resonance in the absence of the Jupiter has been observed in a previous investigation and confirmed here. In this work we add a gas giant on the most external orbit of the system in such a way that its differential migration is convergent with the low-mass planets. We show that the result of this set-up is the speeding up of the migration of the super-Earth and, after that, all three planets become locked in a triple mean-motion resonance. However, this resonance is not maintained due to the low-mass planet eccentricity excitation, a fact that leads to close encounters between planets and eventually to the ejection from the internal orbits of one or both low-mass planets. We have observed that the ejected low-mass planets can leave the system, fall into a star or become the external planet relative to the gas giant. In our simulations the latter situation has been observed for the super-Earth. It follows from the results presented here that the presence of a Jupiter-like planet

  3. A point mutation in the EGF-4 domain of β3 integrin is responsible for the formation of the Seca platelet alloantigen and affects receptor function

    PubMed Central

    Sachs, Ulrich J.; Bakchoul, Tamam; Eva, Olga; Giptner, Astrid; Bein, Gregor; Aster, Richard H.; Gitter, Maria; Peterson, Julie; Santoso, Sentot

    2013-01-01

    Summary Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Seca) residing on αIIbβ3. The location of Seca on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G1818T) in exon 11 of the β3 gene (ITGB3), changing Lys580 (wild-type) to Asn580 (Seca). Two additional members of the family Sec were typed Seca positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn580, but not Lys580 αIIbβ3, bound anti-Seca, which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn580 variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Seca positive platelets, which, however, are heterozygous for the Lys580Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys580Asn mutation responsible for the formation of the Seca antigenic determinant affects αIIbβ3 receptor function. PMID:22116617

  4. Ocean acidification does not affect magnesium composition or dolomite formation in living crustose coralline algae, Porolithon onkodes in an experimental system

    NASA Astrophysics Data System (ADS)

    Nash, M. C.; Uthicke, S.; Negri, A. P.; Cantin, N. E.

    2015-09-01

    There are concerns that Mg-calcite crustose coralline algae (CCA), which are key reef builders on coral reefs, will be most susceptible to increased rates of dissolution under higher pCO2 and ocean acidification. Due to the higher solubility of Mg-calcite, it has been hypothesised that magnesium concentrations in CCA Mg-calcite will decrease as the ocean acidifies, and that this decrease will make their skeletons more chemically stable. In addition to Mg-calcite, CCA Porolithon onkodes, the predominant encrusting species on tropical reefs, can have dolomite (Ca0.5Mg0.5CO3) infilling cell spaces which increases their stability. However, nothing is known about how bio-mineralised dolomite formation responds to higher pCO2. Using P. onkodes grown for 3 and 6 months in tank experiments, we aimed to determine (1) if mol % MgCO3 in new crust and new settlement was affected by increasing CO2 levels (365, 444, 676 and 904 μatm), (2) whether bio-mineralised dolomite formed within these time frames, and (3) if so, whether this was effected by CO2. Our results show that there was no significant effect of CO2 on mol % MgCO3 in any sample set, indicating an absence of a plastic response under a wide range of experimental conditions. Dolomite within the CCA cells formed within 3 months and dolomite abundance did not vary significantly with CO2 treatment. While evidence mounts that climate change will impact many sensitive coral and CCA species, the results from this study indicate that reef-building P. onkodes will continue to form stabilising dolomite infill under near-future acidification conditions, thereby retaining its higher resistance to dissolution.

  5. Enhanced formation of aromatic amino acids increases fragrance without affecting flower longevity or pigmentation in Petunia × hybrida.

    PubMed

    Oliva, Moran; Ovadia, Rinat; Perl, Avichai; Bar, Einat; Lewinsohn, Efraim; Galili, Gad; Oren-Shamir, Michal

    2015-01-01

    Purple Petunia × hybrida V26 plants accumulate fragrant benzenoid-phenylpropanoid molecules and anthocyanin pigments in their petals. These specialized metabolites are synthesized mainly from the aromatic amino acids phenylalanine. Here, we studied the profile of secondary metabolites of petunia plants, expressing a feedback-insensitive bacterial form of 3-deoxy-di-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway, as a tool to stimulate the conversion of primary to secondary metabolism via the aromatic amino acids. We focused on specialized metabolites contributing to flower showy traits. The presence of AroG* protein led to increased aromatic amino acid levels in the leaves and high phenylalanine levels in the petals. In addition, the AroG* petals accumulated significantly higher levels of fragrant benzenoid-phenylpropanoid volatiles, without affecting the flowers' lifetime. In contrast, AroG* abundance had no effect on flavonoids and anthocyanins levels. The metabolic profile of all five AroG* lines was comparable, even though two lines produced the transgene in the leaves, but not in the petals. This implies that phenylalanine produced in leaves can be transported through the stem to the flowers and serve as a precursor for formation of fragrant metabolites. Dipping cut petunia stems in labelled phenylalanine solution resulted in production of labelled fragrant volatiles in the flowers. This study emphasizes further the potential of this metabolic engineering approach to stimulate the production of specialized metabolites and enhance the quality of various plant organs. Furthermore, transformation of vegetative tissues with AroG* is sufficient for induced production of specialized metabolites in organs such as the flowers. PMID:25283446

  6. The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

    PubMed

    Sato, Masahiro; Maeda, Kosuke; Koriyama, Miyu; Inada, Emi; Saitoh, Issei; Miura, Hiromi; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi; Miyoshi, Kazuchika

    2016-01-01

    The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes. PMID:27589724

  7. An Earlier Uterine Environment Favors the In Vivo Development of Fresh Pig Morulae and Blastocysts Transferred by a Nonsurgical Deep-uterine Method

    PubMed Central

    ANGEL, Miguel Angel; GIL, Maria Antonia; CUELLO, Cristina; SANCHEZ-OSORIO, Jonatan; GOMIS, Jesus; PARRILLA, Inmaculada; VILA, Jordi; COLINA, Ignacio; DIAZ, Marta; REIXACH, Josep; VAZQUEZ, Jose Luis; VAZQUEZ, Juan Maria; ROCA, Jordi; MARTINEZ, Emilio A.

    2014-01-01

    This study aimed to evaluate the effect of recipient-donor estrous cycle synchrony on recipient reproductive performance after nonsurgical deep-uterine (NsDU) embryo transfer (ET). The transfers (N=132) were conducted in recipients sows that started estrus 24 h before (–24 h; N=9) or 0 h (synchronous; N=31), 24 h (+24 h; N=74) or 48 h (+48 h; N=18) after the donors. A total of 30 day 5 morulae or day 6 blastocysts (day 0=onset of estrus) were transferred per recipient. The highest farrowing rates (FRs) were achieved when estrus appeared in recipients 24 h later than that in the donors (81.1%), regardless of the embryonic stage used for the transfers. The FR notably decreased (P<0.05) when recipients were –24 h asynchronous (0%), synchronous (61.3%) or +48 h asynchronous (50%) relative to the donors. No differences in litter size (LS) and piglet birth weights were observed among the synchronous and +24 h or +48 h asynchronous groups. While a +24 h asynchronous recipient was suitable for transfers performed with either morulae (FR, 74.3%; LS, 9.2 ± 0.6 piglets) or blastocysts (FR, 84.6%; LS, 9.8 ± 0.6 piglets), a + 48 h asynchronous recipient was adequate for blastocysts (FR, 87.5%; LS, 10.4 ± 0.7 piglets) but not for morulae (FR, 30.0%; LS, 7.3 ± 2.3 piglets). In conclusion, this study confirms the effectiveness of the NsDU-ET technology and shows that porcine embryos tolerate better a less advanced uterine environment if they are nonsurgically transferred deep into the uterine horn. PMID:25030061

  8. Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts.

    PubMed

    Chan, Wen-Hsiung

    2007-06-01

    The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras-->ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 muM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras-->ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

  9. Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts

    PubMed Central

    Chan, Wen-Hsiung

    2007-01-01

    The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras→ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 μM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras→ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

  10. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development.

    PubMed

    Ortiz-Escribano, N; Smits, K; Piepers, S; Van den Abbeel, E; Woelders, H; Van Soom, A

    2016-07-15

    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were vitrified in 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5-M sucrose. Oocytes that survived the vitrification process were fertilized. Denuded oocytes were fertilized with or without supplementation of intact COCs (DOsCOCs). First, survival and embryo development rates were studied. Higher survival rates were obtained for DOs and DOsCOCs (94% and 95%, respectively) compared with COCs (82.7%, P < 0.05). Corona radiata oocytes showed similar survival rates when compared with DOs. The cleavage and blastocyst rates of vitrified DOs were compromised because cumulus cells were not present during the fertilization (34% and 2.7%, respectively). However, the situation could be reverted when DOs were supplemented with intact COCs (DOsCOCs; 62.7% and 12.7%, respectively, P < 0.05). Vitrified CRs showed similar cleavage and blastocyst rate (49.3% and 7.7%, respectively) compared with COCs (54.8% and 4.9%, respectively). In the second experiment, the penetration rate was analyzed. Removing cumulus cells before fertilization reduced the fertilization of vitrified DOs compared with COCs (24.3% vs. 52.8%, P < 0.05). The supplementation of DOs with intact COCs (DOsCOCs) improved the fertilization rate though (49.6%, P < 0.05). No differences in the fertilization rate were found between CRs and COCs. In the third experiment, parthenogenetic activation was examined. Interestingly, the CRs group showed higher cleavage and blastocyst rates (76.8% and 29.6%, respectively) than the COCs (39.1% and 7.5%, respectively, P < 0.05). Furthermore, oocytes from vitrified CRs had the same odds to become a blastocyst as fresh oocytes (1.1 vs. 1.5, respectively). In conclusion, our data

  11. Selection of competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for patients undergoing preimplantation genetic screening: a prospective study with sibling oocytes

    PubMed Central

    2014-01-01

    Background Recent advances in time-lapse monitoring in IVF treatment have provided new morphokinetic markers for embryonic competence. However, there is still very limited information about the relationship between morphokinetic parameters, chromosomal compositions and implantation potential. Accordingly, this study aimed at investigating the effects of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing on pregnancy and implantation outcomes for patients undergoing preimplantation genetic screening (PGS). Methods A total of 1163 metaphase II (MII) oocytes were retrieved from 138 PGS patients at a mean age of 36.6 ± 2.4 years. These sibling MII oocytes were then randomized into two groups after ICSI: 1) Group A, oocytes (n = 582) were cultured in the time-lapse system and 2) Group B, oocytes (n = 581) were cultured in the conventional incubator. For both groups, whole genomic amplification and array CGH testing were performed after trophectoderm biopsy on day 5. One to two euploid blastocysts within the most predictive morphokinetic parameters (Group A) or with the best morphological grade available (Group B) were selected for transfer to individual patients on day 6. Ongoing pregnancy and implantation rates were compared between the two groups. Results There were significant differences in clinical pregnancy rates between Group A and Group B (71.1% vs. 45.9%, respectively, p = 0.037). The observed implantation rate per embryo transfer significantly increased in Group A compared to Group B (66.2% vs. 42.4%, respectively, p = 0.011). Moreover, a significant increase in ongoing pregnancy rates was also observed in Group A compared to Group B (68.9% vs. 40.5%. respectively, p = 0.019). However, there was no significant difference in miscarriage rate between the time-lapse system and the conventional incubator (3.1% vs. 11.8%, respectively, p = 0.273). Conclusions This is the first prospective investigation using

  12. Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation.

    PubMed

    Zheng, Kaijie; Tian, Hainan; Hu, Qingnan; Guo, Hongyan; Yang, Li; Cai, Ling; Wang, Xutong; Liu, Bao; Wang, Shucai

    2016-01-01

    In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation. PMID:26758286

  13. Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation

    PubMed Central

    Zheng, Kaijie; Tian, Hainan; Hu, Qingnan; Guo, Hongyan; Yang, Li; Cai, Ling; Wang, Xutong; Liu, Bao; Wang, Shucai

    2016-01-01

    In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation. PMID:26758286

  14. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides.

    PubMed

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-01-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water. PMID:26394759

  15. Peptide bonds affect the formation of haloacetamides, an emerging class of N-DBPs in drinking water: free amino acids versus oligopeptides

    PubMed Central

    Chu, Wenhai; Li, Xin; Gao, Naiyun; Deng, Yang; Yin, Daqiang; Li, Dongmei; Chu, Tengfei

    2015-01-01

    Haloacetamides (HAcAms), an emerging class of nitrogenous disinfection by-products (N-DBPs) of health concern, have been frequently identified in drinking waters. It has long been appreciated that free amino acids (AAs), accounting for a small fraction of the dissolved organic nitrogen (DON) pool, can form dichloroacetamide (DCAcAm) during chlorination. However, the information regarding the impacts of combined AAs, which contribute to the greatest identifiable DON portion in natural waters, is limited. In this study, we compared the formation of HAcAms from free AAs (tyrosine [Tyr] and alanine [Ala]) and combined AAs (Tyr-Ala, Ala-Tyr, Tyr-Tyr-Tyr, Ala-Ala-Ala), and found that HAcAm formation from the chlorination of AAs in combined forms (oligopeptides) significantly exhibited a different pattern with HAcAm formation from free AAs. Due to the presence of peptide bonds in tripeptides, Tyr-Tyr-Tyr and Ala-Ala-Ala produced trichloroacetamide (TCAcAm) in which free AAs was unable to form TCAcAm during chlorination. Moreover, peptide bond in tripeptides formed more tri-HAcAms than di-HAcAms in the presence of bromide. Therefore, the peptide bond may be an important indicator to predict the formation of specific N-DBPs in chlorination. The increased use of algal- and wastewater-impacted water as drinking water sources will increase health concerns over exposure to HAcAms in drinking water. PMID:26394759

  16. Dicer ablation in osteoblasts by Runx2 driven cre-loxP recombination affects bone integrity, but not glucocorticoid-induced suppression of bone formation.

    PubMed

    Liu, Peng; Baumgart, Mario; Groth, Marco; Wittmann, Jürgen; Jäck, Hans-Martin; Platzer, Matthias; Tuckermann, Jan P; Baschant, Ulrike

    2016-01-01

    Glucocorticoid-induced osteoporosis (GIO) is one of the major side effects of long-term glucocorticoid (GC) therapy mediated mainly via the suppression of bone formation and osteoblast differentiation independently of GC receptor (GR) dimerization. Since microRNAs play a critical role in osteoblast differentiation processes, we investigated the role of Dicer dependent microRNAs in the GC-induced suppression of osteoblast differentiation. MicroRNA sequencing of dexamethasone-treated wild-type and GR dimer-deficient mesenchymal stromal cells revealed GC-controlled miRNA expression in a GR dimer-dependent and GR dimer-independent manner. To determine the functional relevance of mature miRNAs in GC-induced osteoblast suppression, mice with an osteoblast-specific deletion of Dicer (Dicer(Runx2Cre)) were exposed to glucocorticoids. In vitro generated Dicer-deficient osteoblasts were treated with dexamethasone and analyzed for proliferation, differentiation and mineralization capacity. In vivo, abrogation of Dicer-dependent miRNA biogenesis in osteoblasts led to growth retardation and impaired bone formation. However, subjecting these mice to GIO showed that bone formation was similar reduced in Dicer(Runx2Cre) mice and littermate control mice upon GC treatment. In line, differentiation of Dicer deficient osteoblasts was suppressed to the same extent as wild type cells by GC treatment. Therefore, Dicer-dependent small RNA biogenesis in osteoblasts plays only a minor role in the pathogenesis of GC-induced inhibition of bone formation. PMID:27554624

  17. Dicer ablation in osteoblasts by Runx2 driven cre-loxP recombination affects bone integrity, but not glucocorticoid-induced suppression of bone formation

    PubMed Central

    Liu, Peng; Baumgart, Mario; Groth, Marco; Wittmann, Jürgen; Jäck, Hans-Martin; Platzer, Matthias; Tuckermann, Jan P.; Baschant, Ulrike

    2016-01-01

    Glucocorticoid-induced osteoporosis (GIO) is one of the major side effects of long-term glucocorticoid (GC) therapy mediated mainly via the suppression of bone formation and osteoblast differentiation independently of GC receptor (GR) dimerization. Since microRNAs play a critical role in osteoblast differentiation processes, we investigated the role of Dicer dependent microRNAs in the GC-induced suppression of osteoblast differentiation. MicroRNA sequencing of dexamethasone-treated wild-type and GR dimer-deficient mesenchymal stromal cells revealed GC-controlled miRNA expression in a GR dimer-dependent and GR dimer-independent manner. To determine the functional relevance of mature miRNAs in GC-induced osteoblast suppression, mice with an osteoblast-specific deletion of Dicer (DicerRunx2Cre) were exposed to glucocorticoids. In vitro generated Dicer-deficient osteoblasts were treated with dexamethasone and analyzed for proliferation, differentiation and mineralization capacity. In vivo, abrogation of Dicer-dependent miRNA biogenesis in osteoblasts led to growth retardation and impaired bone formation. However, subjecting these mice to GIO showed that bone formation was similar reduced in DicerRunx2Cre mice and littermate control mice upon GC treatment. In line, differentiation of Dicer deficient osteoblasts was suppressed to the same extent as wild type cells by GC treatment. Therefore, Dicer-dependent small RNA biogenesis in osteoblasts plays only a minor role in the pathogenesis of GC-induced inhibition of bone formation. PMID:27554624

  18. Geohydrology of the aquifers that may be affected by the surface mining of coal in the Fruitland Formation in the San Juan Basin, northwestern New Mexico

    USGS Publications Warehouse

    Myers, R.G.; Villanueva, E.D.

