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Sample records for single blastocyst transfer

  1. 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

  2. Clinical outcomes of single versus double blastocyst transfer in fresh and vitrified-warmed cycles

    PubMed Central

    Eum, Jin Hee; Park, Jae Kyun; Kim, So Young; Paek, Soo Kyung; Seok, Hyun Ha; Chang, Eun Mi; Lee, Woo Sik

    2016-01-01

    Objective Assisted reproductive technology has been associated with an increase in multiple pregnancies. The most effective strategy for reducing multiple pregnancies is single embryo transfer. Beginning in October 2015, the National Supporting Program for Infertility in South Korea has limited the number of embryos that can be transferred per in vitro fertilization (IVF) cycle depending on the patient's age. However, little is known regarding the effect of age and number of transferred embryos on the clinical outcomes of Korean patients. Thus, this study was performed to evaluate the effect of the number of transferred blastocysts on clinical outcomes. Methods This study was carried out in the Fertility Center of CHA Gangnam Medical Center from January 2013 to December 2014. The clinical outcomes of 514 women who underwent the transfer of one or two blastocysts on day 5 after IVF and of 721 women who underwent the transfer of one or two vitrified-warmed blastocysts were analyzed retrospectively. Results For both fresh and vitrified-warmed cycles, the clinical pregnancy rate and live birth or ongoing pregnancy rate were not significantly different between patients who underwent elective single blastocyst transfer (eSBT) and patients who underwent double blastocyst transfer (DBT), regardless of age. However, the multiple pregnancy rate was significantly lower in the eSBT group than in the DBT group. Conclusion The clinical outcomes of eSBT and DBT were equivalent, but eSBT had a lower risk of multiple pregnancy and is, therefore, the best option. PMID:27689039

  3. Clinical outcomes of single versus double blastocyst transfer in fresh and vitrified-warmed cycles

    PubMed Central

    Eum, Jin Hee; Park, Jae Kyun; Kim, So Young; Paek, Soo Kyung; Seok, Hyun Ha; Chang, Eun Mi; Lee, Woo Sik

    2016-01-01

    Objective Assisted reproductive technology has been associated with an increase in multiple pregnancies. The most effective strategy for reducing multiple pregnancies is single embryo transfer. Beginning in October 2015, the National Supporting Program for Infertility in South Korea has limited the number of embryos that can be transferred per in vitro fertilization (IVF) cycle depending on the patient's age. However, little is known regarding the effect of age and number of transferred embryos on the clinical outcomes of Korean patients. Thus, this study was performed to evaluate the effect of the number of transferred blastocysts on clinical outcomes. Methods This study was carried out in the Fertility Center of CHA Gangnam Medical Center from January 2013 to December 2014. The clinical outcomes of 514 women who underwent the transfer of one or two blastocysts on day 5 after IVF and of 721 women who underwent the transfer of one or two vitrified-warmed blastocysts were analyzed retrospectively. Results For both fresh and vitrified-warmed cycles, the clinical pregnancy rate and live birth or ongoing pregnancy rate were not significantly different between patients who underwent elective single blastocyst transfer (eSBT) and patients who underwent double blastocyst transfer (DBT), regardless of age. However, the multiple pregnancy rate was significantly lower in the eSBT group than in the DBT group. Conclusion The clinical outcomes of eSBT and DBT were equivalent, but eSBT had a lower risk of multiple pregnancy and is, therefore, the best option.

  4. Prediction of clinical pregnancy in vitrified-warmed single blastocyst transfer cycles by pre-freeze morphology

    PubMed Central

    Zhang, Huan; Zhou, Ying; Li, Yan; Zheng, Yi; Xiao, Shiquan; Wu, Yonggen; Wang, Haiqing; Huang, Xuefeng

    2014-01-01

    Background: The selection of blastocyst warmed for transfer is based on pre-freeze morphology in vitrified-warmed single blastocyst transfer cycles. But, it is controversial which parameter of blastocyst morphology most closely related to the clinical outcomes. Objective: To estimate the effect of blastocoele expansion, trophectoderm (TE) morphology grade, and inner cell mass (ICM) morphology grade on clinical pregnancy in vitrified-warmed single blastocyst transfers. Materials and Methods: There were 172 vitrified-warmed single blastocyst transfer cycles during the year 2012 included in this analysis. Comparison of clinical results between pregnancy and no pregnancy group based on patient and blastocyst morphology characteristics was done. Then stepwise logistic regression analysis was used to select the best morphological predictor for clinical pregnancy. Last, comparison of patient characteristics and clinical outcomes separated by the best independent morphological predictor was done. Results: Comparison of clinical results between pregnancy and no pregnancy group and logistic regression showed the clinical pregnancy rate was affected by ICM. Comparison of patient characteristics separated by ICM grade, ICM grade A cycles got higher clinical pregnancy rate than ICM grade B cycles (54.3% vs. 35.0% respectively, p=0.037). Conclusion: Blastocyst with good ICM morphology could increase clinical pregnancy rate in vitrified-warmed single blastocyst transfer cycles. PMID:25408707

  5. Age-Related Success with Elective Single versus Double Blastocyst Transfer

    PubMed Central

    Friedman, Brooke E.; Davis, Lynn B.; Lathi, Ruth B.; Westphal, Lynn M.; Baker, Valerie L.; Milki, Amin A.

    2011-01-01

    Background. Although the optimal outcome of assisted reproductive technology (ART) is a healthy singleton pregnancy, the rate of twin gestation from ART in women over the age of 35 is persistently high. Methods/Findings. We compared clinical pregnancy rates (PRs), ongoing pregnancy/live birth rates, and multiple gestation rates (MGRs) in 108 women who chose elective single blastocyst transfer (eSBT) to 415 women who chose elective double blastocyst transfer (eDBT) at a hospital-based IVF center. There was no significant difference in PR between eSBT and eDBT (57.4% versus 50.2%, P = 0.47) nor between eSBT and eDBT within each age group: <35, 35–37, 38–40, and >40. The risk of multiple gestations, however, was greatly increased between eSBT and eDBT (1.6 versus 32.4%, P < 0.00005), and this difference did not vary across age groups. Conclusion(s). Women undergoing eDBT are at uniformly high risk of multiple gestation regardless of age. eSBT appears to significantly lower the risk of multiple gestation without compromising PR. PMID:22191047

  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. Controlled natural cycle IVF with antagonist use and blastocyst transfer.

    PubMed

    Trokoudes, K M; Minbattiwalla, M B; Kalogirou, L; Pantelides, K; Mitsingas, P; Sokratous, A; Chrysanthou, A; Fasouliotis, S J

    2005-12-01

    A method of controlled natural cycle IVF (CONCIVF) was sought to provide simpler and shorter treatment without the risks of ovarian hyperstimulation syndrome and multiple pregnancies. A total of 138 couples with normal ovulation and normal sperm parameters, in whom the women were <40 years old, were the candidates for this study. Gonadotrophin-releasing hormone antagonist was used before human chorionic gonadotrophin (HCG) administration if LH increased to a concentration of 10 mIU/ml before HCG injection. Treatment was initiated at > or =16 mm follicular growth and at oestradiol concentrations > or =400 pmol/l with 5000 IU HCG induction. All the embryos were cultured to the blastocyst stage and transferred only if they reached early or advanced blastulation. A total of 126 patients underwent oocyte retrieval. In 102 cases, one oocyte was retrieved; 95% of the oocytes fertilized, 99% cleaved and 47.9% achieved the blastocyst stage. The implantation rate per blastocyst transfer was 53.3% and the live-birth rate per embryo transfer was 40%. Therefore, CONCIVF with blastocyst transfer gives acceptable blastocyst development and implantation rates without the long- or short-term side effects of ovulation induction. PMID:16417731

  8. 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

    2015-09-03

    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.

  9. 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

  10. RNA-seq analysis of single bovine blastocysts

    PubMed Central

    2013-01-01

    Background Use of RNA-Seq presents unique benefits in terms of gene expression analysis because of its wide dynamic range and ability to identify functional sequence variants. This technology provides the opportunity to assay the developing embryo, but the paucity of biological material available from individual embryos has made this a challenging prospect. Results We report here the first application of RNA-Seq for the analysis of individual blastocyst gene expression, SNP detection, and characterization of allele specific expression (ASE). RNA was extracted from single bovine blastocysts (n = 5), amplified, and analyzed using high-throughput sequencing. Approximately 38 million sequencing reads were generated per embryo and 9,489 known bovine genes were found to be expressed, with a high correlation of expression levels between samples (r > 0.97). Transcriptomic data was analyzed to identify SNP in expressed genes, and individual SNP were examined to characterize allele specific expression. Expressed biallelic SNP variants with allelic imbalances were observed in 473 SNP, where one allele represented between 65-95% of a variant’s transcripts. Conclusions This study represents the first application of RNA-seq technology in single bovine embryos allowing a representation of the embryonic transcriptome and the analysis of transcript sequence variation to describe specific allele expression. PMID:23705625

  11. Monochorionic triamniotic triplets following conventional in vitro fertilization and blastocyst transfer

    PubMed Central

    Gurunath, Sumana; Makam, Adinarayana; Vinekar, Sriprada; Biliangady, Reeta H.

    2015-01-01

    Multiple pregnancy in in vitro fertilization (IVF) is on the decline with a reduction in number of embryos transferred. But the risk of monozygotic splitting persists. The risk of monozygotic twinning in women undergoing IVF is reported to be twice that of natural conception, and monochorionic triplets are even rarer at 100 times more than natural conception. We report a case of monochorionic triamniotic (MCTA) triplets following conventional IVF and blastocyst transfer without zona manipulation. This report highlights the possibility of zygotic splitting in IVF in young couples with no family history, in centers with good experience with blastocyst transfer. MCTA triplets carry a high risk of perinatal mortality and morbidity and need multidisciplinary care. Prevention and prediction of zygotic splitting ought to be realized with better reporting and identification of possible risk factors. PMID:25838751

  12. Efficacy of oocyte vitrification combined with blastocyst stage transfer in an egg donation program.

    PubMed

    García, Javier I; Noriega-Portella, Luis; Noriega-Hoces, Luis

    2011-04-01

    BACKGROUND A successful oocyte vitrification program is important for women with various indications for assisted reproduction technology. The objective of this study was to report the outcome of vitrification of oocytes, obtained through an oocyte donation program, by evaluating the embryo development, pregnancy and implantation rates (IRs) after blastocyst transfer. METHODS A total of 1098 oocytes were obtained from 78 donors. There were 312 oocytes used in the study group (vitrified oocytes) and 786 used in the control group (fresh oocytes). There were 34 recipients who received blastocysts obtained from vitrified oocytes and 58 recipients who received blastocysts from fresh oocytes. The fertilization rate, cleavage rate, embryo quality, pregnancy rate (PR) and IR were compared between groups. RESULTS Vitrified oocytes showed a survival rate of 89.4%. There was no difference in the fertilization rate (76.1 and 87.5%), Day 2 cleavage rate (96.3 and 98.0%) or blastocyst formation rate (41.3 and 45.3%) for the study and control groups, respectively. PRs, IRs and miscarriages rates (MRs) were similar for the study group compared with the control group (PR: 61.8 versus 60.0%; IR: 43.9 versus 42.9%; MR: 9.5 versus 5.9%). CONCLUSIONS The developmental competence of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, since they preserve the potential to be fertilized and to develop into high-quality blastocysts, similar to embryos from fresh oocytes. The successful clinical outcome indicates the use of this procedure for oocyte donation programs and for oocyte storage in general.

  13. 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

  14. Development of buffalo (Bubalus bubalis) embryonic stem cell lines from somatic cell nuclear transferred blastocysts.

    PubMed

    Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan Singh

    2015-11-01

    We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES) cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions. PMID:26987926

  15. 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

  16. Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

    PubMed

    Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

    2014-08-01

    Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, p<0.05), compact morulae formation (60.83 vs. 51.30%, p<0.05), and the blastomere apoptosis index (3.70 ± 1.41 vs. 4.43% ± 1.65, p<0.05) of bovine SCNT embryos. However, vitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, p<0.05) on day 7 and the hatching blastocysts formation rate on day 9 (26.51 vs. 50.65%, p<0.05) compared with that of the untreated group. Vitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

  17. 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

  18. Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro.

    PubMed

    Wen, Duan-Cheng; Bi, Chun-Ming; Xu, Ying; Yang, Cai-Xia; Zhu, Zi-Yu; Sun, Qing-Yuan; Chen, Da-Yuan

    2005-08-01

    The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.

  19. Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles

    PubMed Central

    2014-01-01

    Background Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Methods Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. Results A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The

  20. Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

    PubMed Central

    Degrelle, Séverine A.; Jaffrezic, Florence; Campion, Evelyne; Lê Cao, Kim-Anh; Le Bourhis, Daniel; Richard, Christophe; Rodde, Nathalie; Fleurot, Renaud; Everts, Robin E.; Lecardonnel, Jérôme; Heyman, Yvan; Vignon, Xavier; Tian, Xiuchun C.; Lewin, Harris A.; Renard, Jean-Paul; Hue, Isabelle

    2012-01-01

    Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters

  1. 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

  2. Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

    PubMed Central

    Ni, Wuhua; Xiao, Shiquan; Qiu, Xiufang; Jin, Jianyuan; Pan, Chengshuang; Li, Yan; Fei, Qianjin; Yang, Xu; Zhang, Liya; Huang, Xuefeng

    2014-01-01

    During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles. PMID:24733108

  3. 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

  4. A comparative study between cleavage stage embryo transfer at day 3 and blastocyst stage transfer at day 5 in in-vitro fertilization/intra-cytoplasmic sperm injection on clinical pregnancy rates

    PubMed Central

    Kaur, Prabhleen; Swarankar, M. L.; Maheshwari, Manju; Acharya, Veena

    2014-01-01

    OBJECTIVE: To evaluate the efficacy of blastocyst transfer in comparison with cleavage stage transfer. STUDY DESIGN: A randomized, prospective study was conducted in Infertility clinic, Department of Obstetrics and Gynecology, Mahatma Gandhi Hospital, Jaipur on 300 patients aged 25-40 years undergoing in-vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) cycle from May 2010-April 2011. When three or more Grade-I embryos were observed on day 2 of culture, patients were divided randomly into two study groups, cleavage stage transfer and blastocyst transfer group having 150 patients each. Primary outcomes evaluated were, Clinical pregnancy rate and Implantation rate. The results were analyzed using proportions, standard deviation and Chi-square test. RESULTS: Both the groups were similar for age, indication and number of embryos transferred. Clinical pregnancies after blastocyst transfer were significantly higher 66 (44.0%) compared to cleavage stage embryo transfer 44 (29.33%) (P < 0.01). Implantation rate for blastocyst transfer group was also significantly higher (P < 0.001). CONCLUSION: Blastocyst transfer having higher implantation rate and clinical pregnancy rate lead to reduction in multiple pregnancies. PMID:25395745

  5. 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

  6. Intrafollicular Oocyte Transfer (IFOT) of Abattoir-Derived and In Vitro-Matured Oocytes Results in Viable Blastocysts and Birth of Healthy Calves.

    PubMed

    Kassens, Ana; Held, Eva; Salilew-Wondim, Dessie; Sieme, Harald; Wrenzycki, Christine; Tesfaye, Dawit; Schellander, Karl; Hoelker, Michael

    2015-06-01

    There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13-14 mm delivered significantly (P < 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9-10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P < 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa

  7. An earlier uterine environment favors the in vivo development of fresh pig morulae and blastocysts transferred by a nonsurgical deep-uterine method.

    PubMed

    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.

  8. 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

  9. 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

  10. 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

  11. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq

    PubMed Central

    Blakeley, Paul; Fogarty, Norah M. E.; del Valle, Ignacio; Wamaitha, Sissy E.; Hu, Tim Xiaoming; Elder, Kay; Snell, Philip; Christie, Leila; Robson, Paul; Niakan, Kathy K.

    2015-01-01

    Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells. PMID:26293300

  12. 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.

  13. Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer.

    PubMed

    Iwamoto, Masaki; Onishi, Akira; Fuchimoto, Dai-ichiro; Somfai, Tamas; Suzuki, Shun-ichi; Yazaki, Satoko; Hashimoto, Michiko; Takeda, Kumiko; Tagami, Takahiro; Hanada, Hirofumi; Noguchi, Junko; Kaneko, Hiroyuki; Nagai, Takashi; Kikuchi, Kazuhiro

    2005-11-01

    The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus-oocyte complexes (COCs) were cultured initially for 36 h and subsequently with or without 5 mM caffeine for 24 h (in total for 60 h: 60CA+ or 60CA- group, respectively). As a control group, COCs were cultured for 48 h without caffeine (48CA-). The pronuclear formation rates at 10 h after electrical stimulation in the 60CA+ and 60CA- groups decreased significantly (p < 0.05) compared with the 48CA- group. However, the fragmentation rate was significantly higher (p < 0.05) in the 60CA- group than in the 60CA+ and 48CA- groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p < 0.05) than the 48CA- group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1-11 h) before the completion of maturation (48 or 60 h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1-2 h in the 60CA- oocytes was significantly lower (p < 0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA- group tended to be lower (p = 0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA- groups were significantly lower (p < 0.05) than in the 48CA- group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the

  14. 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

  15. Cloned endangered species takin (Budorcas taxicolor) by inter-species nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine.

    PubMed

    Li, Yanxin; Dai, Yunping; Du, Weihua; Zhao, Chunjiang; Wang, Haiping; Wang, Lili; Li, Rong; Liu, Ying; Wan, Rong; Li, Ning

    2006-02-01

    Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of takin-bovine embryos in vitro produced by fusing takin ear fibroblasts with enucleated bovine oocytes and examine the fate of mitochondrial DNA in these embryos. We also compare the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compare the cell numbers of the blastocyst. Our results indicate that: (1) takin-bovine cloned embryos can develop to the blastocyst stage in vitro (5%), (2) blastocyst mitochondria DNA are derived primarily from bovine oocytes in spite of a little takin donor cell mitochondrial DNA, (3) using the same cloned protocol, development efficiency is significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48 vs. 28% vs. 5%, P < 0.01), and (4) cell numbers in the blastocysts of the three species of embryos were not different. These results suggest that the bovine oocytes can reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tends to be higher than inter-species cloning; the more close the species of the donor cell is to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.