    1986-01-01

    A monitoring network of 50 wells was installed from 1975 to 1979 in the surface-mineable coal area. The purpose of the network was to collect hydrologic data and to establish a data base. The hydrologic data consist of water level measurements, water chemistry, and selected aquifer characteristics. The four aquifers considered in this study are: (1) The Pictured Cliffs Sandstone; (2) coal seams and interbedded lithologic units of the Fruitland Formation; (3) overburden of the Kirtland Shale and Fruitland Formation; and (4) the alluvium along the Chaco River and its eastern and northeastern tributaries. Five sites were selected for study. Four of these sites, Western Coal Company 's lease near Fruitland, Bisti West, Kimbeto, and Ojo Encino, have surface-mineable coal. The Chaco River alluvium was selected because it is downstream from anticipated mining activity. The specific conductances of the representative water samples from selected wells in the 4 aquifers in the 5 study areas ranged from 800 to 23,000 microsiemens/cm at 25 C. The dominant cation in all samples was sodium. The water is brackish in the Pictured Cliffs Sandstone and in the coal seams and interbedded lithologic units of the Fruitland Formation. It is fresh to saline in the overburden of the Fruitland Shale and Fruitland Formation, and fresh to brackish in the Chaco River alluvium. (Author 's abstract)

  19. A Novel Factor Controlling Bistability in Bacillus subtilis: the YmdB Protein Affects Flagellin Expression and Biofilm Formation ▿†

    PubMed Central

    Diethmaier, Christine; Pietack, Nico; Gunka, Katrin; Wrede, Christoph; Lehnik-Habrink, Martin; Herzberg, Christina; Hübner, Sebastian; Stülke, Jörg

    2011-01-01

    Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σD-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation. PMID:21856853

  20. Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus

    PubMed Central

    Bachert, Beth A.; Choi, Soo J.; LaSala, Paul R.; Harper, Tiffany I.; McNitt, Dudley H.; Boehm, Dylan T.; Caswell, Clayton C.; Ciborowski, Pawel; Keene, Douglas R.; Flores, Anthony R.; Musser, James M.; Squeglia, Flavia; Marasco, Daniela; Berisio, Rita; Lukomski, Slawomir

    2016-01-01

    The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.

  1. Catechol degradation on hematite/silica–gas interface as affected by gas composition and the formation of environmentally persistent free radicals

    PubMed Central

    Li, Hao; Guo, Huiying; Pan, Bo; Liao, Shaohua; Zhang, Di; Yang, Xikun; Min, Chungang; Xing, Baoshan

    2016-01-01

    Environmentally persistent free radicals (EPFRs) formed on a solid particle surface have received increasing attention because of their toxic effects. However, organic chemical fate regulated by EPFRs has rarely been investigated, and this information may provide the missing link in understanding their environmental behavior. Previous studies have suggested that the reduction of transition metals is involved in EPFRs formation. We thus hypothesize that an oxidative environment may inhibit EPFRs formation in particle-gas interface, which will consequently release free radicals and accelerate organic chemical degradation. Our result indicates that a 1% hematite coating on a silica surface inhibited catechol degradation in N2, especially at low catechol loadings on solid particles (SCT). However, under an O2 environment, catechol degradation decreased when SCT was <1 μg/mg but increased when SCT was >1 μg/mg. Stable organic free radicals were observed in the N2 system with g factors in the 2.0035–2.0050 range, suggesting the dominance of oxygen-centered free radicals. The introduction of O2 into the catechol degradation system substantially decreased the free radical signals and decreased the Fe(II) content. These results were observed in both dark and light irradiation systems, indicating the ubiquitous presence of EPFRs in regulating the fate of organic chemicals. PMID:27079263

  2. Catechol degradation on hematite/silica-gas interface as affected by gas composition and the formation of environmentally persistent free radicals.

    PubMed

    Li, Hao; Guo, Huiying; Pan, Bo; Liao, Shaohua; Zhang, Di; Yang, Xikun; Min, Chungang; Xing, Baoshan

    2016-01-01

    Environmentally persistent free radicals (EPFRs) formed on a solid particle surface have received increasing attention because of their toxic effects. However, organic chemical fate regulated by EPFRs has rarely been investigated, and this information may provide the missing link in understanding their environmental behavior. Previous studies have suggested that the reduction of transition metals is involved in EPFRs formation. We thus hypothesize that an oxidative environment may inhibit EPFRs formation in particle-gas interface, which will consequently release free radicals and accelerate organic chemical degradation. Our result indicates that a 1% hematite coating on a silica surface inhibited catechol degradation in N2, especially at low catechol loadings on solid particles (SCT). However, under an O2 environment, catechol degradation decreased when SCT was <1 μg/mg but increased when SCT was >1 μg/mg. Stable organic free radicals were observed in the N2 system with g factors in the 2.0035-2.0050 range, suggesting the dominance of oxygen-centered free radicals. The introduction of O2 into the catechol degradation system substantially decreased the free radical signals and decreased the Fe(II) content. These results were observed in both dark and light irradiation systems, indicating the ubiquitous presence of EPFRs in regulating the fate of organic chemicals. PMID:27079263

  3. Catechol degradation on hematite/silica–gas interface as affected by gas composition and the formation of environmentally persistent free radicals

    NASA Astrophysics Data System (ADS)

    Li, Hao; Guo, Huiying; Pan, Bo; Liao, Shaohua; Zhang, Di; Yang, Xikun; Min, Chungang; Xing, Baoshan

    2016-04-01

    Environmentally persistent free radicals (EPFRs) formed on a solid particle surface have received increasing attention because of their toxic effects. However, organic chemical fate regulated by EPFRs has rarely been investigated, and this information may provide the missing link in understanding their environmental behavior. Previous studies have suggested that the reduction of transition metals is involved in EPFRs formation. We thus hypothesize that an oxidative environment may inhibit EPFRs formation in particle-gas interface, which will consequently release free radicals and accelerate organic chemical degradation. Our result indicates that a 1% hematite coating on a silica surface inhibited catechol degradation in N2, especially at low catechol loadings on solid particles (SCT). However, under an O2 environment, catechol degradation decreased when SCT was <1 μg/mg but increased when SCT was >1 μg/mg. Stable organic free radicals were observed in the N2 system with g factors in the 2.0035–2.0050 range, suggesting the dominance of oxygen-centered free radicals. The introduction of O2 into the catechol degradation system substantially decreased the free radical signals and decreased the Fe(II) content. These results were observed in both dark and light irradiation systems, indicating the ubiquitous presence of EPFRs in regulating the fate of organic chemicals.

  4. Inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling in the mouse blastocyst

    PubMed Central

    Kono, Kanako; Tamashiro, Dana Ann A.; Alarcon, Vernadeth B.

    2014-01-01

    Specification of the trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst correlates with cell position, as TE derives from outer cells whereas ICM from inner cells. Differences in position are reflected by cell polarization and Hippo signaling. Only in outer cells, the apical-basal cell polarity is established, and Hippo signaling is inhibited in such a manner that LATS1 and 2 (LATS1/2) kinases are prevented from phosphorylating YAP, a key transcriptional co-activator of the TE-specifying gene Cdx2. However, the molecular mechanisms that regulate these events are not fully understood. Here, we showed that inhibition of RHO-ROCK signaling enhances ICM and suppresses TE characteristics through activation of Hippo signaling and disruption of apical-basal polarity. Embryos treated with ROCK inhibitor Y-27632 exhibited elevated expression of ICM marker NANOG and reduced expression of CDX2 at the blastocyst stage. Y-27632-treated embryos failed to accumulate YAP in the nucleus, although it was rescued by concomitant inhibition of LATS1/2. Segregation between apical and basal polarity regulators, namely PARD6B, PRKCZ, SCRIB, and LLGL1, was dampened by Y-27632 treatment, whereas some of the polarization events at the late 8-cell stage such as compaction and apical localization of p-ERM and tyrosinated tubulin occurred normally. Similar abnormalities of Hippo signaling and apical-basal polarization were also observed in embryos that were treated with RHO GTPases inhibitor. These results suggest that RHO-ROCK signaling plays an essential role in regulating Hippo signaling and cell polarization to enable proper specification of the ICM and TE lineages. PMID:24997360

  5. Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass, surface charge, and nanoparticle-cell interaction.

    PubMed

    Gräfe, Christine; Weidner, Andreas; Lühe, Moritz V D; Bergemann, Christian; Schacher, Felix H; Clement, Joachim H; Dutz, Silvio

    2016-06-01

    The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood-brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS-PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30kDa and 100kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30min-incubation. PMID:26556312

  6. Solution Conditions Affect the Ability of the K30D Mutation To Prevent Amyloid Fibril Formation by Apolipoprotein C-II: Insights from Experiments and Theoretical Simulations.

    PubMed

    Mao, Yu; Todorova, Nevena; Zlatic, Courtney O; Gooley, Paul R; Griffin, Michael D W; Howlett, Geoffrey J; Yarovsky, Irene

    2016-07-12

    Apolipoproteins form amphipathic helical structures that bind lipid surfaces. Paradoxically, lipid-free apolipoproteins display a strong propensity to form cross-β structure and self-associate into disease-related amyloid fibrils. Studies of apolipoprotein C-II (apoC-II) amyloid fibrils suggest that a K30-D69 ion pair accounts for the dual abilities to form helix and cross-β structure. Consistent with this is the observation that a K30D mutation prevents fibril formation under standard fibril forming conditions. However, we found that fibril formation by K30D apoC-II proceeded readily at low pH and a higher salt or protein concentration. Structural analysis demonstrated that K30D apoC-II fibrils at pH 7 have a structure similar to that of the wild-type fibrils but are less stable. Molecular dynamics simulations of the wild-type apoC-II fibril model at pH 7 and 3 showed that the loss of charge on D69 at pH 3 leads to greater separation between residues K30 and D69 within the fibril with a corresponding reduction in β-strand content around residue 30. In contrast, in simulations of the K30D mutant model at pH 7 and 3, residues D30 and D69 moved closer at pH 3, accompanied by an increase in β-strand content around residue 30. The simulations also demonstrated a strong dominance of inter- over intramolecular contacts between ionic residues of apoC-II and suggested a cooperative mechanism for forming favorable interactions between the individual strands under different conditions. These observations demonstrate the important role of the buried K30-D69 ion pair in the stability and solution properties of apoC-II amyloid fibrils. PMID:27311794

  7. σ(B) affects biofilm formation under the dual stress conditions imposed by adding salt and low temperature in Listeria monocytogenes.

    PubMed

    Lee, Jin-Ju; Lee, Gilho; Shin, Ji-Hyun

    2014-10-01

    The food-borne pathogenic bacteria Listeria monocytogenes can form biofilms on various surfaces including food-processing equipment. Biofilms offer survival benefits to the organisms entrapped against environmental insults. Moreover, the σ(B) transcription factor of L. monocytogenes plays an important role in its survival under various stress conditions. In this study, we evaluated whether σ(B) contributes to biofilm formation when L. monocytogenes is grown under various temperatures and media. When the wild-type strain was grown under static biofilm culture below ambient temperature (15°C) for 72 h, the difference in viable cell number (in both planktonic and biofilm cells) between the wild-type and ΔsigB mutant increased by adding NaCl to BHI broth (9% salt BHI > 6% salt BHI > BHI, w/v), and the specific activity of β-galactosidase was highly induced in the wild-type strain grown in 6% salt containing BHI broth. Furthermore, we measured surface-adhered biofilm forming ability using the crystal violet staining method. The wild-type strain formed a four times larger biofilm than that of the ΔsigB mutant in 6% salt-BHI medium at 15°C over a 72 h incubation and also showed the highest level of β-galactosidase specific activity. However, both the wild-type and ΔsigB mutant L. monocytogenes were defective for forming a biofilm in 9% salt-BHI medium at 15°C. Our results suggest that σ(B) plays an enhanced role in surface-adhered biofilm formation when L. monocytogenes encounters dual stress conditions, such as 6% NaCl and low temperature. PMID:25269605

  8. Nonlinear electrodynamics and modification of initial singularities, and dark matter and dark energy affecting structure formation in the early and later universe

    NASA Astrophysics Data System (ADS)

    Beckwith, A. W.

    2015-07-01

    We find that having the scale factor close to zero due to a given magnetic field value in an early universe magnetic field affects how we would interpret Mukhanov's chapter on “self reproduction” of the universe in in his reference. The stronger an early-universe magnetic field is, the greater the likelihood of production of about 20 new domains of size 1/H, with H the early-universe Hubble constant, per Planck time interval in evolution. We form DM from considerations as to a minimum time step, and then generate DM via axions. Through Ng's quantum infinite statistics, we compare a DM count, giving entropy. The remainder of the document is in terms of DE as well as comparing entropy in galaxies versus entropy in the universe, through a lens of Mistra's quantum theory of the big bang.

  9. A high proportion of blue light increases the photosynthesis capacity and leaf formation rate of Rosa × hybrida but does not affect time to flower opening.

    PubMed

    Terfa, Meseret Tesema; Solhaug, Knut Asbjørn; Gislerød, Hans Ragnar; Olsen, Jorunn Elisabeth; Torre, Sissel

    2013-05-01

    Alterations in light quality affect plant morphogenesis and photosynthetic responses but the effects vary significantly between species. Roses exhibit an irradiance-dependent flowering control but knowledge on light quality responses is scarce. In this study we analyzed, the responses in morphology, photosynthesis and flowering of Rosa × hybrida to different blue (B) light proportions provided by light-emitting diodes (LED, high B 20%) and high pressure sodium (HPS, low B 5%) lamps. There was a strong morphological and growth effect of the light sources but no significant difference in total dry matter production and flowering. HPS-grown plants had significantly higher leaf area and plant height, yet a higher dry weight proportion was allocated to leaves than stems under LED. LED plants showed 20% higher photosynthetic capacity (Amax ) and higher levels of soluble carbohydrates. The increase in Amax correlated with an increase in leaf mass per unit leaf area, higher stomata conductance and CO2 exchange, total chlorophyll (Chl) content per area and Chl a/b ratio. LED-grown leaves also displayed a more sun-type leaf anatomy with more and longer palisade cells and a higher stomata frequency. Although floral initiation occurred at a higher leaf number in LED, the time to open flowers was the same under both light conditions. Thereby the study shows that a higher portion of B light is efficient in increasing photosynthesis performance per unit leaf area, enhancing growth and morphological changes in roses but does not affect the total Dry Matter (DM) production or time to open flower. PMID:23020549

  10. Dietary strawberry seed oil affects metabolite formation in the distal intestine and ameliorates lipid metabolism in rats fed an obesogenic diet

    PubMed Central

    Jurgoński, Adam; Fotschki, Bartosz; Juśkiewicz, Jerzy

    2015-01-01

    Objective To answer the question whether dietary strawberry seed oil rich in α-linolenic acid and linoleic acid (29.3 and 47.2% of total fatty acids, respectively) can beneficially affect disorders induced by the consumption of an obesogenic diet. Design Thirty-two male Wistar rats were randomly assigned to four groups of eight animals each and fed with a basal or obesogenic (high in fat and low in fiber) diet that contained either strawberry seed oil or an edible rapeseed oil. A two-way analysis of variance was then applied to assess the effects of diet and oil and the interaction between them. Results After 8 weeks of feeding, the obesogenic diet increased the body weight and the liver mass and fat content, whereas decreased the cecal acetate and butyrate concentration. This diet also altered the plasma lipid profile and decreased the liver sterol regulatory element-binding protein 1c (SREBP-1c) content. However, the lowest liver SREBP-1c content was observed in rats fed an obesogenic diet containing strawberry seed oil. Moreover, dietary strawberry seed oil decreased the cecal short-chain fatty acid concentrations (acetate, propionate, and butyrate) regardless of the diet type, whereas the cecal β-glucuronidase activity was considerably increased only in rats fed an obesogenic diet containing strawberry seed oil. Dietary strawberry seed oil also lowered the liver fat content, the plasma triglyceride level and the atherogenic index of plasma. Conclusions Strawberry seed oil has a potent lipid-lowering activity but can unfavorably affect microbial metabolism in the distal intestine. The observed effects are partly due to the synergistic action of the oil and the obesogenic diet. PMID:25636326

  11. Identification of Plants That Inhibit Lipid Droplet Formation in Liver Cells: Rubus suavissimus Leaf Extract Protects Mice from High-Fat Diet-Induced Fatty Liver by Directly Affecting Liver Cells

    PubMed Central

    Takahashi, Tomohiro; Sugawara, Wataru; Takiguchi, Yuya; Takizawa, Kento; Nakabayashi, Ami; Nakamura, Mitsuo; Nagano-Ito, Michiyo; Ichikawa, Shinichi

    2016-01-01

    Fatty liver disease is a condition in which abnormally large numbers of lipid droplets accumulate in liver cells. Fatty liver disease induces inflammation under conditions of oxidative stress and may result in cancer. To identify plants that protect against fatty liver disease, we examined the inhibitory effects of plant extracts on lipid droplet formation in mouse hepatoma cells. A screen of 98 water extracts of plants revealed 4 extracts with inhibitory effects. One of these extracts, Rubus suavissimus S. Lee (Tien-cha or Chinese sweet tea) leaf extract, which showed strong inhibitory effects, was tested in a mouse fatty liver model. In these mouse experiments, intake of the plant extract significantly protected mice against fatty liver disease without affecting body weight gain. Our results suggest that RSE directly affects liver cells and protects them from fatty liver disease. PMID:27429636

  12. Identification of Plants That Inhibit Lipid Droplet Formation in Liver Cells: Rubus suavissimus Leaf Extract Protects Mice from High-Fat Diet-Induced Fatty Liver by Directly Affecting Liver Cells.