  16. 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

  17. Transcriptome Analysis of Pig In Vivo, In Vitro–Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway

    PubMed Central

    Whitworth, Kristin M.; Mao, Jiude; Lee, Kiho; Spollen, William G.; Samuel, Melissa S.; Walters, Eric M.; Spate, Lee D.

    2015-01-01

    Abstract Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro–fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 μM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 μM ISAHA had the same percentage of blastocyst development as Scriptaid (p<0.05). Two treatments, 1.0 μM ISAHA and 1.0 μM SAHA, had higher mean cell number than No HDACi treatment (p<0.021). Embryo transfers performed with 10 μM SAHA- and 1 μM ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos (p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT. PMID:26731590

  18. Blastocyst microinjection automation.

    PubMed

    Mattos, Leonardo S; Grant, Edward; Thresher, Randy; Kluckman, Kimberly

    2009-09-01

    Blastocyst microinjections are routinely involved in the process of creating genetically modified mice for biomedical research, but their efficiency is highly dependent on the skills of the operators. As a consequence, much time and resources are required for training microinjection personnel. This situation has been aggravated by the rapid growth of genetic research, which has increased the demand for mutant animals. Therefore, increased productivity and efficiency in this area are highly desired. Here, we pursue these goals through the automation of a previously developed teleoperated blastocyst microinjection system. This included the design of a new system setup to facilitate automation, the definition of rules for automatic microinjections, the implementation of video processing algorithms to extract feedback information from microscope images, and the creation of control algorithms for process automation. Experimentation conducted with this new system and operator assistance during the cells delivery phase demonstrated a 75% microinjection success rate. In addition, implantation of the successfully injected blastocysts resulted in a 53% birth rate and a 20% yield of chimeras. These results proved that the developed system was capable of automatic blastocyst penetration and retraction, demonstrating the success of major steps toward full process automation.

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

    PubMed

    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.

  20. Heterotopic pregnancy after a single embryo transfer

    PubMed Central

    Lee, Ji Sun; Cha, Hyun-Hwa; Han, Ae Ra; Lee, Seong Goo

    2016-01-01

    Heterotopic pregnancy is a rare and life-threatening condition which is defined as coexistent intrauterine and ectopic gestation. The risk of ectopic and heterotopic pregnancy is increasing due to the increased risk of multiple pregnancies with the aid of assisted reproductive technologies. However, it hardly happens in the setting of single embryo transfer, since single embryo transfer significantly reduces the incidence of multiple pregnancies. Surprisingly, we experienced a case of heterotopic pregnancy after a single embryo transfer caused by coincidental natural pregnancy during assisted reproductive technologies. An infertile woman who underwent, during her natural cycle, transfer of a single embryo that had been cryopreserved for 3 years was found to be heterotopically pregnant. After an early and successful management with laparoscopic right salpingectomy, she finally reached at full-term vaginal delivery. PMID:27462600

  1. Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes.

    PubMed

    Sotomaru, Y; Kato, Y; Tsunoda, Y

    1999-07-15

    Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.

  2. 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

  3. 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

  4. Single Molecule Spectroscopy of Electron Transfer

    SciTech Connect

    Michael Holman; Ling Zang; Ruchuan Liu; David M. Adams

    2009-10-20

    The objectives of this research are threefold: (1) to develop methods for the study electron transfer processes at the single molecule level, (2) to develop a series of modifiable and structurally well defined molecular and nanoparticle systems suitable for detailed single molecule/particle and bulk spectroscopic investigation, (3) to relate experiment to theory in order to elucidate the dependence of electron transfer processes on molecular and electronic structure, coupling and reorganization energies. We have begun the systematic development of single molecule spectroscopy (SMS) of electron transfer and summaries of recent studies are shown. There is a tremendous need for experiments designed to probe the discrete electronic and molecular dynamic fluctuations of single molecules near electrodes and at nanoparticle surfaces. Single molecule spectroscopy (SMS) has emerged as a powerful method to measure properties of individual molecules which would normally be obscured in ensemble-averaged measurement. Fluctuations in the fluorescence time trajectories contain detailed molecular level statistical and dynamical information of the system. The full distribution of a molecular property is revealed in the stochastic fluctuations, giving information about the range of possible behaviors that lead to the ensemble average. In the case of electron transfer, this level of understanding is particularly important to the field of molecular and nanoscale electronics: from a device-design standpoint, understanding and controlling this picture of the overall range of possible behaviors will likely prove to be as important as designing ia the ideal behavior of any given molecule.

  5. 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

  6. Single-embryo transfer versus multiple-embryo transfer.

    PubMed

    Gerris, Jan

    2009-01-01

    Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology. PMID:19406034

  7. Single-embryo transfer versus multiple-embryo transfer.

    PubMed

    Gerris, Jan

    2009-01-01

    Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology.

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

    PubMed

    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

  9. 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

  10. Monozygous double inner cell masses in mouse blastocysts following fertilization in vitro and in vivo.

    PubMed

    Chida, S

    1990-06-01

    The incidence of human identical twins after in vitro fertilization and embryo transfer (IVF-ET) is estimated to be higher than the commonly accepted incidence after conception in vivo. This paper reports that 8 mouse blastocysts which had double inner cell masses in 261 blastocysts (3.1%) were identified after fertilization in vitro and they developed into trophoblastic outgrowth formation with two inner cell masses. In contrast, only 3 monozygous double inner cell masses were observed in 526 blastocysts (0.6%) which were fertilized in vivo and cultured from the two-cell stage in vitro. It is therefore possible that fertilization in vitro predisposes to monozygous twinning.

  11. Single embryo transfer - state of the art.

    PubMed

    De Neubourg, Diane; Gerris, Jan

    2003-12-01

    Every practitioner active in the field of assisted reproduction treatment is aware of the risks and complications related to twin and higher-order multiple pregnancies. Introduction of single embryo transfer (SET) into IVF/intracytoplasmic sperm injection (ICSI) is one of the possible ways of reducing the rate of twin pregnancy. Careful selection of patients, in combination with elective SET, has been shown to decrease the twin pregnancy rate while maintaining a stable ongoing pregnancy rate. The combination of a woman younger than 38 years of age, in her first or second IVF/ICSI cycle and with an embryo with a high implantation potential is the key to successful SET. This article will discuss embryo selection and patient selection and review the data published on SET. In the Centre for Reproductive Medicine at Middelheim Hospital, 39% of all transfers in 2002 were SET; the ongoing pregnancy rate remained stable at 30.6%. The twin (multiple) pregnancy rate declined to 11.7%. Particular attention should be drawn to the augmenting effect of the pregnancy rate of frozen-thawed cycles. Health economic data available so far subscribe the plea for SET.

  12. Avoiding multiple pregnancies in ART: a plea for single embryo transfer.

    PubMed

    Gerris, J; Van Royen, E

    2000-09-01

    It has been generally accepted that triplets after IVF/intracytoplasmic sperm injection (ICSI) can and should be avoided by adopting a standard strategy of replacing no more than two embryos. However, there is an increasing awareness of the risks and costs and of the epidemic size of twin pregnancies after IVF/ICSI. This has resulted in efforts to replace no more than one embryo. However, this approach has been hampered by our relative inability to identify embryos with a very high implantation potential. To identify such embryos, a number of strategies are being considered, both at the two pronuclear (2PN), early cleavage and the blastocyst stages. At the 2PN stage, the polarity characteristics of the nucleoli have been shown to be correlated with a high implantation rate. Similarly, the morphological characteristics at day 2 and 3 have been used to describe top quality embryos in approximately 75% of all IVF/ICSI cycles. Blastocyst culture has resulted in very high implantation rates in the hands of some authors. No approach has shown its superiority at present, but initial experience with single embryo transfer (SET) at the early cleavage stage by Scandinavian and Belgian groups shows that an ongoing pregnancy rate of 35% and more can be achieved. Proper identification of patients at risk of a twin pregnancy after double embryo transfer is equally important. It is clear that mainly young patients (aged <34 years) during their first, perhaps first two, IVF/ICSI cycles constitute the main population at risk (responsible for >80% of all twins) and are the main target group for twin prevention by SET of a top quality embryo at whatever stage. Therefore, in our opinion, although a further fine-tuning of both embryo and patient characteristics relating to a high risk for (twin) pregnancy is desirable, SET should be introduced carefully and progressively in each IVF/ICSI programme from now on. Correct counselling is very important and both public and private insurers

  13. Successful production of piglets derived from expanded blastocysts vitrified using a micro volume air cooling method without direct exposure to liquid nitrogen.

    PubMed

    Misumi, Koji; Hirayama, Yuri; Egawa, Sachiko; Yamashita, Shoko; Hoshi, Hiroyoshi; Imai, Kei

    2013-12-17

    This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN₂). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN₂ for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN₂. PMID:23955236

  14. The precise timing of embryo splitting for monozygotic dichorionic diamniotic twins: when does embryo splitting for monozygotic dichorionic diamniotic twins occur? Evidence for splitting at the morula/blastocyst stage from studies of in vitro fertilization.

    PubMed

    Kyono, Koichi

    2013-08-01

    There is a long-held credo, as illustrated in Langman's Medical Embryology (11th ed., Sadler, 2010), that dichorionic diamniotic (DD) twins develop after embryo splitting in the early stages of embryonic development. However, from our clinical experiences of the examination of data from single-embryo transfers in 16 fertility clinics in Japan and from various reports, the majority of occurrences of DD twins have been found in the blastocyst stages.

  15. 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

  16. Successful cryopreservation of mouse blastocysts using a new vitrification solution.

    PubMed

    Valdez, C A; Abas Mazni, O; Takahashi, Y; Fujikawa, S; Kanagawa, H

    1992-11-01

    Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20-22 degrees C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l-1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0.01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96.2%) or exposed to VSv for 0.5 min (95.4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the

  17. Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion).

    PubMed

    Saadeldin, Islam M; Khoirinaya, Candrani; Kim, Su Jin; Moon, Joon Ho; Almadaly, Essam; Lee, Byeong Chun

    2016-02-01

    This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP-expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two-cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP-expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose "prematuration somatic cell fusion" as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.

  18. A tetrastable naphthalenediimide: anion induced charge transfer, single and double electron transfer for combinational logic gates.

    PubMed

    Ajayakumar, M R; Hundal, Geeta; Mukhopadhyay, Pritam

    2013-09-11

    Herein we demonstrate the formation of the first tetrastable naphthalenediimide (NDI, 1a) molecule having multiple distinctly readable outputs. Differential response of 1a to fluoride anions induces intramolecular charge transfer (ICT), single/double electron transfer (SET/DET) leading to a set of combinational logic gates for the first time with a NDI moiety. PMID:23752683

  19. Correlated Single Quantum Dot Blinking and Interfacial Electron Transfer Dynamics.

    PubMed

    Jin, Shengye; Hsiang, Jung-Cheng; Zhu, Haiming; Song, Nianhui; Dickson, Robert M; Lian, Tianquan

    2010-08-31

    The electron transfer (ET) dynamics from core/multi-shell (CdSe/CdS(3ML)ZnCdS(2ML)ZnS(2ML)) quantum dots (QDs) to adsorbed Fluorescein (F27) molecules have been studied by single particle spectroscopy to probe the relationship between single QD interfacial electron transfer and blinking dynamics. Electron transfer from the QD to F27 and the subsequent recombination were directly observed by ensemble-averaged transient absorption spectroscopy. Single QD-F27 complexes show correlated fluctuation of fluorescence intensity and lifetime, similar to those observed in free QDs. With increasing ET rate (controlled by F27-to-QD ratio), the lifetime of on states decreases and relative contribution of off states increases. It was shown that ET is active for QDs in on states, the excited state lifetime of which reflects the ET rate, whereas in the off state QD excitons decay by Auger relaxation and ET is not a competitive quenching pathway. Thus, the blinking dynamics of single QDs modulate their interfacial ET activity. Furthermore, interfacial ET provides an additional pathway for generating off states, leading to correlated single QD interfacial ET and blinking dynamics in QD-acceptor complexes. Because blinking is a general phenomenon of single QDs, it appears that the correlated interfacial ET and blinking and the resulting intermittent ET activity are general phenomena for single QDs.

  20. Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

    PubMed

    Skrzyszowska, M; Smorag, Z; Katska, L

    1997-09-01

    It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (blastocyst through the perforation and formation of an incomplete demi-embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

  1. Outcomes of elective cryopreserved single or double embryo transfers following failure to conceive after fresh single embryo transfer.

    PubMed

    Monteleone, Pedro Augusto Araújo; Mirisola, R J; Gonçalves, S P; Baracat, Edmund C; Serafini, Paulo C

    2016-08-01

    The main adverse effect of IVF is the high multiple pregnancy rate resulting from the transfer of two or more embryos. The objective was to evaluate pregnancy rates in infertile women with a good prognosis who failed to conceive in a fresh elective single embryo transfer (eSET) and had a second cycle with elective double vitrified-warmed embryo transfer (eDFET) compared with elective single vitrified-warmed embryo transfer (eSFET). A total of 142 intracytoplasmic sperm injection cycles using a conventional protocol were evaluated. Good-prognosis patients underwent eSET in a fresh cycle, and those who failed to conceive underwent a second vitrified-warmed embryo transfer: eDFET (n = 102) or eSFET (n = 40). Embryos were transferred and vitrified on day 5 of development. Patients who received eDFET had fewer implantations (30.9%) than eSFET (52.5%; P = 0.004); pregnancy rates were similar (eDFET: 35.3%, eSFET: 42.5%). Patients with the eSFET had one monozygotic twin (5.9%), and 22.2% of eDFET patients had multiple pregnancies. Patients with a good prognosis who failed to conceive in the first fresh eSET did not have an advantage when receiving eDFET in the second cycle, as pregnancy rates were similar; 22.2% of patients in the eDFET group had multiple pregnancies. PMID:27317130

  2. Aqueous proton transfer across single-layer graphene

    DOE PAGES

    Achtyl, Jennifer L.; Unocic, Raymond R.; Xu, Lijun; Cai, Yu; Raju, Muralikrishna; Zhang, Weiwei; Sacci, Robert L.; Vlassiouk, Ivan V.; Fulvio, Pasquale F.; Ganesh, Panchapakesan; et al

    2015-03-17

    Proton transfer across single-layer graphene proceeds with large computed energy barriers and is thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused ​silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energymore » barriers of 0.61–0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while ​pyrylium-like ether terminations shut down proton exchange. In conclusion, unfavourable energy barriers to helium and ​hydrogen transfer indicate the process is selective for aqueous protons.« less

  3. Aqueous proton transfer across single-layer graphene

    PubMed Central

    Achtyl, Jennifer L.; Unocic, Raymond R.; Xu, Lijun; Cai, Yu; Raju, Muralikrishna; Zhang, Weiwei; Sacci, Robert L.; Vlassiouk, Ivan V.; Fulvio, Pasquale F.; Ganesh, Panchapakesan; Wesolowski, David J.; Dai, Sheng; van Duin, Adri C. T.; Neurock, Matthew; Geiger, Franz M.

    2015-01-01

    Proton transfer across single-layer graphene proceeds with large computed energy barriers and is therefore thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energy barriers of 0.61–0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while pyrylium-like ether terminations shut down proton exchange. Unfavourable energy barriers to helium and hydrogen transfer indicate the process is selective for aqueous protons. PMID:25781149

  4. Aqueous proton transfer across single-layer graphene

    SciTech Connect

    Achtyl, Jennifer L.; Unocic, Raymond R.; Xu, Lijun; Cai, Yu; Raju, Muralikrishna; Zhang, Weiwei; Sacci, Robert L.; Vlassiouk, Ivan V.; Fulvio, Pasquale F.; Ganesh, Panchapakesan; Wesolowski, David J.; Dai, Sheng; van Duin, Adri C. T.; Neurock, Matthew; Geiger, Franz M.

    2015-03-17

    Proton transfer across single-layer graphene proceeds with large computed energy barriers and is thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused ​silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid–base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energy barriers of 0.61–0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while ​pyrylium-like ether terminations shut down proton exchange. In conclusion, unfavourable energy barriers to helium and ​hydrogen transfer indicate the process is selective for aqueous protons.

  5. Aqueous proton transfer across single-layer graphene

    NASA Astrophysics Data System (ADS)

    Achtyl, Jennifer L.; Unocic, Raymond R.; Xu, Lijun; Cai, Yu; Raju, Muralikrishna; Zhang, Weiwei; Sacci, Robert L.; Vlassiouk, Ivan V.; Fulvio, Pasquale F.; Ganesh, Panchapakesan; Wesolowski, David J.; Dai, Sheng; van Duin, Adri C. T.; Neurock, Matthew; Geiger, Franz M.

    2015-03-01

    Proton transfer across single-layer graphene proceeds with large computed energy barriers and is therefore thought to be unfavourable at room temperature unless nanoscale holes or dopants are introduced, or a potential bias is applied. Here we subject single-layer graphene supported on fused silica to cycles of high and low pH, and show that protons transfer reversibly from the aqueous phase through the graphene to the other side where they undergo acid-base chemistry with the silica hydroxyl groups. After ruling out diffusion through macroscopic pinholes, the protons are found to transfer through rare, naturally occurring atomic defects. Computer simulations reveal low energy barriers of 0.61-0.75 eV for aqueous proton transfer across hydroxyl-terminated atomic defects that participate in a Grotthuss-type relay, while pyrylium-like ether terminations shut down proton exchange. Unfavourable energy barriers to helium and hydrogen transfer indicate the process is selective for aqueous protons.