    PubMed

    Takahashi, Tomohiro; Sugawara, Wataru; Takiguchi, Yuya; Takizawa, Kento; Nakabayashi, Ami; Nakamura, Mitsuo; Nagano-Ito, Michiyo; Ichikawa, Shinichi

    2016-01-01

    Fatty liver disease is a condition in which abnormally large numbers of lipid droplets accumulate in liver cells. Fatty liver disease induces inflammation under conditions of oxidative stress and may result in cancer. To identify plants that protect against fatty liver disease, we examined the inhibitory effects of plant extracts on lipid droplet formation in mouse hepatoma cells. A screen of 98 water extracts of plants revealed 4 extracts with inhibitory effects. One of these extracts, Rubus suavissimus S. Lee (Tien-cha or Chinese sweet tea) leaf extract, which showed strong inhibitory effects, was tested in a mouse fatty liver model. In these mouse experiments, intake of the plant extract significantly protected mice against fatty liver disease without affecting body weight gain. Our results suggest that RSE directly affects liver cells and protects them from fatty liver disease. PMID:27429636

  13. Cleavage of E-cadherin and β-catenin by calpain affects Wnt signaling and spheroid formation in suspension cultures of human pluripotent stem cells.

    PubMed

    Konze, Sarah A; van Diepen, Laura; Schröder, Anke; Olmer, Ruth; Möller, Hanna; Pich, Andreas; Weißmann, Robert; Kuss, Andreas W; Zweigerdt, Robert; Buettner, Falk F R

    2014-04-01

    The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell-cell and cell-extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell-cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that pharmacological

  14. Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells*

    PubMed Central

    Konze, Sarah A.; van Diepen, Laura; Schröder, Anke; Olmer, Ruth; Möller, Hanna; Pich, Andreas; Weißmann, Robert; Kuss, Andreas W.; Zweigerdt, Robert; Buettner, Falk F. R.

    2014-01-01

    The envisioned clinical and industrial use of human pluripotent stem cells and their derivatives has given major momentum to the establishment of suspension culture protocols that enable the mass production of cells. Understanding molecular changes accompanying the transfer from adherent to suspension culture is of utmost importance because this information can have a direct effect on the development of optimized culture conditions. In this study we assessed the gene expression of human embryonic stem cells and induced pluripotent stem cells grown in surface-adherent culture (two-dimensional) versus free-floating suspension culture spheroids (three-dimensional). We combined a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture with deep-sequencing-based transcriptomics. Cells in three-dimensional culture showed reduced expression of proteins forming structural components of cell–cell and cell–extracellular matrix junctions. However, fully unexpected, we found up-regulation of secreted inhibitors of the canonical Wnt signaling pathway and, concomitantly, a reduction in the level of active β-catenin and in the expression of Wnt target genes. In Western blot analyses the cysteine protease calpain was shown to cleave E-cadherin and β-catenin under three-dimensional culture conditions. Our data allowed the development of a model in which calpain cleavage of E-cadherin induces the disintegration of focal cell contacts and generates a 100-kDa E-cadherin fragment required for the formation of three-dimensional cell–cell contacts in spheroids. The parallel release of β-catenin and its potential activation by calpain cleavage are counterbalanced by the overexpression of soluble Wnt pathway inhibitors. According to this model, calpain has a key function in the interplay between E-cadherin and β-catenin-mediated intercellular adhesion and the canonical Wnt signaling pathway. Supporting this model, we show that

  15. Critical factors affecting the 3D microstructural formation in hybrid conductive adhesive materials studied by X-ray nano-tomography

    NASA Astrophysics Data System (ADS)

    Chen-Wiegart, Yu-Chen Karen; Figueroa-Santos, Miriam Aileen; Petrash, Stanislas; Garcia-Miralles, Jose; Wang, Jun

    2014-12-01

    Conductive adhesives are found favorable in a wide range of applications including a lead-free solder in micro-chips, flexible and printable electronics and enhancing the performance of energy storage devices. Composite materials comprised of metallic fillers and a polymer matrix are of great interest to be implemented as hybrid conductive adhesives. Here we investigated a cost-effective conductive adhesive material consisting of silver-coated copper as micro-fillers using synchrotron-based three-dimensional (3D) X-ray nano-tomography. The key factors affecting the quality and performance of the material were quantitatively studied in 3D on the nanometer scale for the first time. A critical characteristic parameter, defined as a shape-factor, was determined to yield a high-quality silver coating, leading to satisfactory performance. A `stack-and-screen' mechanism was proposed to elaborate such a phenomenon. The findings and the technique developed in this work will facilitate the future advancement of conductive adhesives to have a great impact in micro-electronics and other applications.Conductive adhesives are found favorable in a wide range of applications including a lead-free solder in micro-chips, flexible and printable electronics and enhancing the performance of energy storage devices. Composite materials comprised of metallic fillers and a polymer matrix are of great interest to be implemented as hybrid conductive adhesives. Here we investigated a cost-effective conductive adhesive material consisting of silver-coated copper as micro-fillers using synchrotron-based three-dimensional (3D) X-ray nano-tomography. The key factors affecting the quality and performance of the material were quantitatively studied in 3D on the nanometer scale for the first time. A critical characteristic parameter, defined as a shape-factor, was determined to yield a high-quality silver coating, leading to satisfactory performance. A `stack-and-screen' mechanism was proposed to

  16. Prediction and characterization of heat-affected zone formation in tin-bismuth alloys due to nickel-aluminum multilayer foil reaction

    NASA Astrophysics Data System (ADS)

    Hooper, R. J.; Davis, C. G.; Johns, P. M.; Adams, D. P.; Hirschfeld, D.; Nino, J. C.; Manuel, M. V.

    2015-06-01

    Reactive multilayer foils have the potential to be used as local high intensity heat sources for a variety of applications. Most of the past research effort concerning these materials have focused on understanding the structure-property relationships of the foils that govern the energy released during a reaction. To improve the ability of researchers to more rapidly develop technologies based on reactive multilayer foils, a deeper and more predictive understanding of the relationship between the heat released from the foil and microstructural evolution in the neighboring materials is needed. This work describes the development of a numerical model for the purpose of predicting heat affected zone size in substrate materials. The model is experimentally validated using a commercially available Ni-Al multilayer foils and alloys from the Sn-Bi binary system. To accomplish this, phenomenological models for predicting the variation of physical properties (i.e., thermal conductivity, density, and heat capacity) with temperature and composition in the Sn-Bi system were utilized using literature data.

  17. Prediction and characterization of heat-affected zone formation in tin-bismuth alloys due to nickel-aluminum multilayer foil reaction

    DOE PAGESBeta

    Hooper, R. J.; Davis, C. G.; Johns, P. M.; Adams, D. P.; Hirschfeld, D.; Nino, J. C.; Manuel, M. V.

    2015-06-26

    Reactive multilayer foils have the potential to be used as local high intensity heat sources for a variety of applications. In this study, most of the past research effort concerning these materials have focused on understanding the structure-property relationships of the foils that govern the energy released during a reaction. To improve the ability of researchers to more rapidly develop technologies based on reactive multilayer foils, a deeper and more predictive understanding of the relationship between the heat released from the foil and microstructural evolution in the neighboring materials is needed. This work describes the development of a numerical modelmore » for the purpose of predicting heat affected zone size in substrate materials. The model is experimentally validated using a commercially available Ni-Al multilayer foils and alloys from the Sn-Bi binary system. To accomplish this, phenomenological models for predicting the variation of physical properties (i.e., thermal conductivity, density, and heat capacity) with temperature and composition in the Sn-Bi system were utilized using literature data.« less

  18. Altered Subcellular Localization of Tumor-Specific Cyclin E Isoforms Affects Cyclin-Dependent Kinase 2 Complex Formation and Proteasomal Regulation

    PubMed Central

    Delk, Nikki A.; Hunt, Kelly K.; Keyomarsi, Khandan

    2009-01-01

    In tumors, alternative translation and posttranslational proteolytic cleavage of full-length cyclin E (EL) produces tumorigenic low molecular weight cyclin E (LMW-E) isoforms that lack a portion of the EL amino-terminus containing a nuclear localization sequence. Therefore, we hypothesized that LMW-E isoforms have altered subcellular localization. To explore our hypothesis, we compared EL versus LMW-E localization in cell lysates and in vivo using fractionation and protein complementation assays. Our results reveal that LMW-E isoforms preferentially accumulate in the cytoplasm where they bind the cyclin E kinase partner, cyclin-dependent kinase 2 (Cdk2), and have associated kinase activity. The nuclear ubiquitin ligase Fbw7 targets Cdk2-bound cyclin E for degradation; thus, we examined if altered subcellular localization affected LMW-E degradation. We found that cytoplasmic LMW-E/Cdk2 was less susceptible to Fbw7-mediated degradation. One implication of our findings is that altered LMW-E and LMW-E/Cdk2 subcellular localization may lead to aberrant LMW-E protein interactions, regulation, and activity, ultimately contributing to LMW-E tumorigenicity. PMID:19318554

  19. Critical factors affecting the 3D microstructural formation in hybrid conductive adhesive materials studied by X-ray nano-tomography.

    PubMed

    Chen-Wiegart, Yu-chen Karen; Figueroa-Santos, Miriam Aileen; Petrash, Stanislas; Garcia-Miralles, Jose; Wang, Jun

    2015-01-21

    Conductive adhesives are found favorable in a wide range of applications including a lead-free solder in micro-chips, flexible and printable electronics and enhancing the performance of energy storage devices. Composite materials comprised of metallic fillers and a polymer matrix are of great interest to be implemented as hybrid conductive adhesives. Here we investigated a cost-effective conductive adhesive material consisting of silver-coated copper as micro-fillers using synchrotron-based three-dimensional (3D) X-ray nano-tomography. The key factors affecting the quality and performance of the material were quantitatively studied in 3D on the nanometer scale for the first time. A critical characteristic parameter, defined as a shape-factor, was determined to yield a high-quality silver coating, leading to satisfactory performance. A 'stack-and-screen' mechanism was proposed to elaborate such a phenomenon. The findings and the technique developed in this work will facilitate the future advancement of conductive adhesives to have a great impact in micro-electronics and other applications. PMID:25474162

  20. Prediction and characterization of heat-affected zone formation in tin-bismuth alloys due to nickel-aluminum multilayer foil reaction

    SciTech Connect

    Hooper, R. J.; Davis, C. G.; Johns, P. M.; Adams, D. P.; Hirschfeld, D.; Nino, J. C.; Manuel, M. V.

    2015-06-26

    Reactive multilayer foils have the potential to be used as local high intensity heat sources for a variety of applications. In this study, most of the past research effort concerning these materials have focused on understanding the structure-property relationships of the foils that govern the energy released during a reaction. To improve the ability of researchers to more rapidly develop technologies based on reactive multilayer foils, a deeper and more predictive understanding of the relationship between the heat released from the foil and microstructural evolution in the neighboring materials is needed. This work describes the development of a numerical model for the purpose of predicting heat affected zone size in substrate materials. The model is experimentally validated using a commercially available Ni-Al multilayer foils and alloys from the Sn-Bi binary system. To accomplish this, phenomenological models for predicting the variation of physical properties (i.e., thermal conductivity, density, and heat capacity) with temperature and composition in the Sn-Bi system were utilized using literature data.

  1. Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions

    PubMed Central

    Karja, Ni Wayan Kurniani; Kikuchi, Kazuhiro; Fahrudin, Mokhamad; Ozawa, Manabu; Somfai, Tamás; Ohnuma, Katsuhiko; Noguchi, Junko; Kaneko, Hiroyuki; Nagai, Takashi

    2006-01-01

    Background Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined. Methods In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6. Results Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA

  2. Vitamin A deprivation affects the progression of the spermatogenic wave and initial formation of the blood-testis barrier, resulting in irreversible testicular degeneration in mice.

    PubMed

    Chihara, Masataka; Otsuka, Saori; Ichii, Osamu; Kon, Yasuhiro

    2013-12-17

    The blood testis-barrier (BTB) is essential for maintaining homeostasis in the seminiferous epithelium. Although many studies have reported that vitamin A (VA) is required for the maintenance of spermatogenesis, the relationships between the BTB, spermatogenesis and VA have not been elucidated. In this study, we analyzed BTB assembly and spermatogenesis in the testes of mice fed the VA-deficient (VAD) diet from the prepubertal period to adulthood. During the prepubertal period, no changes were observed in the initiation and progression of the first spermatogenic wave in mice fed the VAD diet. However, the numbers of preleptotene/leptotene spermatocytes derived from the second spermatogenic wave onwards were decreased, and initial BTB formation was also delayed, as evidenced by the decreased expression of mRNAs encoding BTB components and VA signaling molecules. From 60 days postpartum, mice fed the VAD diet exhibited apoptosis of germ cells, arrest of meiosis, disruption of the BTB, and dramatically decreased testis size. Furthermore, vacuolization and calcification were observed in the seminiferous epithelium of adult mice fed the VAD diet. Re-initiation of spermatogenesis by VA replenishment in adult mice fed the VAD diet rescued BTB assembly after when the second spermatogenic wave initiated from the arrested spermatogonia reached the preleptotene/leptotene spermatocytes. These results suggested that BTB integrity was regulated by VA metabolism with meiotic progression and that the impermeable BTB was required for persistent spermatogenesis rather than meiotic initiation. In conclusion, consumption of the VAD diet led to critical defects in spermatogenesis progression and altered the dynamics of BTB assembly. PMID:23934320

  3. Vitamin A Deprivation Affects the Progression of the Spermatogenic Wave and Initial Formation of the Blood-testis Barrier, Resulting in Irreversible Testicular Degeneration in Mice

    PubMed Central

    CHIHARA, Masataka; OTSUKA, Saori; ICHII, Osamu; KON, Yasuhiro

    2013-01-01

    Abstract The blood testis-barrier (BTB) is essential for maintaining homeostasis in the seminiferous epithelium. Although many studies have reported that vitamin A (VA) is required for the maintenance of spermatogenesis, the relationships between the BTB, spermatogenesis and VA have not been elucidated. In this study, we analyzed BTB assembly and spermatogenesis in the testes of mice fed the VA-deficient (VAD) diet from the prepubertal period to adulthood. During the prepubertal period, no changes were observed in the initiation and progression of the first spermatogenic wave in mice fed the VAD diet. However, the numbers of preleptotene/leptotene spermatocytes derived from the second spermatogenic wave onwards were decreased, and initial BTB formation was also delayed, as evidenced by the decreased expression of mRNAs encoding BTB components and VA signaling molecules. From 60 days postpartum, mice fed the VAD diet exhibited apoptosis of germ cells, arrest of meiosis, disruption of the BTB, and dramatically decreased testis size. Furthermore, vacuolization and calcification were observed in the seminiferous epithelium of adult mice fed the VAD diet. Re-initiation of spermatogenesis by VA replenishment in adult mice fed the VAD diet rescued BTB assembly after when the second spermatogenic wave initiated from the arrested spermatogonia reached the preleptotene/leptotene spermatocytes. These results suggested that BTB integrity was regulated by VA metabolism with meiotic progression and that the impermeable BTB was required for persistent spermatogenesis rather than meiotic initiation. In conclusion, consumption of the VAD diet led to critical defects in spermatogenesis progression and altered the dynamics of BTB assembly. PMID:23934320

  4. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Main, Heather; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1-60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346003

  5. On the actual variety of plate dynamical mechanisms and how mantle evolution affected them through time, from core formation to the Indian collision

    NASA Astrophysics Data System (ADS)

    Osmaston, Miles

    2010-05-01

    If horizontal plate motions were driven by thermal convection of the mantle, they would display the action of slow-to-change body forces. Yet rapid changes of spreading rate and direction, and ridge jumps, are well-documented for the past 130Ma. Also convection cannot readily cause rotations of a plate (e.g. Africa) about a pole within the plate or near it. And plate motions, especially that of India, scarcely fit a convective pattern. To address these problems we look first at mantle motivation at both ends of earth history, beginning with core formation. I then introduce 3 important properties of mantle materials, whose neglect by mantle modellers has surely impaired the value of their work, but whose recognition illuminates the present plate dynamical situation and provides the Earth with a heat engine that is not thermal convection. Finally I sketch the intervening changes in behaviour over time, the sharpest of which brought about the rise of atmospheric oxygen at ~2.25Ga. Core formation. As the very high specific angular momentum of mean planetary material (>105-fold relative to solar) can only be achieved if the planets were wholly accreted in presence of the nebula [1], the iron percolation model is ruled out, because it takes too long. This validates the A.E.Ringwood model (1960-1978) involving nebular H reaction with erupting FeO. The iron then loads the downgoing limb of what is then not a truly thermal convection system. Huge volumes of reaction water were produced, giving the early Earth a wet mantle, a (diminishing) feature that we'll see has constrained mantle behaviour ever since. Plate dynamics since 150Ma. Multiple plate dynamical evidence [2], which will be rapidly re-presented here, shows that currently (a) the Earth has a 2-layer mantle system with a boundary at ~660km and (b) that most cratons have tectospheric keels that reach right to that boundary, or nearly so. The argument is the simple and persuasive one (even to seismologists) of mantle

  6. Effects of long-term treatment with bovine somatotropin on follicular dynamics and subsequent oocyte and blastocyst yield in an OPU-IVF program.