  6. Role of Nuclear Receptors in Blastocyst Implantation

    PubMed Central

    Vasquez, YM; DeMayo, FJ

    2013-01-01

    The regulation of blastocyst implantation in the uterus is orchestrated by the ovarian hormones estrogen and progesterone. These hormones act via their nuclear receptors to direct the transcriptional activity of the endometrial compartments and create a defined period in which the uterus is permissive to embryo implantation termed the “window of receptivity”. Additional members of the nuclear receptor family have also been described to have a potential role in endometrial function. Much of what we know about the function of these nuclear receptors during implantation we have learned from the use of mouse models. Transgenic murine models with targeted gene ablation have allowed us to identify a complex network of paracrine signaling between the endometrial epithelium and stroma. While some of the critical molecules have been identified, the mechanism underlying the intricate communication between endometrial compartments during the implantation window has not been fully elucidated. Defining this mechanism will help identify markers of a receptive uterine environment, ultimately providing a useful tool to help improve the fertility outlook for reproductively challenged couples. The aim of this review is to outline our current understanding of how nuclear receptors and their effector molecules regulate blastocyst implantation in the endometrium. PMID:23994285

  7. Dissipation-enabled efficient excitation transfer from a single photon to a single quantum emitter

    NASA Astrophysics Data System (ADS)

    Trautmann, N.; Alber, G.

    2016-05-01

    We propose a scheme for triggering a dissipation-dominated highly efficient excitation transfer from a single-photon wave packet to a single quantum emitter. This single-photon-induced optical pumping turns dominant dissipative processes, such as spontaneous photon emission by the emitter or cavity decay, into valuable tools for quantum information processing and quantum communication. It works for an arbitrarily shaped single-photon wave packet with sufficiently small bandwidth provided a matching condition is satisfied which balances the dissipative rates involved. Our scheme does not require additional laser pulses or quantum feedback and does not rely on high finesse optical resonators. In particular, it can be used to enhance significantly the coupling of a single photon to a single quantum emitter implanted in a one-dimensional waveguide or even in a free space scenario. We demonstrate the usefulness of our scheme for building a deterministic quantum memory and a deterministic frequency converter between photonic qubits of different wavelengths.

  8. Differential gene expression profile in bovine blastocysts resulting from hyperglycemia exposure during early cleavage stages.

    PubMed

    Cagnone, Gaël L M; Dufort, Isabelle; Vigneault, Christian; Sirard, Marc-André

    2012-02-01

    To understand the compromised survival of embryos derived from assisted reproductive techniques, transcriptome survey of early embryonic development has shown the impact of in vitro culture environment on gene expression in bovine or other living species. However, how the differentially expressed genes translate into developmentally compromised embryos is unresolved. We therefore aimed to characterize transcriptomic markers expressed by bovine blastocysts cultured in conditions that are known to impair embryo development. As increasing glucose concentrations has been shown to be stressful for early cleavage stages of mammalian embryos and to decrease subsequent blastocyst survival, in vitro-matured/fertilized bovine zygotes were cultured in control (0.2 mM) or high-glucose (5 mM) conditions until the 8- to 16-cell stage, and then transferred to control media until they reached the blastocyst stage. The concentration of 5 mM glucose was chosen as a stress treatment because there was a significant effect on blastocyst rate without the treatment's being lethal as with 10 mM. Microarray analysis revealed gene expression differences unrelated to embryo sex or hatching. Overrepresented processes among differentially expressed genes in treated blastocysts were extracellular matrix signalling, calcium signaling, and energy metabolism. On a pathophysiological level, higher glucose treatment impacts pathways associated with diabetes and tumorigenesis through genes controlling the Warburg effect, i.e., emphasis on use of anaerobic glycolysis rather than oxidative phosphorylation. These results allowed us to conclude that disruption of in vitro preattachment development is concomitant with gene expression modifications involved in metabolic control.

  9. Single and multiple jet impingement heat transfer on rotating disks

    NASA Astrophysics Data System (ADS)

    Metzger, D. E.; Partipilo, V. A.

    1989-01-01

    In some gas turbine engine designs cooling air jets are directed at the rotating disk in an atempt to enhance the convection coefficients and reduce the amount of gas flow required for cooling. The jet-impingement scheme is particularly attractive for achieving intense cooling at a specific radial location, such as the blade attachment region. In earlier single-jet studies, the interaction between an impinging jet and rotating disk has been found to involve a flow regime transition. The present study extends the previously acquired data base with new results from both heat-transfer and flow-visualization testing, including effects of hub size, jet travel distance, and the number of jets. Results include a superposition scheme for predicting heat transfer for multiple jets and a criterion for the minimum amount of flow required through each jet nozzle to assure enhancement of the disk convection.

  10. Condensation heat transfer of steam on a single horizontal tube

    NASA Astrophysics Data System (ADS)

    Graber, K. A.

    1983-06-01

    An experimental apparatus was designed, constructed and instrumented in an effort to systematically and carefully study the condensation heat-transfer coefficient on a single, horizontal tube. A smooth, thick-walled copper tube of length 133.5 mm, with an outside diameter of 15.9 mm and an inside diameter of 12.7 mm was instrumented with six wall thermocouples. The temperature rise across the test section was measured accurately using quartz crystal thermometers. The inside heat-transfer coefficient was determined using the Sieder-Tate correlation with leading coefficient of 0.029. Initial steam side data were taken at atmospheric pressure to test the data acquisition/reduction computer programs.

  11. Mass transfer experiments on single irregular-shaped particles

    SciTech Connect

    Ramezan, M. ); Kale, S.R. ); Anderson, R.J. )

    1991-01-01

    Mass transfer from irregular-shaped naphthalene particles (100-200 {mu}m in size) was studied in an electrodynamic balance. Charged particles were suspended in an electrostatic field directly in line with a calibrated air jet. Mass and size change histories were obtained under ambient conditions, and under steady- and pulsed-flow conditions. For natural convection, the time-averaged Sherwood number was similar to that for spheres. Forced-convection Sherwood number under steady-flow conditions was strongly dependent on particle shape and particle Reynolds number, and was consistently higher than values predicted for spheres at comparable Reynolds numbers. This paper validates the technique and indicates the shape effect on mass transfer from single particles.

  12. Single Electron Transfer Living Radical Polymerization via a New Initiator

    NASA Astrophysics Data System (ADS)

    Bai, Xiongxiong; Hu, Ying; Zhang, Xu; Ai, Lingling; Cheng, Chuanjie

    2014-08-01

    Research and development of novel initiating system such as single electron transfer living radical polymerization (SET-LRP) is of high importance in polymer chemistry. A new SET-LRP initiator was synthesized and applied to prepare end-functionalized poly(methyl methacrylate) (PMMA) in this study. α-Trichloromethyl benzyl alcohol was firstly synthesized, followed by preparation of PMMA under SET-LRP conditions. Conversion of MMA was 81.9%, and the molecular weight of PMMA was about 2.5 kDa at 60 °C for 1 h. Consistency of the number-average molecular weight of PMMA from NMR, GPC and theoretical calculation indicated that the polymerization featured controllable property. Broad molecular weight distribution (MWD) may be ascribed to branched polymers formed by initiation and chain transfer.

  13. 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

  14. Single photon time transfer link model for GNSS satellites

    NASA Astrophysics Data System (ADS)

    Vacek, Michael; Michalek, Vojtech; Peca, Marek; Prochazka, Ivan; Blazej, Josef

    2015-05-01

    The importance of optical time transfer serving as a complement to traditional microwave links, has been attested for GNSSes and for scientific missions. Single photon time transfer (SPTT) is a process, allowing to compare (subtract) time readings of two distant clocks. Such a comparison may be then used to synchronize less accurate clock to a better reference, to perform clock characterization and calibration, to calculate mean time out of ensemble of several clocks, displaced in space. The single-photon time transfer is well established in field of space geodesy, being supported by passive retro-reflectors within space segment of five known GNSSes. A truly two-way, active terminals work aboard of Jason-2 (T2L2) - multiphoton operation, GNSS Beidou (Compass) - SPTT, and are going to be launched within recent ACES project (ELT) - SPTT, and GNSS GLONASS - multiphoton operation. However, there is still missing comprehensive theoretical model of two-way (using satellite receiver and retroreflector) SPTT link incorporating all crucial parameters of receiver (both ground and space segment receivers), transmitter, atmosphere effects on uplink and downlink path, influence of retroreflector. The input to calculation of SPTT link performance will be among others: link budget (distance, power, apertures, beam divergence, attenuation, scattering), propagating medium (atmosphere scintillation, beam wander, etc.), mutual Tx/Rx velocity, wavelength. The SPTT model will be evaluated without the properties of real components. These will be added in the further development. The ground-to-space SPTT link performance of typical scenarios are modeled. This work is a part of the ESA study "Comparison of optical time-transfer links."

  15. Single phase channel flow forced convection heat transfer

    SciTech Connect

    Hartnett, J.P.

    1999-04-01

    A review of the current knowledge of single phase forced convection channel flow of liquids (Pr > 5) is presented. Two basic channel geometries are considered, the circular tube and the rectangular duct. Both laminar flow and turbulent flow are covered. The review begins with a brief overview of the heat transfer behavior of Newtonian fluids followed by a more detailed presentation of the behavior of purely viscous and viscoelastic Non-Newtonian fluids. Recent developments dealing with aqueous solutions of high molecular weight polymers and aqueous solutions of surfactants are discussed. The review concludes by citing a number of challenging research opportunities.

  16. Extended culture up to the blastocyst stage: a strategy to avoid multiple pregnancies in assisted reproductive technologies.

    PubMed

    Sepúlveda, Soledad J; Portella, Jimmy R; Noriega, Luis P; Escudero, Ernesto L; Noriega, Luis H

    2011-01-01

    The aim of this study was to review the experience and outcomes of assisted reproduction cycles with embryos grown up to day 5 of development, comparing different parameters according to the ages of the patients. We retrospectively studied 1,874 assisted reproduction cycles where embryo culture was extended up to the fifth or sixth day of development. All IVF and ICSI cycles were included, comparing, according to patient age, the following rates: blastocyst formation, pregnancy, implantation and abortion. As control, we analyzed cycles with donated oocytes from young donors (OD). The number of embryos reaching the blastocyst stage is similar in all groups of patients. Only the OD group was different in terms of blastocyst formation, pregnancy and implantation rates. Patients over 39 years of age had an abortion rate of 59.1 %, which is significantly higher than the other groups. Extended embryo culture up to the blastocyst stage can be implemented in programs of assisted reproduction in order to increase the pregnancy rate. The potential of blastocyst implantation is high, allowing us to transfer fewer embryos and reduce the probability of multiple pregnancies.

  17. 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

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

    PubMed

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

    2015-12-01

    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.

  19. Pregnancies established from handmade cloned blastocysts reconstructed using skin fibroblasts in buffalo (Bubalus bubalis).

    PubMed

    Shah, R A; George, A; Singh, M K; Kumar, D; Anand, T; Chauhan, M S; Manik, R S; Palta, P; Singla, S K

    2009-05-01

    Handmade cloning (HMC), a simple, micromanipulation-free cloning technique, has been applied for the production of cloned embryos and offspring in many livestock species. The objective of the present study was to compare the effect of donor cell type on developmental competence of HMC embryos and to explore the possibility of establishing pregnancies using these embryos in buffalo. After technical optimization of the HMC procedure for in vitro development of cloned blastocysts, various donor cells were compared for their developmental efficiency. Using buffalo fetal-, newborn-, adult fibroblasts and cumulus cells, blastocyst production rates obtained from reconstructed embryos were 24.0+/-1.8% (35/145), 33.0+/-8.0% (56/163), 21.0+/-9.3% (29/133) and 49.6+/-1.9% (77/154), respectively. Blastocyst rates were higher (P<0.05) in cumulus cell reconstructed embryos in comparison to those derived from fetal or adult fibroblasts. Pregnancy diagnosis (transrectal ultrasonography) was carried out at Day 40 of gestation. Following transfer of HMC embryos reconstructed using newborn fibroblasts 25% (2/8) buffaloes were pregnant and are at Days 201 and 94 of gestation, whereas after transfer of HMC embryos reconstructed using fetal fibroblasts, 20% (1/5) buffaloes were pregnant and are at Day 73 of gestation. In conclusion, HMC could be a simple and efficient technique for the production of cloned embryos for establishing pregnancies in buffalo.

  20. Global gene expression profiles reveal significant nuclear reprogramming by the blastocyst stage after cloning.

    PubMed

    Smith, Sadie L; Everts, Robin E; Tian, X Cindy; Du, Fuliang; Sung, Li-Ying; Rodriguez-Zas, Sandra L; Jeong, Byeong-Seon; Renard, Jean-Paul; Lewin, Harris A; Yang, Xiangzhong

    2005-12-01

    Nuclear transfer (NT) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. Here, we compared global gene expression profiles of individual bovine NT blastocysts with their somatic donor cells and fertilized control embryos using cDNA microarray technology. The NT embryos' gene expression profiles were drastically different from those of their donor cells and closely resembled those of the naturally fertilized embryos. Our findings demonstrate that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, problems may occur during redifferentiation for tissue genesis and organogenesis, and small reprogramming errors may be magnified downstream in development.

  1. Fabrication and single-electron-transfer operation of a triple-dot single-electron transistor

    SciTech Connect

    Jo, Mingyu Uchida, Takafumi; Tsurumaki-Fukuchi, Atsushi; Arita, Masashi; Takahashi, Yasuo; Fujiwara, Akira; Nishiguchi, Katsuhiko; Ono, Yukinori; Inokawa, Hiroshi

    2015-12-07

    A triple-dot single-electron transistor was fabricated on silicon-on-insulator wafer using pattern-dependent oxidation. A specially designed one-dimensional silicon wire having small constrictions at both ends was converted to a triple-dot single-electron transistor by means of pattern-dependent oxidation. The fabrication of the center dot involved quantum size effects and stress-induced band gap reduction, whereas that of the two side dots involved thickness modulation because of the complex edge structure of two-dimensional silicon. Single-electron turnstile operation was confirmed at 8 K when a 100-mV, 1-MHz square wave was applied. Monte Carlo simulations indicated that such a device with inhomogeneous tunnel and gate capacitances can exhibit single-electron transfer.

  2. Production of calves by transfer of nuclei from cultured inner cell mass cells.

    PubMed

    Sims, M; First, N L

    1994-06-21

    We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days. PMID:8016127

  3. Production of calves by transfer of nuclei from cultured inner cell mass cells.

    PubMed

    Sims, M; First, N L

    1994-06-21

    We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.

  4. Production of calves by transfer of nuclei from cultured inner cell mass cells.

    PubMed Central

    Sims, M; First, N L

    1994-01-01

    We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days. Images PMID:8016127

  5. Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts.

    PubMed

    Pinyopummintr, T; Bavister, B D

    1996-01-01

    Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

  6. Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

    PubMed Central

    Chan, Wen-Hsiung

    2014-01-01

    We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5–10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole. PMID:24933639

  7. Quality analysis of buffalo blastocysts derived from oocytes vitrified before or after enucleation and reconstructed with somatic cell nuclei.

    PubMed

    Muenthaisong, S; Laowtammathron, C; Ketudat-Cairns, M; Parnpai, R; Hochi, S

    2007-03-01

    We investigated the potential of vitrified-warmed buffalo oocytes to develop to blastocysts after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). In vitro-matured oocytes before and after enucleation (M-II oocytes and enucleated oocytes, respectively) were put in 7.5% DMSO and 7.5% ethylene glycol (EG) for 4, 7 and 10 min, and then vitrified (Cryotop device) after 1-min equilibration in 15% DMSO, 15% EG and 0.5M sucrose. Following 4-, 7- and 10-min exposure, proportions of the post-warm oocytes with a normal vitelline membrane were similar (66-71% in M-II oocytes and 69-71% in enucleated oocytes). However, 18-20% of the normal M-II oocytes had no detectable first polar body in their perivitelline space (no potential for subsequent enucleation). When the post-warm M-II oocytes were treated for PA by 7% ethanol, 10 microg/mL cycloheximide and 1.25 microg/mL cytochalasin-D, parthenogenetic development into Day-7 blastocysts occurred in 10-13% of cultured oocytes, lower (P<0.05) than fresh (control) oocytes (24%). In the absence of the cooling and warming, blastocyst rates in the 4-min exposure group (22%), but not in the 7-min and 10-min exposure groups (14-15%), were similar to that in the fresh group (23%). The total cell number (group average 117-132 cells) and the ICM ratio (22-24%) of the PA blastocysts derived from vitrified M-II oocytes were comparable with fresh oocytes (127 cells and 25%). After SCNT (with fibroblast cells and vitrified-warmed oocytes), blastocyst rates were similar for the three exposure periods for M-II oocytes (8-10%) and enucleated oocytes (7-9%), but were lower (P<0.05) than in the fresh group (15%). The total cell number of the SCNT blastocysts derived from vitrified M-II and enucleated oocytes (80-90 and 82-101 cells) was smaller (P<0.05) than from fresh oocytes (135 cells); the ICM ratio of blastocysts derived from the M-II and enucleated oocytes after vitrification in 7- or 10-min exposure groups (20

  8. 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.

  9. 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

  10. Enhanced histone acetylation in somatic cells induced by a histone deacetylase inhibitor improved inter-generic cloned leopard cat blastocysts.