    PubMed

    Bols, P E; Ysebaert, M T; Lein, A; Coryn, M; Van Soom, A; de Kruif, A

    1998-04-01

    Fourteen Holstein-Friesian heifers between 15 and 22 months of age with normal reproductive tracts were used in an experimental set up to investigate the effect of a long term rBST-treatment on the follicular population prior to transvaginal ovum pick-up (OPU). The estrous cycle of the animals was synchronized by means of a double injection of 2 ml cloprostenol 11 days apart. The heifers were divided in 2 equal groups (n = 7) in which the animals had the same average body weight, one group receiving a weekly subcutaneous injection of 640 mg recombinantly derived bovine somatotropin (rBST) on Mondays (rBST-treatment group) and a control group, being injected with 10 ml of saline. Heifers in both groups were submitted to OPU twice a week on Mondays and Thursdays using a 5 Mhz transducer and a disposable, 55 mm long, 20-g short bevelled needle at a vacuum pressure corresponding to approximately 13 ml water/min. The experimental period lasted for 10 weeks (April to June), each animal receiving a total of 10 injections and being submitted to OPU for 20 times. Oocytes were subsequently matured and cultured in a separate drop per cow following conventional IVF procedures. A blood sample was taken on heparin immediately after each OPU session, for determination of blood progesterone concentrations to assess the influence of treatment and OPU procedure on the cow's estrous cycle. Although results show a significant increase in the total number of follicles and medium sized follicles in the rBST-treated group, no statistically significant different number of retrieved oocytes between the rBST-treated and nontreated group could be detected. The average number of retrieved oocytes per session per cow was comparable, being 6.4 for the treated and 6.0 for the control group. Additionally, the average number of blastocysts per cow per session did not differ significantly between groups, being 1.41 in the rBST-treated group and 1.53 in the control group. The number of cultured

  7. Hormad1 mutation disrupts synaptonemal complex formation, recombination, and chromosome segregation in mammalian meiosis.

    PubMed

    Shin, Yong-Hyun; Choi, Youngsok; Erdin, Serpil Uckac; Yatsenko, Svetlana A; Kloc, Malgorzata; Yang, Fang; Wang, P Jeremy; Meistrich, Marvin L; Rajkovic, Aleksandar

    2010-11-01

    Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell-specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1(-/) (-)) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1(-/) (-) testes and ovaries as shown by the drastic decrease in the γH2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with γH2AX to the sex body during pachytene. BRCA1, ATR, and γH2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes γH2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1(-/) (-) ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1(-/) (-) oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing. PMID:21079677

  8. Plant Hormones: How They Affect Root Formation.

    ERIC Educational Resources Information Center

    Reinhard, Diana Hereda

    This science study aid, produced by the U.S. Department of Agriculture, includes a series of plant rooting activities for secondary science classes. The material in the pamphlet is written for students and includes background information on plant hormones, a vocabulary list, and five learning activities. Objectives, needed materials, and…

  9. Cryosurvival of in vitro produced embryos as affected by health status effect of oocyte donor cow.

    PubMed

    Hosseini, S M; Hajian, M; Asgari, V; Forouzanfar, M; Ostadhosseini, S; Moulavi, F; Abedi, P; Kiani, M; Vash N, N T; Safahani-Langroodi, M; Nasr-Esfahani, M H

    2013-01-01

    In vitro embryo production and embryo vitrification of genetically superior cows that culled inevitably due to health problems can accelerate genetic progress. This study was carried out to investigate whether maternal age and health status effects of high genetic merit cows affect cryosurvival and developmental competence of IVP embryos. In this sense, the effects of ageing and four common culling causes of dairy cows [repeat breeding (RPB), udder problems (UPM), chronic endometritis (CRE), and lameness (LAM)] on in vitro embryo development, and in vivo developmental competence after embryo vitrification were evaluated. The mean number of oocytes obtained per cow did not vary significantly between donors indifferent groups. Cleavage rates in RPB (86.0+/-4.2%), SEN (81.3+/-2.5%) and CRE (77.6+/-6.3%) cows which were comparable to control (95.9+/-1.5%) but were significantly higher than the related rate of UPM donors (50.6+/-2.6%). Importantly, there was no significant difference between the blastocyst rates of different groups. Mean overall survival rate was not different between the groups and was not affected by the blastocyst production rate. There was no significant difference between pregnancy rates of different groups. The results of the present study indicated that in cattle, neither ageing, nor these four diseases affect ovarian potential in terms of the yield and quality of in vitro embryo development. PMID:24441373

  10. Microtubule organisation, pronuclear formation and embryonic development of mouse oocytes after intracytoplasmic sperm injection or parthenogenetic activation and then slow-freezing with 1, 2-propanediol.

    PubMed

    Li, Dun-Gao; Zhu, Yan; Xing, Feng-Ying; Li, Shan-Gang; Chen, Xue-Jin; Jiang, Man-Xi

    2013-01-01

    The goal of this study was to investigate the effect of cryopreservation on oocytes at different times after intracytoplasmic sperm injection (ICSI) and parthenogenetic activation. The study was performed in mouse oocytes fertilised by ICSI, or in artificially-activated oocytes, which were cryopreserved immediately, one hour or five hours later through slow-freezing. After thawing, the rates of survival, fertilisation-activation, embryonic development of oocytes-zygotes and changes in the cytoskeleton and ploidy were observed. Our results reveal a significant difference in survival rates of 0-, 1- and 5-h cryopreserved oocytes following ICSI and artificial activation. Moreover, significant differences in two pronuclei (PN) development existed between the 0-, 1- and 5-h groups of oocytes frozen after ICSI, while the rates of two-PN development of activated oocytes were different between the 1-h and 5-h groups. Despite these initial differences, there was no difference in the rate of blastocyst formation from two-PN zygotes following ICSI or artificial activation. However, compared with ICSI or artificially-activated oocytes cryopreserved at 5h, many oocytes from the 0- and 1-h cryopreservation groups developed to zygotes with abnormal ploidy; this suggests that too little time before cryopreservation can result in some activated oocytes forming abnormal ploidy. However, our results also demonstrate that spermatozoa can maintain normal fertilisation capacity in frozen ICSI oocytes and the procedure of freeze-thawing did not affect the later development of zygotes. PMID:23594385

  11. Affective Learning.

    ERIC Educational Resources Information Center

    Brown, Charles T.

    This paper addresses itself to the question, "What does feeling have to do with knowing?" Two movements in affective education are discussed which have come into focus in recent years and which attempt to define the relationship between knowing and feeling. The first, a conscious application of the role of arousal in learning, emphasizes arousal…

  12. [Affective dependency].

    PubMed

    Scantamburlo, G; Pitchot, W; Ansseau, M

    2013-01-01

    Affective dependency is characterized by emotional distress (insecure attachment) and dependency to another person with a low self-esteem and reassurance need. The paper proposes a reflection on the definition of emotional dependency and the confusion caused by various denominations. Overprotective and authoritarian parenting, cultural and socio-environmental factors may contribute to the development of dependent personality. Psychological epigenetic factors, such as early socio-emotional trauma could on neuronal circuits in prefronto-limbic regions that are essential for emotional behaviour.We also focus on the interrelations between dependent personality, domestic violence and addictions. The objective for the clinician is to propose a restoration of self-esteem and therapeutic strategies focused on autonomy. PMID:23888587

  13. Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor.

    PubMed

    Choi, Bom-Ie; Harvey, Alexandra J; Green, Mark P

    2016-01-01

    Increasing evidence supports an association between exposure to endocrine disruptors, such as the xenoestrogen bisphenol A (BPA), a commonly used plasticiser, and the developmental programming of offspring health. To date however animal studies to investigate a direct causal have mainly focussed on supra-environmental BPA concentrations, without investigating the effect on the early embryo. In this study we investigated the effect of acute BPA exposure (days 3.5 to 7.5 post-fertilisation) at environmentally relevant concentrations (1 and 10 ng/mL) on in vitro bovine embryo development, quality and metabolism. We then examined whether culturing embryos in the presence of the oestrogen receptor inhibitor fulvestrant could negate effects of BPA and 17β-oestradiol (E2). Exposure to BPA or E2 (10 ng/mL) decreased blastocyst rate and the percentage of transferrable quality embryos, without affecting cell number, lineage allocation or metabolic gene expression compared to untreated embryos. Notably, blastocysts exposed to BPA and E2 (10 ng/mL) displayed an increase in glucose consumption. The presence of fulvestrant however negated the adverse developmental and metabolic effects, suggesting BPA elicits its effects via oestrogen-mediated pathways. This study demonstrates that even acute exposure to an environmentally relevant BPA concentration can affect early embryo development and metabolism. These may have long-term health consequences on an individual. PMID:27384909

  14. Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor

    PubMed Central

    Choi, Bom-Ie; Harvey, Alexandra J.; Green, Mark P.

    2016-01-01

    Increasing evidence supports an association between exposure to endocrine disruptors, such as the xenoestrogen bisphenol A (BPA), a commonly used plasticiser, and the developmental programming of offspring health. To date however animal studies to investigate a direct causal have mainly focussed on supra-environmental BPA concentrations, without investigating the effect on the early embryo. In this study we investigated the effect of acute BPA exposure (days 3.5 to 7.5 post-fertilisation) at environmentally relevant concentrations (1 and 10 ng/mL) on in vitro bovine embryo development, quality and metabolism. We then examined whether culturing embryos in the presence of the oestrogen receptor inhibitor fulvestrant could negate effects of BPA and 17β-oestradiol (E2). Exposure to BPA or E2 (10 ng/mL) decreased blastocyst rate and the percentage of transferrable quality embryos, without affecting cell number, lineage allocation or metabolic gene expression compared to untreated embryos. Notably, blastocysts exposed to BPA and E2 (10 ng/mL) displayed an increase in glucose consumption. The presence of fulvestrant however negated the adverse developmental and metabolic effects, suggesting BPA elicits its effects via oestrogen-mediated pathways. This study demonstrates that even acute exposure to an environmentally relevant BPA concentration can affect early embryo development and metabolism. These may have long-term health consequences on an individual. PMID:27384909

  15. Derivation of Huntington Disease affected Genea017 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea017 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 40 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, genetic analysis confirmed a 46, XY karyotype and male allele pattern through CGH and STR analysis. The hESC line had pluripotent cell morphology, 87% of cells expressed Nanog, 95% Oct4, 88% Tra1-60 and 99% SSEA4, gave a PluriTest pluripotency score of 34.74, novelty of 1.27, demonstrated alkaline phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346022

  16. Derivation of FSHD1 affected human embryonic stem cell line Genea050.

    PubMed

    Dumevska, Biljana; Chami, Omar; Main, Heather; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea050 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying a deletion in 4q35 with only 5 D4Z4 repeats by PGD linkage analysis, indicative of FSHD1. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 92% of cells expressed Nanog, 97% Oct4, 79% Tra1-60 and 99% SSEA4, gave a Pluritest Pluripotency score of 25.45, Novelty of 1.45 demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346025

  17. Derivation of Huntington Disease affected Genea018 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Main, Heather; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea018 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 46 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 75% of cells expressed Nanog, 91% Oct4, 73% Tra1-60 and 96% SSEA4, gave a Pluritest pluripotency score of 31.12, Novelty of 1.45, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346005

  18. Affect, Citizenship, Politics: Implications for Education

    ERIC Educational Resources Information Center

    Zembylas, Michalinos

    2009-01-01

    This article analyses the role of emotions in the constitution of political identity and boundary formation, and discusses the educational implications of that analysis in the context of citizenship education. The author begins by examining how affect is fundamental to the formation of borders, nationhood and citizenship, and discusses the role of…

  19. Oxycline formation induced by Fe(II) oxidation in a water reservoir affected by acid mine drainage modeled using a 2D hydrodynamic and water quality model - CE-QUAL-W2.

    PubMed

    Torres, Ester; Galván, Laura; Cánovas, Carlos Ruiz; Soria-Píriz, Sara; Arbat-Bofill, Marina; Nardi, Albert; Papaspyrou, Sokratis; Ayora, Carlos

    2016-08-15

    The Sancho reservoir is an acid mine drainage (AMD)-contaminated reservoir located in the Huelva province (SW Spain) with a pH close to 3.5. The water is only used for a refrigeration system of a paper mill. The Sancho reservoir is holomictic with one mixing period per year in the winter. During this mixing period, oxygenated water reaches the sediment, while under stratified conditions (the rest of the year) hypoxic conditions develop at the hypolimnion. A CE-QUAL-W2 model was calibrated for the Sancho Reservoir to predict the thermocline and oxycline formation, as well as the salinity, ammonium, nitrate, phosphorous, algal, chlorophyll-a, and iron concentrations. The version 3.7 of the model does not allow simulating the oxidation of Fe(II) in the water column, which limits the oxygen consumption of the organic matter oxidation. However, to evaluate the impact of Fe(II) oxidation on the oxycline formation, Fe(II) has been introduced into the model based on its relationship with labile dissolved organic matter (LDOM). The results show that Fe oxidation is the main factor responsible for the oxygen depletion in the hypolimnion of the Sancho Reservoir. The limiting factors for green algal growth have also been studied. The model predicted that ammonium, nitrate, and phosphate were not limiting factors for green algal growth. Light appeared to be one of the limiting factors for algal growth, while chlorophyll-a and dissolved oxygen concentrations could not be fully described. We hypothesize that dissolved CO2 is one of the limiting nutrients due to losses by the high acidity of the water column. The sensitivity tests carried out support this hypothesis. Two different remediation scenarios have been tested with the calibrated model: 1) an AMD passive treatment plant installed at the river, which removes completely Fe, and 2) different depth water extractions. If no Fe was introduced into the reservoir, water quality would significantly improve in only two years

  20. Formative Assessment

    ERIC Educational Resources Information Center

    Technology & Learning, 2005

    2005-01-01

    In today's climate of high-stakes testing and accountability, educators are challenged to continuously monitor student progress to ensure achievement. This article details how formative assessment helps educators meet this challenge and to ensure achievement. Formative assessment can influence learning and support achievement, allowing teachers…

  1. Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells.

    PubMed Central

    Gutgsell, N; Englund, N; Niu, L; Kaya, Y; Lane, B G; Ofengand, J

    2000-01-01

    Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102-112) established that TruB, a pseudouridine (psi) synthase from Escherichia coli, was able to make psi55 in tRNA transcripts but not in transcripts of full-length or fragmented 16S or 23S ribosomal RNAs. By deletion of the truB gene, we now show that TruB is the only protein in E. coli able to make psi55 in vivo. Lack of TruB and psi55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type. The negative selection did not appear to be acting at either the exponential or stationary phase. Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 degrees C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to approximately 0.2% of its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB. Direct analysis showed that this mutant did not make psi55 under the conditions of the competition experiment. Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of psi55. PMID:11142385

  2. How Obesity Affects Tendons?

    PubMed

    Abate, Michele; Salini, Vincenzo; Andia, Isabel

    2016-01-01

    Several epidemiological and clinical observations have definitely demonstrated that obesity has harmful effects on tendons. The pathogenesis of tendon damage is multi-factorial. In addition to overload, attributable to the increased body weight, which significantly affects load-bearing tendons, systemic factors play a relevant role. Several bioactive peptides (chemerin, leptin, adiponectin and others) are released by adipocytes, and influence tendon structure by means of negative activities on mesenchymal cells. The ensuing systemic state of chronic, sub-clinic, low-grade inflammation can damage tendon structure. Metabolic disorders (diabetes, impaired glucose tolerance, and dislipidemia), frequently associated with visceral adiposity, are concurrent pathogenetic factors. Indeed, high glucose levels increase the formation of Advanced Glycation End-products, which in turn form stable covalent cross-links within collagen fibers, modifying their structure and functionality.Sport activities, so useful for preventing important cardiovascular complications, may be detrimental for tendons if they are submitted to intense acute or chronic overload. Therefore, two caution rules are mandatory: first, to engage in personalized soft training program, and secondly to follow regular check-up for tendon pathology. PMID:27535258

  3. Affective processing requires awareness.

    PubMed

    Lähteenmäki, Mikko; Hyönä, Jukka; Koivisto, Mika; Nummenmaa, Lauri

    2015-04-01

    Studies using backward masked emotional stimuli suggest that affective processing may occur outside visual awareness and imply primacy of affective over semantic processing, yet these experiments have not strictly controlled for the participants' awareness of the stimuli. Here we directly compared the primacy of affective versus semantic categorization of biologically relevant stimuli in 5 experiments (n = 178) using explicit (semantic and affective discrimination; Experiments 1-3) and implicit (semantic and affective priming; Experiments 4-5) measures. The same stimuli were used in semantic and affective tasks. Visual awareness was manipulated by varying exposure duration of the masked stimuli, and subjective level of stimulus awareness was measured after each trial using a 4-point perceptual awareness scale. When participants reported no awareness of the stimuli, semantic and affective categorization were at chance level and priming scores did not differ from zero. When participants were even partially aware of the stimuli, (a) both semantic and affective categorization could be performed above chance level with equal accuracy, (b) semantic categorization was faster than affective categorization, and (c) both semantic and affective priming were observed. Affective categorization speed was linearly dependent on semantic categorization speed, suggesting dependence of affective processing on semantic recognition. Manipulations of affective and semantic categorization tasks revealed a hierarchy of categorization operations beginning with basic-level semantic categorization and ending with superordinate level affective categorization. We conclude that both implicit and explicit affective and semantic categorization is dependent on visual awareness, and that affective recognition follows semantic categorization. PMID:25559654

  4. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

    PubMed

    Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

    2016-06-01

    Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene. PMID:26197710

  5. Galaxy formation

    SciTech Connect

    Silk, J.

    1984-11-01

    Implications of the isotropy of the cosmic microwave background on large and small angular scales for galaxy formation are reviewed. In primeval adiabatic fluctuations, a universe dominated by cold, weakly interacting nonbaryonic matter, e.g., the massive photino is postulated. A possible signature of photino annihilation in our galactic halo involves production of cosmic ray antiprotons. If the density is near its closure value, it is necessary to invoke a biasing mechanism for suppressing galaxy formation throughout most of the universe in order to reconcile the dark matter density with the lower astronomical determinations of the mean cosmological density. A mechanism utilizing the onset of primordial massive star formation to strip gaseous protogalaxies is described. Only the densest, early collapsing systems form luminous galaxies. (ESA)

  6. Comet formation

    NASA Astrophysics Data System (ADS)

    Blum, J.

    2014-07-01

    There has been vast progress in our understanding of planetesimal formation over the past decades, owing to a number of laboratory experiments as well as to refined models of dust and ice agglomeration in protoplanetary disks. Coagulation rapidly forms cm-sized ''pebbles'' by direct sticking in collisions at low velocities (Güttler et al. 2010; Zsom et al. 2010). For the further growth, two model approaches are currently being discussed: (1) Local concentration of pebbles in nebular instabilities until gravitational instability occurs (Johansen et al. 2007). (2) A competition between fragmentation and mass transfer in collisions among the dusty bodies, in which a few ''lucky winners'' make it to planetesimal sizes (Windmark et al. 2012a,b; Garaud et al. 2013). Predictions of the physical properties of the resulting bodies in both models allow a distinction of the two formation scenarios of planetesimals. In particular, the tensile strength (i.e, the inner cohesion) of the planetesimals differ widely between the two models (Skorov & Blum 2012; Blum et al. 2014). While model (1) predicts tensile strengths on the order of ˜ 1 Pa, model (2) results in rather compactified dusty bodies with tensile strengths in the kPa regime. If comets are km-sized survivors of the planetesimal-formation era, they should in principle hold the secret of their formation process. Water ice is the prime volatile responsible for the activity of comets. Thermophysical models of the heat and mass transport close to the comet-nucleus surface predict water-ice sublimation temperatures that relate to maximum sublimation pressures well below the kPa regime predicted for formation scenario (2). Model (1), however, is in agreement with the observed dust and gas activity of comets. Thus, a formation scenario for cometesimals involving gravitational instability is favored (Blum et al. 2014).

  7. Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

    PubMed Central

    THONGKITTIDILOK, Chommanart; THARASANIT, Theerawat; SONGSASEN, Nucharin; SANANMUANG, Thanida; BUARPUNG, Sirirak; TECHAKUMPHU, Mongkol

    2015-01-01

    This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages. PMID:25985792

  8. Planet Formation

    NASA Astrophysics Data System (ADS)

    Klahr, Hubert; Brandner, Wolfgang

    2011-02-01

    1. Historical notes on planet formation Bodenheimer; 2. The formation and evolution of planetary systems Bouwman et al.; 3. Destruction of protoplanetary disks by photoevaporation Richling, Hollenbach and Yorke; 4. Turbulence in protoplanetary accretion disks Klahr, Rozyczka, Dziourkevitch, Wunsch and Johansen; 5. The origin of solids in the early solar system Trieloff and Palme; 6. Experiments on planetesimal formation Wurm and Blum; 7. Dust coagulation in protoplanetary disks Henning, Dullemond, Wolf and Dominik; 8. The accretion of giant planet cores Thommes and Duncan; 9. Planetary transits: direct vision of extrasolar planets Lecavelier des Etangs and Vidal-Madjar; 10. The core accretion - gas capture model Hubickyj; 11. Properties of exoplanets Marcy, Fischer, Butler and Vogt; 12. Giant planet formation: theories meet observations Boss; 13. From hot Jupiters to hot Neptures … and below Lovis, Mayor and Udry; 14. Disk-planet interaction and migration Masset and Kley; 15. The Brown Dwarf - planet relation Bate; 16. From astronomy to astrobiology Brandner; 17. Overview and prospective Lin.

  9. Can Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    In quiescent environments (microgravity, capillary tubes, gels) formation of a depletion zone is to be expected, due either to limited sedimentation, density driven convection or a combination of both. The formation of a depletion zone can: Modify solution supersaturation near crystal; Give rise to impurity partitioning. It is conjectured that both supersaturation and impurity partitioning affect protein crystal quality and size. Further detailed investigations on various proteins are needed to assess above hypothesis.

  10. Affective Dynamics in Psychopathology

    PubMed Central

    Trull, Timothy J.; Lane, Sean P.; Koval, Peter; Ebner-Priemer, Ulrich W.

    2016-01-01

    We discuss three varieties of affective dynamics (affective instability, emotional inertia, and emotional differentiation). In each case, we suggest how these affective dynamics should be operationalized and measured in daily life using time-intensive methods, like ecological momentary assessment or ambulatory assessment, and recommend time-sensitive analyses that take into account not only the variability but also the temporal dependency of reports. Studies that explore how these affective dynamics are associated with psychological disorders and symptoms are reviewed, and we emphasize that these affective processes are within a nexus of other components of emotion regulation.

  11. Formation testers

    SciTech Connect

    Brieger, E.

    1980-07-01

    A description is given of a method for use in obtaining multiple pressure tests of an earth formation traversed by a well bore by use of a sidewall fluid sampler well tool which has a fluid pressure sampling chamber in the well tool in open fluid communication with a pad sealing means, comprising the steps of: for one selected level in a well bore, moving a pad sealing means on the well tool into engagement with the wall of a well bore and isolating a wall segment of the earth formation; after the pad sealing means engges the wall segment of the earth formation, generating a hydraulic pressure in the well tool and applying said hydraulic pressure to said fluid pressure sampling chamber for increasing the volume of said fluid pressure sampling chamber thereby to dray a fluid sample from the earth formation engaged by the pad sealing means into the fluid pressure sampling chamber, sensing the pressure of said fluid sample as it is drawn into the fluid pressure sampling chamber while the volume of the sampling chamber is being increased, relieving the hydraulic pressure in the well tool with respect to said fluid pressur sampling chamber for decreasing the volume of said fluid pressure sampling chamber thereby to contact the sampling chamber to dischrge the fluid sample through the pad sealing means; retracting the sealing pad means and, after retrction of sealing pad means from engagement from the wall of the well bore, moving the well tool to a second location at another level in the well bore and, at the second location, repeating the steps of the method performed at the one selected level for obtaining another fluid sample and pressure sensing at said second location.

  12. Drugs affecting the eye.

    PubMed

    Taylor, F

    1985-08-01

    This discussion reviews drugs that affect the eye, including antihyperglycemic agents; corticosteroids; antirheumatic drugs (quinolines, indomethacin, and allopurinol); psychiatric drugs (phenothiazine, thioridazine, and chlorpromazine); drugs used in cardiology (practolol, amiodarone, and digitalis gylcosides); drugs implicated in optic neuritis and atrophy, drugs with an anticholinergic action; oral contraceptives (OCs); and topical drugs and systemic effects. Refractive changes, either myopic or hypermetropic, can occur as a result of hyperglycemia, and variation in vision is sometimes a presenting symptom in diabetes mellitus. If it causes a change in the refraction, treatment of hyperglycemia almost always produces a temporary hypermetropia. A return to the original refractive state often takes weeks, sometimes months. There is some evidence that patients adequately treated with insulin improve more rapidly than those taking oral medication. Such patients always should be referred for opthalmological evaluation as other factors might be responsible, but it might not be possible to order the appropriate spectacle correction for some time. The most important ocular side effect of the systemic adiministration of corticosteroids is the formation of a posterior subcapsular cataract. Glaucoma also can result from corticosteroids, most often when they are applied topically. Corticosteroids have been implicated in the production of benign intracranial hypertension, which is paradoxical because they also are used in its treatment. The most important side effect of drugs such as chloroquine and hydroxychloroquine is an almost always irreversible maculopathy with resultant loss of central vision. Corneal and retinal changes similar to those caused by the quinolines have been reported with indomethacin, but there is some question about a cause and effect relationship. The National Registry of Drug Induced Ocular Side Effects in the US published 30 case histories of

  13. Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes.

    PubMed

    Fukui, Y; Ono, H

    1989-07-01

    Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro. PMID:2760879

  14. Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

    PubMed Central

    Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

    2016-01-01

    High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

  15. Hail Formation in Florida

    NASA Astrophysics Data System (ADS)

    Stanley, Matthew

    Hail poses a substantial threat to life and property in the state of Florida. These losses could be minimized through better understanding of the relationships between atmospheric variables that impact hail formation in Florida. Improving hail forecasting in Florida requires analyzing a number of meteorological parameters and synoptic data related to hail formation. NOAA archive data was retrieved to create a database that was used to categorize text files of hail days. The text files were entered into the National Oceanic and Atmospheric Administration Earth System Research Laboratory website to create National Centers for Environmental Prediction/National Center for Atmospheric Research Reanalysis maps of atmospheric variables for Florida hail days as well as days leading to the hail event. These data were then analyzed to determine the relationship between variables that affect hail formation, in general, across different regions and seasons in Florida using Statistical Product and Service Solutions. The reasoning for the differing factors affecting hail formation between regions, seasons and hail sizes were discussed, as well as forecasting suggestions relating to region and month in Florida. The study found that the majority of all hail that occurs in Florida is during the wet season. A low Lifted Index, high Precipitable Water and lower than average Sea Level Pressure, in most cases, is present during hail days in Florida. Furthermore, results show that Vector Wind magnitude increases as hail size increases. Additionally, several atmospheric variables useful to studying hail events, such as Lifted Index, Precipitable Water, Sea Level Pressure, Vector Wind and Temperature have significant correlations with each other depending on the region and season being observed. Strong correlations between low Lifted Index, high Precipitable Water values and the occurrence of hail events are discussed, as well as the relationship between temperature anomalies at various

  16. Amphiplex Formation

    NASA Astrophysics Data System (ADS)

    Petersen, Shannon; Laaser, Jennifer; Lodge, Timothy

    2015-03-01

    Polymer-micelle complexes are currently under heavy investigation due to their potential applications in targeted drug delivery and gene therapy, yet the dynamics of the complex formation is still relatively unstudied. By varying the ratios of poly(styrene sulfonate) chains and cationic poly(dimethylaminoethyl methacrylate)-b-poly(styrene) micelles and the ionic strength of the system, we created a variety of complex configurations of different sizes and charges. The complexes were characterized dynamic light scattering and zeta potential measurements which provided information regarding the hydrodynamic radius, distribution of sizes, and effective charge.

  17. Isolating Triggered Star Formation

    SciTech Connect

    Barton, Elizabeth J.; Arnold, Jacob A.; Zentner, Andrew R.; Bullock, James S.; Wechsler, Risa H.; /KIPAC, Menlo Park /SLAC

    2007-09-12

    Galaxy pairs provide a potentially powerful means of studying triggered star formation from galaxy interactions. We use a large cosmological N-body simulation coupled with a well-tested semi-analytic substructure model to demonstrate that the majority of galaxies in close pairs reside within cluster or group-size halos and therefore represent a biased population, poorly suited for direct comparison to 'field' galaxies. Thus, the frequent observation that some types of galaxies in pairs have redder colors than 'field' galaxies is primarily a selection effect. We use our simulations to devise a means to select galaxy pairs that are isolated in their dark matter halos with respect to other massive subhalos (N= 2 halos) and to select a control sample of isolated galaxies (N= 1 halos) for comparison. We then apply these selection criteria to a volume-limited subset of the 2dF Galaxy Redshift Survey with M{sub B,j} {le} -19 and obtain the first clean measure of the typical fraction of galaxies affected by triggered star formation and the average elevation in the star formation rate. We find that 24% (30.5 %) of these L* and sub-L* galaxies in isolated 50 (30) h{sup -1} kpc pairs exhibit star formation that is boosted by a factor of {approx}> 5 above their average past value, while only 10% of isolated galaxies in the control sample show this level of enhancement. Thus, 14% (20 %) of the galaxies in these close pairs show clear triggered star formation. Our orbit models suggest that 12% (16%) of 50 (30) h{sup -1} kpc close pairs that are isolated according to our definition have had a close ({le} 30 h{sup -1} kpc) pass within the last Gyr. Thus, the data are broadly consistent with a scenario in which most or all close passes of isolated pairs result in triggered star formation. The isolation criteria we develop provide a means to constrain star formation and feedback prescriptions in hydrodynamic simulations and a very general method of understanding the importance of

  18. Assessing Student Affect

    ERIC Educational Resources Information Center

    Popham, W. James

    2009-01-01

    Student affect--the attitudes, interests, and values that students exhibit and acquire in school--can play a profoundly important role in students' postschool lives, possibly an even more significant role than that played by students' cognitive achievements. If student affect is so crucial, then why don't teachers assess it? One deterrent is that…

  19. Affective Involvement Instrument.

    ERIC Educational Resources Information Center

    Lemlech, Johanna K.

    1970-01-01

    The Affective Involvement Instrument (AII) describes and classifies affective involvement in the process of decision-making as it occurs during classroom activities such as role-playing or group discussions. The thirty-celled instrument behaviorizes the six processes involved in decision-making and combines them with the taxonomic levels of the…

  20. Affectional Patterns of Adolescents.

    ERIC Educational Resources Information Center

    O'Donnell, William J.

    1979-01-01

    This study sought to determine if there is a shift with age in affection (1) from parents to friends, (2) from one parent to the other, and (3) from same-sex to opposite-sex friends. Subjects, eighth graders and eleventh graders, completed the Measurement of Family Affective Structure. (Author)

  1. Habit formation.

    PubMed

    Smith, Kyle S; Graybiel, Ann M

    2016-03-01

    Habits, both good ones and bad ones, are pervasive in animal behavior. Important frameworks have been developed to understand habits through psychological and neurobiological studies. This work has given us a rich understanding of brain networks that promote habits, and has also helped us to understand what constitutes a habitual behavior as opposed to a behavior that is more flexible and prospective. Mounting evidence from studies using neural recording methods suggests that habit formation is not a simple process. We review this evidence and take the position that habits could be sculpted from multiple dissociable changes in neural activity. These changes occur across multiple brain regions and even within single brain regions. This strategy of classifying components of a habit based on different brain signals provides a potentially useful new way to conceive of disorders that involve overly fixed behaviors as arising from different potential dysfunctions within the brain's habit network. PMID:27069378

  2. Habit formation

    PubMed Central

    Smith, Kyle S.; Graybiel, Ann M.