    PubMed

    Lee, Hyo-Sang; Yu, Xian-Feng; Bang, Jae-Il; Cho, Su-Jin; Deb, Gautam Kumar; Kim, Byeong-Woo; Kong, Il-Keun

    2010-11-01

    The objective was to determine whether alterations of histone acetylation status in donor cells affected inter-generic SCNT (igSCNT)-cloned embryo development. Leopard cat cells were treated with trichostatin A (TSA; a histone deacetylase inhibitor) for 48 h, and then donor cells were transferred into enucleated oocytes from domestic cats. Compared to non-treated cells, the acetylated histone 3 at lysine 9 (AcH3K9) and histone 4 at lysine 5 (AcH4K5) in the TSA group increased for up to 48 h (P < 0.05). The AcH3K9 signal ratios of igSCNT group was higher than control group 3 h after activation (P < 0.05). Treatment with TSA significantly increased total cell number of blastocysts (109.1 ± 6.9 vs. 71.8 ± 2.9, mean ± SEM), with no significant effects on rates of cleavage or blastocyst development (71.1 ± 2.8 vs. 67.6 ± 2.9 and 12.2 ± 2.6 vs. 11.0 ± 2.6, respectively). When igSCNT cloned embryos were transferred into a domestic cat oviduct and recovered after 8 d, blastocyst development rates and total cell numbers were greater in the TSA-igSCNT group (20.7 ± 3.0% and 2847.6 ± 37.2) than in the control igSCNT group (5.7 ± 2.2% and 652.1 ± 17.6, P < 0.05). Average total cell numbers of blastocysts were approximately 4.4-fold higher in the TSA-igSCNT group (2847.6 ± 37.2, n = 10) than in the control group (652.1 ± 17.6, n = 8; P < 0.05), but were ∼2.9-fold lower than in vivo cat blastocysts produced by intrauterine insemination (8203.8 ± 29.6, n = 5; P < 0.001). Enhanced histone acetylation levels of donor cells improved in vivo developmental competence and quality of inter-generic cloned embryos; however, fewer cells in blastocysts derived from igSCNT than blastocysts produced by insemination may reduce development potential following intergeneric cloning (none of the cloned embryos were maintained to term).

  11. Single-collision studies of energy transfer and chemical reaction

    SciTech Connect

    Valentini, J.J.

    1993-12-01

    The research focus in this group is state-to-state dynamics of reaction and energy transfer in collisions of free radicals such as H, OH, and CH{sub 3} with H{sub 2}, alkanes, alcohols and other hydrogen-containing molecules. The motivation for the work is the desire to provide a detailed understanding of the chemical dynamics of prototype reactions that are important in the production and utilization of energy sources, most importantly in combustion. The work is primarily experimental, but with an important and growing theoretical/computational component. The focus of this research program is now on reactions in which at least one of the reactants and one of the products is polyatomic. The objective is to determine how the high dimensionality of the reactants and products differentiates such reactions from atom + diatom reactions of the same kinematics and energetics. The experiments use highly time-resolved laser spectroscopic methods to prepare reactant states and analyze the states of the products on a single-collision time scale. The primary spectroscopic tool for product state analysis is coherent anti-Stokes Raman scattering (CARS) spectroscopy. CARS is used because of its generality and because the extraction of quantum state populations from CARS spectra is straightforward. The combination of the generality and easy analysis of CARS makes possible absolute cross section measurements (both state-to-state and total), a particularly valuable capability for characterizing reactive and inelastic collisions. Reactant free radicals are produced by laser photolysis of appropriate precursors. For reactant vibrational excitation stimulated Raman techniques are being developed and implemented.

  12. Expression of proto-oncogenes in bovine preimplantation blastocysts.

    PubMed

    Tetens, F; Kliem, A; Tscheudschilsuren, G; Navarrete Santos, A; Fischer, B

    2000-05-01

    Proto-oncogenes are involved in the regulation of gene expression, for example after ligand binding to growth factor receptors. Expression of the proto-oncogenes c-fos, c-jun, c-ha-ras and c-myc was studied in in vivo grown and in vitro cultured bovine preimplantation blastocysts employing RT-PCR, ribonuclease protection assay and immunohistochemistry. Thirteen- and 14- day-old preimplantation blastocysts, i.e. stages before and during trophoblast elongation, were used. In in vivo-grown blastocysts c-fos, c-jun and c-ha-ras transcripts as well as c-Fos, c-Jun and c-Myc proteins were detected in all stages studied. Cultured blastocysts were treated with 10 nM epidermal growth factor and 10 nM transforming growth factor-alpha simultaneously. Epidermal growth factor and transforming growth factor-alpha treatment induced c-fos mRNA and c-Myc protein expression. The induction of downstream targets of the epidermal growth factor receptor by epidermal growth factor and transforming growth factor-alpha indicates a functional epidermal growth factor signal transduction pathway in elongating bovine blastocysts.

  13. 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

  14. 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

  15. The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs

    PubMed Central

    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

  16. Morphological observations on the diapausing blastocyst of some macropodid marsupials.

    PubMed

    Smith, M J

    1981-03-01

    Diapausing blastocysts from the uteri of females of 11 macropodid species were fixed, torn, flattened and stained for examination of the protoderm. The protoderm remained undifferentiated and unilaminar throughout diapause. The number of cells in the protoderm did not vary with age in Macropus rufus or M. eugenii. Protoderm cell number decreased slightly with age in M. rufogriseus banksianus and appeared to decrease in Potorous tridactylus. The diameter of the shell did not change appreciably with age, and the sizes of the blastocysts of different species were similar.

  17. Effects of fifth day (D5) or sixth day (D6) frozen-thawed blastocysts on neonatal outcomes.

    PubMed

    Wang, Xue; Zhen, Jingran; Sun, Zhengyi; Yu, Qi; Deng, Chengyan; Zhou, Yuanzheng; Wang, Hanbi; He, Fangfang

    2016-10-01

    This study aimed to compare the neonatal outcomes of transplanted fifth day (D5) or sixth day (D6) frozen-thawed blastocysts, aiming to provide guidance when choosing blastocyst recovery methods. The clinical data of 1109 patients that were performed for recovered blastocyst transplantation, as well as the data of 515 patients who had already delivered were analyzed retrospectively. The patients were divided into single- or twins-birth groups according to blastocyst formation time (D5 or D6) and the number of fetus(es). The implantation rate, clinical pregnancy rate and live birth rate of the D5 group were all significantly higher than those in the D6 group (P 0.05). In conclusion, the implantation rate, clinical pregnancy rate and live birth rate in the D5 group were all significantly higher than that in the D6 group. The birth weight at D6 was prone to be heavier no matter the birth group, and the sex ratio in the D5 group was higher than that at D6, the difference in neonatal outcomes between the two groups was not statistically significant.

  18. Single-molecule interfacial electron transfer dynamics manipulated by an external electric current.

    PubMed

    Zhang, Guofeng; Xiao, Liantuan; Chen, Ruiyun; Gao, Yan; Wang, Xiaobo; Jia, Suotang

    2011-08-14

    Interfacial electron transfer (IET) dynamics in a 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) dye molecule/indium tin oxide (ITO) film system have been probed at the ensemble and single-molecule levels. By comparing the difference in the external electric current (EEC) dependence of the fluorescence intensities and lifetimes of the ensembles and single molecules, it is shown that the single-molecule probe can effectively demonstrate IET dynamics. The backward electron transfer and electron transfer from the ground state induce single-molecule fluorescence quenching when an EEC is applied to the DiD/ITO film system.

  19. Variations in mass transfer to single endothelial cells.

    PubMed

    Van Doormaal, Mark A; Zhang, Ji; Wada, Shigeo; Shaw, James E; Won, Doyon; Cybulsky, Myron I; Yip, Chris M; Ethier, C Ross

    2009-06-01

    Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.

  20. 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

  1. The rate of blastocysts production following vitrification with step-wise equilibration of immature mouse oocytes

    PubMed Central

    Mahmoudi, Reza; Rajaei, Farzad; Ragardi Kashani, Iraj; Abbasi, Mehdi; Amidi, Fardin; Sobhani, Aligholi; Amiri, Iraj

    2012-01-01

    Background: Cryopreservation and in vitro maturation (IVM) of oocyte is becoming an important technique in infertility treatment and fertility preservation. Also it has been proposed to establish a genetic resource bank for endangered or commercially important animal species. Objective: The aim of this study was to evaluate viability, maturation and fertilization rate of mouse immature oocytes after single and stepwise vitrification procedure. Materials and Methods: Oocytes were obtained from 4 weeks old female mice 48h after intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin (PMSG). Collected oocytes before vitrification were exposed to cryoprotectant, which was composed of 30% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were warmed and washed two times in medium TCM199 and then subjected to IVM, fertilization and subsequent development to blastocysts. Results: The oocytes survival rates after vitrifying-warming (88.96%), maturation rate (73.23%), the capacity of fertilization (57.80%) and embryonic development to blastocyst (16.41%) in the step-wise exposure were significantly higher (p<0.001) compared with corresponding rate in the single step procedure. Conclusion: The results suggest that vitrification with step-wise procedure has positive effects on maturation and developmental capacity of mice germinal vesicle oocytes in compare with single step vitrification procedure. PMID:25246911

  2. In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up.

    PubMed

    Manjunatha, B M; Gupta, P S P; Ravindra, J P; Devaraj, M; Nandi, S

    2008-03-01

    The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.

  3. Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies

    PubMed Central

    Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

    2015-01-01

    Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6% P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS. PMID:25351780

  4. Mutation-free baby born from a mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome carrier after blastocyst trophectoderm preimplantation genetic diagnosis.