    2016-01-01

    Habits, both good ones and bad ones, are pervasive in animal behavior. Important frameworks have been developed to understand habits through psychological and neurobiological studies. This work has given us a rich understanding of brain networks that promote habits, and has also helped us to understand what constitutes a habitual behavior as opposed to a behavior that is more flexible and prospective. Mounting evidence from studies using neural recording methods suggests that habit formation is not a simple process. We review this evidence and take the position that habits could be sculpted from multiple dissociable changes in neural activity. These changes occur across multiple brain regions and even within single brain regions. This strategy of classifying components of a habit based on different brain signals provides a potentially useful new way to conceive of disorders that involve overly fixed behaviors as arising from different potential dysfunctions within the brain's habit network. PMID:27069378

  3. Sequence Affects the Cyclization of DNA Minicircles.

    PubMed

    Wang, Qian; Pettitt, B Montgomery

    2016-03-17

    Understanding how the sequence of a DNA molecule affects its dynamic properties is a central problem affecting biochemistry and biotechnology. The process of cyclizing short DNA, as a critical step in molecular cloning, lacks a comprehensive picture of the kinetic process containing sequence information. We have elucidated this process by using coarse-grained simulations, enhanced sampling methods, and recent theoretical advances. We are able to identify the types and positions of structural defects during the looping process at a base-pair level. Correlations along a DNA molecule dictate critical sequence positions that can affect the looping rate. Structural defects change the bending elasticity of the DNA molecule from a harmonic to subharmonic potential with respect to bending angles. We explore the subelastic chain as a possible model in loop formation kinetics. A sequence-dependent model is developed to qualitatively predict the relative loop formation time as a function of DNA sequence. PMID:26938490

  4. [Affect and mimetic behavior].

    PubMed

    Zepf, S; Ullrich, B; Hartmann, S

    1998-05-01

    The relationship between facial expression and experienced affect presents many problems. The two diametrically opposed positions proposing solutions to this problem are exemplified using the conceptions of Mandler u. Izard. The underlying premises of both conceptions still prevail in various forms. The authors reject the concepts according to which facial expression is merely correlated to the affects (see Mandler 1975) as well as the view that facial expression controls the affects (see Izard 1977). The relationship between affect and facial expression is reexamined, subjecting it to a semiotic, essentially semantic analysis similar to the Ogden and Richards' language and meaning approach. This analysis involves a critical discussion of Scherer's attempt of a purely communicational interpretation using Bühler's organon model. In the author's approach, facial expression is seen not simply as a system of signals, but as a system of representative signs which signify the affects and refer to the emotive meaning of things for the subject. The authors develop the thesis that human beings are not born simply with the ability to speak, but also with the abstract possibility of performing facial expressions. This ability develops by way of coordinating patterns of expressions, which are presumably phylogenetically determined, with affects that take on a socially determined individual form, similar to language acquisition during socialisation. The authors discuss the methodological implications arising for studies investigating the affective meaning of facial expressions. PMID:9632951

  5. N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle.

    PubMed

    Oseikria, Mouhamad; Elis, Sébastien; Maillard, Virginie; Corbin, Emilie; Uzbekova, Svetlana

    2016-06-01

    The positive effect of n-3 polyunsaturated fatty acids (FAs) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 polyunsaturated fatty acids) during IVM has an effect on oocyte maturation and in vitro embryo development in cattle. We reported that DHA (0, 1, 10, or 100 μM) had no effect on oocyte viability or maturation rate after 22-hour IVM. Incubation of oocyte-cumulus complexes with 1-μM DHA during IVM significantly increased (P < 0.05) oocyte cleavage rate as compared with control (86.1% vs. 78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (39.1% vs. 29.7%, respectively). Supplementation with 1 μM DHA during IVM also induced a significant increase in the blastocyst rate at Day 7 after IVF as compared with control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; P = 0.08). On the contrary, 10-μM DHA had no effects, whereas 100-μM DHA significantly decreased the cleavage rate compared with control (69.5% vs.78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (19.5% vs. 29.7%). As was shown by real-time polymerase chain reaction, negative effects of 100-μM DHA were associated with significant increase of progesterone synthesis by oocyte-cumulus complexes, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CCs) of corresponding oocytes. Docosahexaenoic acid at 1 and 10 μM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1 μM) during IVM may have beneficial effects on oocyte developmental

  6. Tetrahedron Formation Control

    NASA Technical Reports Server (NTRS)

    Guzman, Jose J.

    2003-01-01

    Spacecraft flying in tetrahedron formations are excellent instrument platforms for electromagnetic and plasma studies. A minimum of four spacecraft - to establish a volume - is required to study some of the key regions of a planetary magnetic field. The usefulness of the measurements recorded is strongly affected by the tetrahedron orbital evolution. This paper considers the preliminary development of a general optimization procedure for tetrahedron formation control. The maneuvers are assumed to be impulsive and a multi-stage optimization method is employed. The stages include targeting to a fixed tetrahedron orientation, rotating and translating the tetrahedron and/or varying the initial and final times. The number of impulsive maneuvers citn also be varied. As the impulse locations and times change, new arcs are computed using a differential corrections scheme that varies the impulse magnitudes and directions. The result is a continuous trajectory with velocity discontinuities. The velocity discontinuities are then used to formulate the cost function. Direct optimization techniques are employed. The procedure is applied to the Magnetospheric Multiscale Mission (MMS) to compute preliminary formation control fuel requirements.

  7. Barrier Formation

    PubMed Central

    Lyaruu, D.M.; Medina, J.F.; Sarvide, S.; Bervoets, T.J.M.; Everts, V.; DenBesten, P.; Smith, C.E.; Bronckers, A.L.J.J.

    2014-01-01

    Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b (Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl− for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b−/− mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b−/− mice and was strongly correlated with Cl−. Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl− levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins. PMID:24170372

  8. Pattern Formation

    NASA Astrophysics Data System (ADS)

    Hoyle, Rebecca

    2006-03-01

    From the stripes of a zebra and the spots on a leopard's back to the ripples on a sandy beach or desert dune, regular patterns arise everywhere in nature. The appearance and evolution of these phenomena has been a focus of recent research activity across several disciplines. This book provides an introduction to the range of mathematical theory and methods used to analyse and explain these often intricate and beautiful patterns. Bringing together several different approaches, from group theoretic methods to envelope equations and theory of patterns in large-aspect ratio-systems, the book also provides insight behind the selection of one pattern over another. Suitable as an upper-undergraduate textbook for mathematics students or as a fascinating, engaging, and fully illustrated resource for readers in physics and biology, Rebecca Hoyle's book, using a non-partisan approach, unifies a range of techniques used by active researchers in this growing field. Accessible description of the mathematical theory behind fascinating pattern formation in areas such as biology, physics and materials science Collects recent research for the first time in an upper level textbook Features a number of exercises - with solutions online - and worked examples

  9. Compounds affecting cholesterol absorption

    NASA Technical Reports Server (NTRS)

    Hua, Duy H. (Inventor); Koo, Sung I. (Inventor); Noh, Sang K. (Inventor)

    2004-01-01

    A class of novel compounds is described for use in affecting lymphatic absorption of cholesterol. Compounds of particular interest are defined by Formula I: ##STR1## or a pharmaceutically acceptable salt thereof.

  10. Liver condition affects bovine oocyte qualities by changing the characteristics of follicular fluid and plasma.

    PubMed

    Tanaka, H; Shibano, K; Monji, Y; Kuwayama, T; Iwata, H

    2013-08-01

    The liver is an important organ that contributes to milk production in dairy cows. The aim of this study was to examine whether liver conditions affect the characteristics of blood plasma and follicular fluid (FF) and whether supplementing in vitro maturation medium with FF from either cows with damaged livers (DL) or those with healthy livers (HL) affects oocyte developmental competence. Biochemical characteristics of FF were significantly correlated with those in plasma. As such, the characteristics of both plasma and FF were similarly affected by liver conditions in that the concentrations of total protein and inorganic phosphorus were higher for the DL cow group than for the HL cow group, whereas the concentrations of albumin, lactate dehydrogenase and calcium were lower for DL cows than for HL cows. In addition, supplementing the medium with DL-FF retarded the progression of the nuclear maturation of oocytes collected from the HL cows. On culturing oocytes in maturation medium containing HL-FF, DL-FF or foetal calf serum, the highest developmental rate to the blastocyst stage was observed in the HL-FF group, while the lowest developmental ratio was observed in the DL-FF group. The growth factor array of the FFs revealed that 10 growth factors were significantly downregulated in the DL-FF compared with those in HL-FF. In conclusion, the characteristics of plasma and FF are affected by liver conditions in a similar way. Concentrations of several growth factors were low in DL-FF, as was the ability of DL-FF to support oocyte maturation compared with that of HL-FF. PMID:23281835

  11. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    SciTech Connect

    Taru Sharma, G.; Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G.

    2012-08-03

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it

  12. Affective responses to dance.

    PubMed

    Christensen, Julia F; Pollick, Frank E; Lambrechts, Anna; Gomila, Antoni

    2016-07-01

    The objective of the present work was the characterization of mechanisms by which affective experiences are elicited in observers when watching dance movements. A total of 203 dance stimuli from a normed stimuli library were used in a series of independent experiments. The following measures were obtained: (i) subjective measures of 97 dance-naïve participants' affective responses (Likert scale ratings, interviews); and (ii) objective measures of the physical parameters of the stimuli (motion energy, luminance), and of the movements represented in the stimuli (roundedness, impressiveness). Results showed that (i) participants' ratings of felt and perceived affect differed, (ii) felt and perceived valence but not arousal ratings correlated with physical parameters of the stimuli (motion energy and luminance), (iii) roundedness in posture shape was related to the experience of more positive emotion than edgy shapes (1 of 3 assessed rounded shapes showed a clear effect on positiveness ratings while a second reached trend level significance), (iv) more impressive movements resulted in more positive affective responses, (v) dance triggered affective experiences through the imagery and autobiographical memories it elicited in some people, and (vi) the physical parameters of the video stimuli correlated only weakly and negatively with the aesthetics ratings of beauty, liking and interest. The novelty of the present approach was twofold; (i) the assessment of multiple affect-inducing mechanisms, and (ii) the use of one single normed stimulus set. The results from this approach lend support to both previous and present findings. Results are discussed with regards to current literature in the field of empirical aesthetics and affective neuroscience. PMID:27235953

  13. Drugs affecting glycosaminoglycan metabolism.

    PubMed

    Ghiselli, Giancarlo; Maccarana, Marco

    2016-07-01

    Glycosaminoglycans (GAGs) are charged polysaccharides ubiquitously present at the cell surface and in the extracellular matrix. GAGs are crucial for cellular homeostasis, and their metabolism is altered during pathological processes. However, little consideration has been given to the regulation of the GAG milieu through pharmacological interventions. In this review, we provide a classification of small molecules affecting GAG metabolism based on their mechanism of action. Furthermore, we present evidence to show that clinically approved drugs affect GAG metabolism and that this could contribute to their therapeutic benefit. PMID:27217160

  14. Affective Factors: Anxiety

    ERIC Educational Resources Information Center

    Tasnimi, Mahshad

    2009-01-01

    Affective factors seem to play a crucial role in success or failure in second language acquisition. Negative attitudes can reduce learners' motivation and harm language learning, while positive attitudes can do the reverse. Discovering students' attitudes about language will help both teacher and student in teaching learning process. Anxiety is…

  15. How Technology Affects Teaching.

    ERIC Educational Resources Information Center

    Wiske, Martha Stone; And Others

    This study presents composite profiles of teachers who were interviewed in order to assess how they are being affected by the challenges and opportunities presented by computer technology use. In-depth interviews were held with 76 teachers from 10 sites around the country, and the interview data were analyzed to identify themes and to construct…

  16. Factors affecting soil cohesion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil erodibility is a measure of a soil’s resistance against erosive forces and is affected by both intrinsic (or inherent) soil property and the extrinsic condition at the time erodibility measurement is made. Since soil erodibility is usually calculated from results obtained from erosion experimen...

  17. What Variables Affect Solubility?

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn

    2003-01-01

    Helps middle school students understand the concept of solubility through hands-on experience with a variety of liquids and solids. As they explore factors that affect solubility and saturation, students gain content mastery and an understanding of the inquiry process. Also enables teachers to authentically assess student performance on several…

  18. How Body Affects Brain.

    PubMed

    Suzuki, Wendy A

    2016-08-01

    Studies show that physical exercise can affect a range of brain and cognitive functions. However, little is known about the peripheral signals that initiate these central changes. Moon et al. (2016) provide exciting new evidence that a novel myokine, cathepsin B (CTSB), released with exercise is associated with improved memory. PMID:27508865

  19. Food Affects Human Behavior.

    ERIC Educational Resources Information Center

    Kolata, Gina

    1982-01-01

    A conference on whether food and nutrients affect human behavior was held on November 9, 1982 at the Massachusetts Institute of Technology. Various research studies on this topic are reviewed, including the effects of food on brain biochemistry (particularly sleep) and effects of tryptophane as a pain reducer. (JN)

  20. Affective Dynamics of Leadership: An Experimental Test of Affect Control Theory

    ERIC Educational Resources Information Center

    Schroder, Tobias; Scholl, Wolfgang

    2009-01-01

    Affect Control Theory (ACT; Heise 1979, 2007) states that people control social interactions by striving to maintain culturally shared feelings about the situation. The theory is based on mathematical models of language-based impression formation. In a laboratory experiment, we tested the predictive power of a new German-language ACT model with…

  1. Dynamic Synchronization of Teacher-Students Affection in Affective Instruction

    ERIC Educational Resources Information Center

    Zhang, Wenhai; Lu, Jiamei

    2011-01-01

    Based on Bower's affective network theory, the article links the dynamic analysis of affective factors in affective instruction, and presents affective instruction strategic of dynamic synchronization between teacher and students to implement the best ideal mood that promotes students' cognition and affection together. In the process of teaching,…

  2. Microphysical Processes Affecting the Pinatubo Volcanic Plume

    NASA Technical Reports Server (NTRS)

    Hamill, Patrick; Houben, Howard; Young, Richard; Turco, Richard; Zhao, Jingxia

    1996-01-01

    In this paper we consider microphysical processes which affect the formation of sulfate particles and their size distribution in a dispersing cloud. A model for the dispersion of the Mt. Pinatubo volcanic cloud is described. We then consider a single point in the dispersing cloud and study the effects of nucleation, condensation and coagulation on the time evolution of the particle size distribution at that point.

  3. Values and Affectivity from a Piagetian Point of View.

    ERIC Educational Resources Information Center

    Brown, Terrance

    This paper, which analyzes the evaluative aspect of affectivity, argues that the functional internalization of organic evolution for the formation of the intellectual system of human beings required that some means of selecting behavioral variations be devised, and that the selective mechanism devised was the affective system. From this…

  4. Formation of giant planets

    NASA Astrophysics Data System (ADS)

    Magni, G.; Coradini, A.

    2003-04-01

    In this presentation we address the problem of the formation of giant planets and their regular satellites. We study in particular the problem of formation of the Jupiter System comparing the results of the model with the present characteristics of the system, in order to identify what are those better represented by our approach. In fact here, using a 3-D hydro-dynamical code, we study the modalities of gas accretion onto a solid core, believed to be the seed from which Jupiter started. To do that we have modelled three main regions: the central planet, a turbulent accretion disk surrounding it and an extended region from which the gas is collected. In the extended region we treat the gas as a frictionless fluid. Our main goal is to identify what are the characteristics of the planet during its growth and the physical parameters affecting its growth at the expenses of the nebular gas present in the feeding zone. Moreover we want to understand what are the thermodynamical parameters characterizing the gas captured by the planet and swirling around it. Finally, we check if a disk can be formed in prograde rotation around the planet and if this disk can survive the final phases of the planet formation. Due to the interaction between the accreting planet and the disk it has been necessary to develop a complete model of the Jupiter’s structure. In fact the radiation emitted by the growing planet heats up the surrounding gas. In turn the planet’s thermodynamic structure depend on the mass accretion rate onto it. When the accretion is rapid, shock waves in the gas are formed close to the planet. This region cannot be safely treated by a numerical code; for this reason we have developed a semi-analytically model of a a turbulent accretion disk to be considered as transition between the planet and the surrounding disk.

  5. Granuloma Formation in Pulmonary Sarcoidosis

    PubMed Central

    Broos, Caroline E.; van Nimwegen, Menno; Hoogsteden, Henk C.; Hendriks, Rudi W.; Kool, Mirjam; van den Blink, Bernt

    2013-01-01

    Sarcoidosis is a granulomatous disorder of unknown cause, affecting multiple organs, but mainly the lungs. The exact order of immunological events remains obscure. Reviewing current literature, combined with careful clinical observations, we propose a model for granuloma formation in pulmonary sarcoidosis. A tight collaboration between macrophages, dendritic cells, and lymphocyte subsets, initiates the first steps toward granuloma formation, orchestrated by cytokines and chemokines. In a substantial part of pulmonary sarcoidosis patients, granuloma formation becomes an on-going process, leading to debilitating disease, and sometimes death. The immunological response, determining granuloma sustainment is not well understood. An impaired immunosuppressive function of regulatory T cells has been suggested to contribute to the exaggerated response. Interestingly, therapeutical agents commonly used in sarcoidosis, such as glucocorticosteroids and anti-TNF agents, interfere with granuloma integrity and restore the immune homeostasis in autoimmune disorders. Increasing insight into their mechanisms of action may contribute to the search for new therapeutical targets in pulmonary sarcoidosis. PMID:24339826

  6. Affective Processes and Academic Achievement.

    ERIC Educational Resources Information Center

    Feshbach, Norma Deitch; Feshbach, Seymour

    1987-01-01

    Data indicate that for girls, affective dispositional factors (empathy, depressive affectivity, aggression, and self-concept) are intimately linked to cognitive development and academic achievement. (PCB)

  7. l-Carnitine affects preimplantation embryo development toward infertility in mice.

    PubMed

    Kyvelidou, Christiana; Sotiriou, Dimitris; Antonopoulou, Tania; Tsagkaraki, Margarita; Tserevelakis, George J; Filippidis, George; Athanassakis, Irene

    2016-10-01

    l-Carnitine (l-Cn), despite the beneficial role as energy-generating substance delivering long-chain fatty acids to the β-oxidation pathway in mitochondria, has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal cavity and uterine walls and most importantly, correlating to infertility. Exploring this type of infertility, the effect of l-Cn on preimplantation embryo development, ovarian integrity and systemic maternal immunity was studied. Using nonlinear microscopy analysis, which was shown to be a powerful tool for determining embryo quality by quantitatively estimating the lipid body (LB) content of the cells, it was shown that in vitro and in vivo administration of l-Cn significantly decreased LB mean area in zygotes. Daily intraperitoneal administration of 2.5mg l-Cn for 3, 4 and 7days to mice significantly decreased the percent of normal zygotes. However, only the 7-day treatment persisted by affecting 2- and 8-cell stage embryos, while almost abolishing blastocyst development. Such effects were accompanied by abnormal ovarian histology, showing increased numbers of corpora luteus and elevated progesterone concentration in the serum. In addition, it was shown that the 7-day l-Cn treatment pushed maternal systemic immunity toward inflammation and immunosuppression by increasing CD11b-, CD25- and CD11bGr1-positive cells in spleen, which opposed the necessity for immunostimulation at these early stages of pregnancy. In conclusion, the results presented here demonstrated that elevated doses of l-Cn affect early stages of embryo development, leading to infertility. PMID:27402869

  8. Does Commuting Affect Health?

    PubMed

    Künn-Nelen, Annemarie

    2016-08-01

    This paper analyzes the relation between commuting time and health in the UK. I focus on four different types of health outcomes: subjective health measures, objective health measures, health behavior, and healthcare utilization. Fixed effect models are estimated with British Household Panel Survey data. I find that whereas objective health and health behavior are barely affected by commuting time, subjective health measures are clearly lower for people who commute longer. A longer commuting time is, moreover, related to more visits to the general practitioner. Effects turn out to be more pronounced for women and for commuters driving a car. For women, commuting time is also negatively related to regular exercise and positively to calling in sick. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26010157

  9. Factors Affecting Wound Healing

    PubMed Central

    Guo, S.; DiPietro, L.A.

    2010-01-01

    Wound healing, as a normal biological process in the human body, is achieved through four precisely and highly programmed phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. Many factors can interfere with one or more phases of this process, thus causing improper or impaired wound healing. This article reviews the recent literature on the most significant factors that affect cutaneous wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include oxygenation, infection, age and sex hormones, stress, diabetes, obesity, medications, alcoholism, smoking, and nutrition. A better understanding of the influence of these factors on repair may lead to therapeutics that improve wound healing and resolve impaired wounds. PMID:20139336

  10. Star formation - An overview

    NASA Technical Reports Server (NTRS)

    Evans, N. J., II

    1985-01-01

    Methods for studying star formation are reviewed. Stellar clusters and associations, as well as field stars, provide a fossil record of the star formation process. Regions of current star formation provide a series of snapshots of different epochs of star formation. A simplified picture of individual star formation as it was envisioned in the late 1970s is contrasted with the results of recent observations, in particular the outflow phenomenon.

  11. Formate Formation and Formate Conversion in Biological Fuels Production

    PubMed Central

    Crable, Bryan R.; Plugge, Caroline M.; McInerney, Michael J.; Stams, Alfons J. M.

    2011-01-01

    Biomethanation is a mature technology for fuel production. Fourth generation biofuels research will focus on sequestering CO2 and providing carbon-neutral or carbon-negative strategies to cope with dwindling fossil fuel supplies and environmental impact. Formate is an important intermediate in the methanogenic breakdown of complex organic material and serves as an important precursor for biological fuels production in the form of methane, hydrogen, and potentially methanol. Formate is produced by either CoA-dependent cleavage of pyruvate or enzymatic reduction of CO2 in an NADH- or ferredoxin-dependent manner. Formate is consumed through oxidation to CO2 and H2 or can be further reduced via the Wood-Ljungdahl pathway for carbon fixation or industrially for the production of methanol. Here, we review the enzymes involved in the interconversion of formate and discuss potential applications for biofuels production. PMID:21687599

  12. Pteridines and affective disorders.

    PubMed

    Hoekstra, R; Fekkes, D

    2002-06-01

    The pteridine tetrahydrobiopterin (BH4) is an essential cofactor in the biosynthesis of dopamine, (nor)epinephrine, serotonin and nitric oxide (NO). Furthermore, BH4 has a direct influence on release mechanisms of these neurotransmitters and on serotonin receptor binding activity immunology. The synthesis of BH4 is stimulated by interferon-gamma and hence there is a close relationship with the immune system HPA-axis. In animal experiments it was also found that the hypothalamus-pituitary-adrenal axis influences the pteridine metabolism. In clinical studies, so far, no evidence has been found for this relationship diseases. A congenital biopterin deficiency results in atypical phenylketonuria with severe neuropsychiatric symptoms. In several neurological diseases, such as Parkinson's disease, decreased levels of BH4 are found depression. Since 1984 there have been reports on decreased biopterin and increased neopterin levels in urine and plasma of depressed patients. Conflicting results have also been found, however, due probably to methodological problems therapy. Until now, oral administration of BH4 to depressed patients has been performed by two investigators, which resulted in mainly temporal clinical improvement discussion. Understanding of biochemical mechanisms in which pteridines are involved may contribute to our knowledge of the pathogenesis and treatment of affective disorders. This paper aims to provide an overview of the relevant literature and warrant for further research on this intriguing compound. PMID:26984153

  13. Neonatal Phytoestrogen Exposure Alters Oviduct Mucosal Immune Response to Pregnancy and Affects Preimplantation Embryo Development in the Mouse1

    PubMed Central

    Jefferson, Wendy N.; Padilla-Banks, Elizabeth; Phelps, Jazma Y.; Cantor, Amy M.; Williams, Carmen J.

    2012-01-01

    ABSTRACT Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are “posteriorized” as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype. PMID:22553218

  14. Affective Incoherence: When Affective Concepts and Embodied Reactions Clash

    PubMed Central

    Centerbar, David B.; Clore, Gerald L.; Schnall, Simone; Garvin, Erika

    2008-01-01

    In five studies, we examined the effects on cognitive performance of coherence and incoherence between conceptual and experiential sources of affective information. The studies crossed the priming of happy and sad concepts with affective experiences. In different experiments, these included: approach or avoidance actions, happy or sad feelings, and happy or sad expressive behaviors. In all studies, coherence between affective concepts and affective experiences led to better recall of a story than affective incoherence. We suggested that the experience of such experiential affective cues serves as evidence of the appropriateness of affective concepts that come to mind. The results suggest that affective coherence has epistemic benefits, and that incoherence is costly, for cognitive performance. PMID:18361672

  15. Intracytoplasmic sperm injection affects embryo developmental potential and gene expression in cattle.

    PubMed

    Arias, María Elena; Risopatrón, Jennie; Sánchez, Raúl; Felmer, Ricardo

    2015-03-01

    Some reports have linked intracytoplasmic sperm injection (ICSI) with chromosomal abnormalities, low developmental potential and altered gene expression in embryos. ICSI has also been linked with obesity, early aging and increased incidence of tumors in offspring. Other reports have demonstrated that some of these complications disappeared within a few weeks of life or even showed a lack of such associations. The aim of this study was to evaluate and compare embryo development, quality and gene expression in bovine embryos generated by ICSI and by conventional in vitro fertilization (IVF) insemination. The results showed differences in cleavage (88.5% in IVF and 64.1% in ICSI) and blastocyst formation rates (36.1% in IVF and 22.3% in ICSI). The proportion of ICM cells to total cell count was higher in ICSI (39.2%) than in IVF embryos (29.5%). However, no differences were observed in the total embryonic cell numbers (159.3±28.5 and 161.2±56.2 for IVF and ICSI, respectively) or in the proportion of apoptotic nuclei to the total embryonic cell numbers (2.12 and 2.64% for IVF and ICSI, respectively). Gene expression analysis showed a down-regulation of insulin-like growth factor 2 (IGF2) and overexpression of bcl-2-like protein 4 (BAX), octamer-binding transcription factor four (OCT4), interferon-tau (IFNt), Mn-superoxide dismutase in the mitochondria (SOD2), and catalase (CAT) in embryos generated by ICSI. In conclusion, our study demonstrated differences in the morphological development of bovine embryos as well as in the expression of genes involved in early development between ICSI and IVF embryos. The results may indicate lower developmental potential of ICSI embryos compared with that of IVF. PMID:25726375

  16. Identifying Occupationally Specific Affective Behaviors.

    ERIC Educational Resources Information Center

    Pucel, David J.

    1993-01-01

    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  17. Individual difference variables, affective differentiation, and the structures of affect.

    PubMed

    Terracciano, Antonio; McCrae, Robert R; Hagemann, Dirk; Costa, Paul T

    2003-10-01

    Methodological arguments are usually invoked to explain variations in the structure of affect. Using self-rated affect from Italian samples (N=600), we show that individual difference variables related to affective differentiation can moderate the observed structure. Indices of circumplexity and congruence coefficients to the hypothesized target were used to quantify the observed structures. Results did not support the circumplex model as a universal structure. A circular structure with axes of activation and valence was approximated only among more affectively differentiated groups: students and respondents with high scores on Openness to Feelings and measures of negative emotionality. A different structure, with unipolar Positive Affect and Negative Affect factors, was observed among adults and respondents with low Openness to Feelings and negative emotionality. The observed structure of affect will depend in part on the nature of the sample studied. PMID:12932207

  18. Individual Difference Variables, Affective Differentiation, and the Structures of Affect

    PubMed Central

    Terracciano, Antonio; McCrae, Robert R.; Hagemann, Dirk; Costa, Paul T.

    2008-01-01

    Methodological arguments are usually invoked to explain variations in the structure of affect. Using self-rated affect from Italian samples (N = 600), we show that individual difference variables related to affective differentiation can moderate the observed structure. Indices of circumplexity (Browne, 1992) and congruence coefficients to the hypothesized target were used to quantify the observed structures. Results did not support the circumplex model as a universal structure. A circular structure with axes of activation and valence was approximated only among more affectively differentiated groups: students and respondents with high scores on Openness to Feelings and measures of negative emotionality. A different structure, with unipolar Positive Affect and Negative Affect factors, was observed among adults and respondents with low Openness to Feelings and negative emotionality. The observed structure of affect will depend in part on the nature of the sample studied. PMID:12932207

  19. Magnetic fields and galactic star formation rates

    SciTech Connect

    Loo, Sven Van; Tan, Jonathan C.; Falle, Sam A. E. G.

    2015-02-10

    The regulation of galactic-scale star formation rates (SFRs) is a basic problem for theories of galaxy formation and evolution: which processes are responsible for making observed star formation rates so inefficient compared to maximal rates of gas content divided by dynamical timescale? Here we study the effect of magnetic fields of different strengths on the evolution of giant molecular clouds (GMCs) within a kiloparsec patch of a disk galaxy and resolving scales down to ≃0.5 pc. Including an empirically motivated prescription for star formation from dense gas (n{sub H}>10{sup 5} cm{sup −3}) at an efficiency of 2% per local free-fall time, we derive the amount of suppression of star formation by magnetic fields compared to the nonmagnetized case. We find GMC fragmentation, dense clump formation, and SFR can be significantly affected by the inclusion of magnetic fields, especially in our strongest investigated B-field case of 80 μG. However, our chosen kiloparsec-scale region, extracted from a global galaxy simulation, happens to contain a starbursting cloud complex that is only modestly affected by these magnetic fields and likely requires internal star formation feedback to regulate its SFR.

  20. Structure Formation in Astrophysics

    NASA Astrophysics Data System (ADS)

    Chabrier, Gilles

    2009-01-01

    Part I. Physical Processes and Numerical Methods Common to Structure Formations in Astrophysics: 1. The physics of turbulence E. Levêque; 2. The numerical simulation of turbulence W. Schmidt; 3. Numerical methods for radiation magnetohydrodynamics in astrophysics R. Klein and J. Stone; 4. The role of jets in the formation of planets, stars, and galaxies R. Banerjee, R. Pudritz and R. Ouyed; 5. Advanced numerical methods in astrophysical fluid dynamics A. Hujeirat and F. Heitsch; Part II. Structure and Star Formation in the Primordial Universe: 6. New frontiers in cosmology and galaxy formation challenges for the future R. Ellis and J. Silk; 7. Galaxy formation physics T. Abel, G. Bryan and R. Teyssier; 8. First stars formation, evolution, feedback effects V. Bromm, A. Ferrara and A. Heger; Part III. Contemporary Star and Brown Dwarf Formation: a) Cloud Formation and Fragmentation: 9. Diffuse interstellar medium and the formation of molecular clouds P. Hennebelle, M. Mac Low and E. Vazquez-Semadeni; 10. The formation of distributed and clustered stars in molecular clouds T. Megeath, Z. -Y. Li and A. Nordlund; b) Core Fragmentation and Star Formation: 11. The formation and evolution of prestellar cores P. André, S. Basu and S. Inutsuka; 12. Models for the formation of massive stars; Part IV. Protoplanetary Disks and Planet Formation M. Krumholz and I. Bonnell: 13. Observational properties of disks and young stellar objects G. Duchêne, F. Ménard, J. Muzzerolle and S. Mohanty; 14. Structure and dynamics of protoplanetary disks C. Dullemond, R. Durisen and J. Papaloizou; 15. Planet formation and evolution theory and observation Y. Alibert, I. Baraffe, W. Benz, G. Laughlin and S. Udry; 16. Planet formation assembling the puzzle G. Wurm and T. Guillot; Part V. Summary: 17. Open issues in small- and large-scale structure formation R. Klessen and M. Mac Low; 18. Final word E. Salpeter.

  1. Structure Formation in Astrophysics

    NASA Astrophysics Data System (ADS)

    Chabrier, Gilles

    2011-02-01

    Part I. Physical Processes and Numerical Methods Common to Structure Formations in Astrophysics: 1. The physics of turbulence E. Levêque; 2. The numerical simulation of turbulence W. Schmidt; 3. Numerical methods for radiation magnetohydrodynamics in astrophysics R. Klein and J. Stone; 4. The role of jets in the formation of planets, stars, and galaxies R. Banerjee, R. Pudritz and R. Ouyed; 5. Advanced numerical methods in astrophysical fluid dynamics A. Hujeirat and F. Heitsch; Part II. Structure and Star Formation in the Primordial Universe: 6. New frontiers in cosmology and galaxy formation challenges for the future R. Ellis and J. Silk; 7. Galaxy formation physics T. Abel, G. Bryan and R. Teyssier; 8. First stars formation, evolution, feedback effects V. Bromm, A. Ferrara and A. Heger; Part III. Contemporary Star and Brown Dwarf Formation: a) Cloud Formation and Fragmentation: 9. Diffuse interstellar medium and the formation of molecular clouds P. Hennebelle, M. Mac Low and E. Vazquez-Semadeni; 10. The formation of distributed and clustered stars in molecular clouds T. Megeath, Z. -Y. Li and A. Nordlund; b) Core Fragmentation and Star Formation: 11. The formation and evolution of prestellar cores P. André, S. Basu and S. Inutsuka; 12. Models for the formation of massive stars; Part IV. Protoplanetary Disks and Planet Formation M. Krumholz and I. Bonnell: 13. Observational properties of disks and young stellar objects G. Duchêne, F. Ménard, J. Muzzerolle and S. Mohanty; 14. Structure and dynamics of protoplanetary disks C. Dullemond, R. Durisen and J. Papaloizou; 15. Planet formation and evolution theory and observation Y. Alibert, I. Baraffe, W. Benz, G. Laughlin and S. Udry; 16. Planet formation assembling the puzzle G. Wurm and T. Guillot; Part V. Summary: 17. Open issues in small- and large-scale structure formation R. Klessen and M. Mac Low; 18. Final word E. Salpeter.

  2. Explorations of Affection and Aggression.

    ERIC Educational Resources Information Center

    Shuntich, Richard J.; Shapiro, Richard

    Considerable effort has been devoted to investigating various aspects of love and affection, but there have been few studies about direct expressions of affection. Relationships between gender composition of a dyad and the affection/aggression expressed by the dyad were examined as was the possibility of increasing the amount of affectionate…

  3. Affective Productions of Mathematical Experience

    ERIC Educational Resources Information Center

    Walshaw, Margaret; Brown, Tony

    2012-01-01

    In underscoring the affective elements of mathematics experience, we work with contemporary readings of the work of Spinoza on the politics of affect, to understand what is included in the cognitive repertoire of the Subject. We draw on those resources to tell a pedagogical tale about the relation between cognition and affect in settings of…

  4. Affective Induction and Creative Thinking

    ERIC Educational Resources Information Center

    Fernández-Abascal, Enrique G.; Díaz, María D. Martín

    2013-01-01

    Three studies explored the relation between affect and production of creative divergent thinking, assessed with the Torrance Tests of Creative Thinking (Figural TTCT). In the first study, general, positive, and negative affect, assessed with the Positive and Negative Affect Scale (PANAS) were compared with creative production. In the second study,…

  5. 76 FR 41705 - Approval and Promulgation of Implementation Plans; New York; Revised Format of Materials Being...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-15

    ...The Environmental Protection Agency (EPA) is revising the format of materials submitted by the State of New York that have been incorporated by reference (IBR) into its State Implementation Plan (SIP). The regulations and other materials affected by this format change have all been previously submitted by New York and approved by EPA as SIP revisions. This format revision will primarily affect......

  6. Star Formation in Galaxies

    NASA Technical Reports Server (NTRS)

    1987-01-01

    Topics addressed include: star formation; galactic infrared emission; molecular clouds; OB star luminosity; dust grains; IRAS observations; galactic disks; stellar formation in Magellanic clouds; irregular galaxies; spiral galaxies; starbursts; morphology of galactic centers; and far-infrared observations.

  7. The Format Dilemma.

    ERIC Educational Resources Information Center

    Oder, Norman

    2002-01-01

    Reports results of a survey of public libraries that investigated trends in audiovisual materials. Highlights include format issues; audiobooks; media budgets for various formats; video collections; DVDs; circulation; collection sizes; music CDs; and future possibilities. (LRW)

  8. Simple Ontology Format (SOFT)

    SciTech Connect

    Sorokine, Alexandre

    2011-10-01

    Simple Ontology Format (SOFT) library and file format specification provides a set of simple tools for developing and maintaining ontologies. The library, implemented as a perl module, supports parsing and verification of the files in SOFt format, operations with ontologies (adding, removing, or filtering of entities), and converting of ontologies into other formats. SOFT allows users to quickly create ontologies using only a basic text editor, verify it, and portray it in a graph layout system using customized styles.

  9. Affect as a Psychological Primitive

    PubMed Central

    Barrett, Lisa Feldman; Bliss-Moreau, Eliza

    2009-01-01

    In this article, we discuss the hypothesis that affect is a fundamental, psychologically irreducible property of the human mind. We begin by presenting historical perspectives on the nature of affect. Next, we proceed with a more contemporary discussion of core affect as a basic property of the mind that is realized within a broadly distributed neuronal workspace. We then present the affective circumplex, a mathematical formalization for representing core affective states, and show that this model can be used to represent individual differences in core affective feelings that are linked to meaningful variation in emotional experience. Finally, we conclude by suggesting that core affect has psychological consequences that reach beyond the boundaries of emotion, to influence learning and consciousness. PMID:20552040

  10. Encountering science education's capacity to affect and be affected

    NASA Astrophysics Data System (ADS)

    Alsop, Steve

    2015-12-01

    What might science education learn from the recent affective turn in the humanities and social sciences? Framed as a response to Michalinos Zembylas's article, this essay draws from selected theorizing in affect theory, science education and science and technology studies, in pursuit of diverse and productive ways to talk of affect within science education. These discussions are framed by desires to transcend traditional epistemic boundaries and practices. The article concludes offering some associated ambiguities and tensions involved.

  11. Factors affecting dwell times on digital displaying

    NASA Technical Reports Server (NTRS)

    Williams, A. J.; Harris, R. L., Sr.

    1985-01-01

    A series of exploratory tests were conducted to investigate the effects of advanced display formats and display media on pilot scanning behavior using Langley's oculometer, a desktop flight simulator, a conventional electro-mechanical meter, and various digital displays. The primary task was for the test subject to maintain level flight, on a specific course heading, during moderate turbulence. A secondary task of manually controlling the readout of a display was used to examine the effects of the display format on a subject's scan behavior. Secondary task scan parameters that were evaluated were average dwell time, dwell time histograms, and number of dwells per meter change. The round dial meter demonstrated shorter dwell times and fewer dwells per meter change than the digital displays. The following factors affected digital display scanning behavior: (1) the number of digits; (2) the update rate of the digits; (3) the display media; and (4) the character font. The size of the digits used in these tests (0.28 to 0.50 inches) did not affect scan behavior measures.

  12. Undergraduate Students' Mental Models of Hailstone Formation

    ERIC Educational Resources Information Center

    Cin, Mustafa

    2013-01-01

    The objective of this research is to investigate students' mental models of the hailstone formation and explore factors that may affect their mental models. The sample chosen for the study was composed of a total of 84 students. The students attended the 1st to 4 th grade classes of the Social Studies Teaching Programme at Giresun University in…

  13. Formative Evaluation of a Neuroanatomy Course

    ERIC Educational Resources Information Center

    Sterret, Patrick R.; Littlefield, John H.

    1976-01-01

    Student cognitive performance data and affective reactions provided the basis for a formative evaluation of this neuroanatomy curriculum for freshmen medical students. The cerebral hempispheres topic area was marked by poor cognitive performance and low ratings in lecture quality. Videotapes designed to augment neurophysiology also received low…

  14. Boundaries for Biofilm Formation: Humidity and Temperature

    PubMed Central

    Else, Terry Ann; Pantle, Curtis R.; Amy, Penny S.

    2003-01-01

    Environmental conditions which define boundaries for biofilm production could provide useful ecological information for biofilm models. A practical use of defined conditions could be applied to the high-level nuclear waste repository at Yucca Mountain. Data for temperature and humidity conditions indicate that decreases in relative humidity or increased temperature severely affect biofilm formation on three candidate canister metals. PMID:12902302

  15. Testing the Grandchildren's Received Affection Scale using Affection Exchange Theory.

    PubMed

    Mansson, Daniel H

    2013-04-01

    The purpose of this study was to test the Grandchildren's Received Affection Scale (GRAS) using Affection Exchange Theory (Floyd, 2006). In accordance with Affection Exchange Theory, it was hypothesized that grandchildren's scores on the Trait Affection Received Scale (i.e., the extent to which individuals by nature receive affection) would be related significantly and positively to their reports of received affection from their grandparents (i.e., their scores on the GRAS). Additionally, a research question was asked to explore if grandchildren's received affection from their grandparents is dependent on their grandparent's biological sex or lineage (i.e., maternal vs paternal). Thus, young adult grandchildren (N = 422) completed the GRAS and the Trait Affection Received Scale. The results of zero-order Pearson correlational analyses provided support for the hypothesis, whereas the results of MANOVAs tests only partially support extant grandparent-grandchild theory and research. These findings broaden the scope of Affection Exchange Theory and also bolster the GRAS's utility in future grandparent-grandchild affectionate communication research. PMID:23833883

  16. Medical image file formats.

    PubMed

    Larobina, Michele; Murino, Loredana

    2014-04-01

    Image file format is often a confusing aspect for someone wishing to process medical images. This article presents a demystifying overview of the major file formats currently used in medical imaging: Analyze, Neuroimaging Informatics Technology Initiative (Nifti), Minc, and Digital Imaging and Communications in Medicine (Dicom). Concepts common to all file formats, such as pixel depth, photometric interpretation, metadata, and pixel data, are first presented. Then, the characteristics and strengths of the various formats are discussed. The review concludes with some predictive considerations about the future trends in medical image file formats. PMID:24338090

  17. Positive affect and psychobiological processes

    PubMed Central

    Dockray, Samantha; Steptoe, Andrew

    2010-01-01

    Positive affect has been associated with favourable health outcomes, and it is likely that several biological processes mediate the effects of positive mood on physical health. There is converging evidence that positive affect activates the neuroendocrine, autonomic and immune systems in distinct and functionally meaningful ways. Cortisol, both total output and the awakening response, has consistently been shown to be lower among individuals with higher levels of positive affect. The beneficial effects of positive mood on cardiovascular function, including heart rate and blood pressure, and the immune system have also been described. The influence of positive affect on these psychobiological processes are independent of negative affect, suggesting that positive affect may have characteristic biological correlates. The duration and conceptualisation of positive affect may be important considerations in understanding how different biological systems are activated in association with positive affect. The association of positive affect and psychobiological processes has been established, and these biological correlates may be partly responsible for the protective effects of positive affect on health outcomes. PMID:20097225

  18. Can Solution Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

  19. Star Formation for Predictive Primordial Galaxy Formation

    NASA Astrophysics Data System (ADS)

    Milosavljević, Miloš; Safranek-Shrader, Chalence

    The elegance of inflationary cosmology and cosmological perturbation theory ends with the formation of the first stars and galaxies, the initial sources of light that launched the phenomenologically rich process of cosmic reionization. Here we review the current understanding of early star formation, emphasizing unsolved problems and technical challenges. We begin with the first generation of stars to form after the Big Bang and trace how they influenced subsequent star formation. The onset of chemical enrichment coincided with a sharp increase in the overall physical complexity of star forming systems. Ab-initio computational treatments are just now entering the domain of the predictive and are establishing contact with local observations of the relics of this ancient epoch.

  20. [Development of the affect system].

    PubMed

    Moser, U; Von Zeppelin, I

    1996-01-01

    The authors show that the development of the affect system commences with affects of an exclusively communicative nature. These regulate the relationship between subject and object. On a different plane they also provide information on the feeling of self deriving from the interaction. Affect is seen throughout as a special kind of information. One section of the article is given over to intensity regulation and early affect defenses. The development of cognitive processes leads to the integration of affect systems and cognitive structures. In the pre-conceptual concretistic phase, fantasies change the object relation in such a way as to make unpleasant affects disappear. Only at a later stage do fantasies acquire the capacity to deal with affects. Ultimately, the affect system is grounded on an invariant relationship feeling. On a variety of different levels it displays the features typical of situation theory and the theory of the representational world, thus making it possible to entertain complex object relations. In this process the various planes of the affect system are retained and practised. Finally, the authors discuss the consequences of their remarks for the understanding of psychic disturbances and the therapies brought to bear on them. PMID:8584745

  1. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    PubMed

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  2. Abnormal Early Cleavage Events Predict Early Embryo Demise: Sperm Oxidative Stress and Early Abnormal Cleavage

    PubMed Central

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M.; Pera, Renee Reijo; Meyers, Stuart

    2014-01-01

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors. PMID:25307782

  3. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

    PubMed Central

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  4. PLUME FORMATION, TRANSPORT AND MODELING, PRESENTED IN RENO, NEVADA

    EPA Science Inventory

    This presentation presents information on plume formation, transport and modeling. The outline for the presentation is: conceptualization of hydrocarbon releases; factors affecting plume length and concentration; modeling considerations; and uncertainty in model calculations.

  5. Affective and motivational influences in person perception.

    PubMed

    Kuzmanovic, Bojana; Jefferson, Anneli; Bente, Gary; Vogeley, Kai

    2013-01-01

    Interpersonal impression formation is highly consequential for social interactions in private and public domains. These perceptions of others rely on different sources of information and processing mechanisms, all of which have been investigated in independent research fields. In social psychology, inferences about states and traits of others as well as activations of semantic categories and corresponding stereotypes have attracted great interest. On the other hand, research on emotion and reward demonstrated affective and motivational influences of social cues on the observer, which in turn modulate attention, categorization, evaluation, and decision processes. While inferential and categorical social processes have been shown to recruit a network of cortical brain regions associated with mentalizing and evaluation, the affective influence of social cues has been linked to subcortical areas that play a central role in detection of salient sensory input and reward processing. In order to extend existing integrative approaches to person perception, both the inferential-categorical processing of information about others, and affective and motivational influences of this information on the beholder should be taken into account. PMID:23781188

  6. How High Glucose Levels Affect Tendon Homeostasis.

    PubMed

    Snedeker, Jess G

    2016-01-01

    Among the many factors playing a role in tendon disease, unregulated biochemical reactions between glucose and the collagen extracellular matrix are coming increasingly into focus. We have shown that formation of advanced glycation end-products that cross-link the collagen extracellular matrix can drastically affect cellular level mechanical properties of the matrix, and in turn affect cell-level biomechanical stimuli during physiological loading of the tissue. We suggest that these may adversely affect tendon cell response to matrix damage, as well as the quality of the consequent repair. If such mechanical feedback loops are altered, the ability of tendon cells to maintain tissue in a functional, healthy state may be compromised. Although key foundational elements of biochemical, biomechanical, and biological understanding are now in place, the full extent of how these aspects interact, including the precise mechanisms by which advanced glycation end-products pathologically disrupt connective tissue homeostasis and damage repair, are only beginning to be adequately appreciated. PMID:27535261

  7. Providing Psychoeducational Support for Children Affected by AIDS.

    ERIC Educational Resources Information Center

    Black, Sandra

    This practicum evaluated a psychoeducational group format developed to support children living in families affected by HIV and AIDS. The major goals were to help children cope with stressful events, with chronic strain, and with role transitions. There were five objectives for the children: (1) decrease feelings of isolation, confusion, anger and…

  8. Negative Affect Impairs Associative Memory but Not Item Memory

    ERIC Educational Resources Information Center

    Bisby, James A.; Burgess, Neil

    2014-01-01

    The formation of associations between items and their context has been proposed to rely on mechanisms distinct from those supporting memory for a single item. Although emotional experiences can profoundly affect memory, our understanding of how it interacts with different aspects of memory remains unclear. We performed three experiments to examine…

  9. FACTORS ADVERSELY AFFECTING AMPHIBIAN POPULATIONS IN THE US

    EPA Science Inventory

    Factors known or suspected to be adversely affecting native amphibian populations in the US were identified using information from species accounts written in a standardized format by multiple authors in a forthcoming book. Specific adverse factors were identified for 53 (58%) of...

  10. Produce Surface Characteristics Affect Product Quality and Safety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The surface characteristics of fresh-cut fruits and vegetables vary largely among produce types, maturities and processing procedure. Studies have shown that the surface topography of produce significantly affected adherence, attachment, and biofilm formation of bacteria, as well as their removal a...

  11. A Digest of Supreme Court Decisions Affecting Education.

    ERIC Educational Resources Information Center

    Zirkel, Perry A., Ed.

    This digest is a reference tool documenting 144 of the most important Supreme Court cases affecting education from 1859 to 1977, with emphasis on the decisions made during the past 25 years. Each case is written up in a three-part format that provides the facts and background of the case. The cases are organized into five sections: school district…

  12. Tropical cyclone formation

    SciTech Connect

    Montgomery, M.T.; Farrell, B.F. )

    1993-01-15

    The physics of tropical cyclone formation is not well understood, and more is known about the mature hurricane than the formative mechanisms that produce it. It is believed part of the reason for this can be traced to insufficient upper-level atmospheric data. Recent observations suggest that tropical cyclones are initiated by asymmetric interactions associated with migratory upper-level potential vorticity disturbances and low-level disturbances. Favored theories of cyclones formation, however, focus on internal processes associated with cumulus convection and/or air-sea interaction. This work focuses on external mechanisms of cyclone formation and, using both a two- and three-dimensional moist geostrophic momentum model, investigates the role of upper-level potential vorticity disturbances on the formation process. A conceptual model of tropical cyclone formation is proposed, and implications of the theory are discussed. 71 refs., 5 figs., 1 tab.

  13. Blood plasma collected after adrenocorticotropic hormone administration during the preovulatory period in the sow negatively affects in vitro fertilization by disturbing spermatozoa function.

    PubMed

    González, R; Kumaresan, A; Bergqvist, A S; Sjunnesson, Y C B

    2015-04-15

    Successful fertilization is essential for reproduction and might be negatively affected by stressful events, which could alter the environment where fertilization occurs. The aim of the study was to determine whether an altered hormonal profile in blood plasma caused by adrenocorticotropic hormone (ACTH) administration could affect in vitro fertilization in the pig model. In experiment 1, gametes were exposed for 24 hours to plasma from ACTH-treated, non-ACTH-treated sows, or medium with BSA. Fertilization, cleavage, and blastocyst rates were lower in the ACTH group compared with the no ACTH or BSA control groups (P < 0.01). In experiment 2, the exposure of matured oocytes for