    PubMed

    Heindryckx, Björn; Neupane, Jitesh; Vandewoestyne, Mado; Christodoulou, Christodoulos; Jackers, Yens; Gerris, Jan; Van den Abbeel, Etienne; Van Coster, Rudy; Deforce, Dieter; De Sutter, Petra

    2014-09-01

    To investigate the applicability of preimplantation genetic diagnosis (PGD), we used trophectoderm (TE) biopsy to determine the mutation load in a 35-year-old female with mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome (MELAS). Transfer of a mutation-free blastocyst gave birth to a healthy boy with undetectable mutation in any of the analyzed tissues. We found strong correlation among TE cells (r=0.90) within blastocysts and also between cytoplasmic fragments and TE (r=0.95). This is the first case of mutation-free baby born from a MELAS patient after TE biopsy and supports the applicability of blastocyst PGD for patients with mtDNA disorders to establish healthy offspring.

  5. Single-node orbit analsyis with radiation heat transfer only

    NASA Technical Reports Server (NTRS)

    Peoples, J. A.

    1977-01-01

    The steady-state temperature of a single node which dissipates energy by radiation only is discussed for a nontime varying thermal environment. Relationships are developed to illustrate how shields can be utilized to represent a louver system. A computer program is presented which can assess periodic temperature characteristics of a single node in a time varying thermal environment having energy dissipation by radiation only. The computer program performs thermal orbital analysis for five combinations of plate, shields, and louvers.

  6. Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

    PubMed

    Velasquez, Alejandra E; Castro, Fidel O; Veraguas, Daniel; Cox, Jose F; Lara, Evelyn; Briones, Mario; Rodriguez-Alvarez, Lleretny

    2016-02-01

    Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

  7. Reversible State Transfer between Light and a Single Trapped Atom

    SciTech Connect

    Boozer, A. D.; Boca, A.; Miller, R.; Northup, T. E.; Kimble, H. J.

    2007-05-11

    We demonstrate the reversible mapping of a coherent state of light with a mean photon number n{approx_equal}1.1 to and from the hyperfine states of an atom trapped within the mode of a high-finesse optical cavity. The coherence of the basic processes is verified by mapping the atomic state back onto a field state in a way that depends on the phase of the original coherent state. Our experiment represents an important step toward the realization of cavity QED-based quantum networks, wherein coherent transfer of quantum states enables the distribution of quantum information across the network.

  8. Ultrafast Single and Multiexciton Energy Transfer in Semiconductor Nanoplatelets

    NASA Astrophysics Data System (ADS)

    Schaller, Richard

    Photophysical processes such as fluorescence resonance energy transfer (FRET) enable optical antennas, wavelength down-conversion in light-emitting diodes (LEDs), and optical bio-sensing schemes. The rate and efficiency of this donor to acceptor transfer of excitation between chromophores dictates the utility of FRET and can unlock new device operation motifs including quantum-funnel solar cells and reduced gain thresholds. However, the fastest reported FRET time constants involving spherical quantum dots (QDs) (0.12-1 ns), do not outpace biexciton Auger recombination (0.01-0.1 ns), which impedes multiexciton-driven applications including electrically-pumped lasers and carrier-multiplication-enhanced photovoltaics. Precisely controlled, few-monolayer thick semiconductor nano-platelets with tens-of-nanometer diameters exhibit intense optical transitions and hundreds-of-picosecond Auger recombination, but heretofore lack FRET characterizations. We examine binary CdSe NPL solids and show that inter-plate FRET (~6-23 ps, presumably for co-facial arrangements) can occur 15-50 times faster than Auger recombination and demonstrate multiexcitonic FRET, making such materials ideal candidates for advanced technologies. This work was performed at the Center for Nanoscale Materials, a U.S. Department of Energy Office of Science User Facility under Contract No. DE-AC02-06CH11357.

  9. Characterization of human PGD blastocysts with unbalanced chromosomal translocations and human embryonic stem cell line derivation?

    PubMed

    Frydman, N; Féraud, O; Bas, C; Amit, M; Frydman, R; Bennaceur-Griscelli, A; Tachdjian, G

    2009-01-01

    Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

  10. Chemical reaction fouling model for single-phase heat transfer

    SciTech Connect

    Panchal, C.B.; Watkinson, A.P.

    1993-08-01

    A fouling model was developed on the premise that the chemical reaction for generation of precursor can take place in the bulk fluid, in the thermalboundary layer, or at the fluid/wall interface, depending upon the interactive effects of flu id dynamics, heat and mass transfer, and the controlling chemical reaction. The analysis was used to examine the experimental data for fouling deposition of polyperoxides produced by autoxidation of indene in kerosene. The effects of fluid and wall temperatures for two flow geometries were analyzed. The results showed that the relative effects of physical parameters on the fouling rate would differ for the three fouling mechanisms; therefore, it is important to identify the controlling mechanism in applying the closed-flow-loop data to industrial conditions.

  11. Scalable Transfer of Suspended Two-Dimensional Single Crystals.

    PubMed

    Li, Bo; He, Yongmin; Lei, Sidong; Najmaei, Sina; Gong, Yongji; Wang, Xin; Zhang, Jing; Ma, Lulu; Yang, Yingchao; Hong, Sanghyun; Hao, Ji; Shi, Gang; George, Antony; Keyshar, Kunttal; Zhang, Xiang; Dong, Pei; Ge, Liehui; Vajtai, Robert; Lou, Jun; Jung, Yung Joon; Ajayan, Pulickel M

    2015-08-12

    Large-scale suspended architectures of various two-dimensional (2D) materials (MoS2, MoSe2, WS2, and graphene) are demonstrated on nanoscale patterned substrates with different physical and chemical surface properties, such as flexible polymer substrates (polydimethylsiloxane), rigid Si substrates, and rigid metal substrates (Au/Ag). This transfer method represents a generic, fast, clean, and scalable technique to suspend 2D atomic layers. The underlying principle behind this approach, which employs a capillary-force-free wet-contact printing method, was studied by characterizing the nanoscale solid-liquid-vapor interface of 2D layers with respect to different substrates. As a proof-of-concept, a photodetector of suspended MoS2 has been demonstrated with significantly improved photosensitivity. This strategy could be extended to several other 2D material systems and open the pathway toward better optoelectronic and nanoelectromechnical systems.

  12. Epitaxial film transfer technique for producing single crystal Si film on an insulating substrate

    NASA Astrophysics Data System (ADS)

    Kimura, M.; Egami, K.; Kanamori, M.; Hamaguchi, T.

    1983-08-01

    Epitaxial film transfer, a new technique for producing a single crystal Si film with both large size and high quality on an insulating substrate, is demonstrated. The technique in which an epitaxial Si film is transferred to a secondary substrate by using three fundamental processes of epitaxial growth, bonding of two wafers, and substrate elimination, can produce a 2-in. single crystal Si film as thin as 1.5 μm on a insulating substrate. Thickness variation can be controlled to ±0.06 μm across a 2-in. wafer. An epitaxial Si film is transferred without significant degradation in quality although a fine film waving exists.

  13. Single cell activity reveals direct electron transfer in methanotrophic consortia

    NASA Astrophysics Data System (ADS)

    McGlynn, Shawn E.; Chadwick, Grayson L.; Kempes, Christopher P.; Orphan, Victoria J.

    2015-10-01

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

  14. Single cell activity reveals direct electron transfer in methanotrophic consortia.

    PubMed

    McGlynn, Shawn E; Chadwick, Grayson L; Kempes, Christopher P; Orphan, Victoria J

    2015-10-22

    Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer. PMID:26375009

  15. Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats.

    PubMed

    Kapawa, A; Giannakis, D; Tsoukanelis, K; Kanakas, N; Baltogiannis, D; Agapitos, E; Loutradis, D; Miyagawa, I; Sofikitis, N

    2004-04-01

    We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, androgen-binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke-exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation.

  16. Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study

    PubMed Central

    Ubaldi, Filippo Maria; Capalbo, Antonio; Colamaria, Silvia; Ferrero, Susanna; Maggiulli, Roberta; Vajta, Gábor; Sapienza, Fabio; Cimadomo, Danilo; Giuliani, Maddalena; Gravotta, Enrica; Vaiarelli, Alberto; Rienzi, Laura

    2015-01-01

    STUDY QUESTION Is an elective single-embryo transfer (eSET) policy an efficient approach for women aged >35 years when embryo selection is enhanced via blastocyst culture and preimplantation genetic screening (PGS)? SUMMARY ANSWER Elective SET coupled with enhanced embryo selection using PGS in women older than 35 years reduced the multiple pregnancy rates while maintaining the cumulative success rate of the IVF programme. WHAT IS KNOWN ALREADY Multiple pregnancies mean an increased risk of premature birth and perinatal death and occur mainly in older patients when multiple embryos are transferred to increase the chance of pregnancy. A SET policy is usually recommended in cases of good prognosis patients, but no general consensus has been reached for SET application in the advanced maternal age (AMA) population, defined as women older than 35 years. Our objective was to evaluate the results in terms of efficacy, efficiency and safety of an eSET policy coupled with increased application of blastocyst culture and PGS for this population of patients in our IVF programme. STUDY DESIGN, SIZE, DURATION In January 2013, a multidisciplinary intervention involving optimization of embryo selection procedure and introduction of an eSET policy in an AMA population of women was implemented. This is a retrospective 4-year (January 2010–December 2013) pre- and post-intervention analysis, including 1161 and 499 patients in the pre- and post-intervention period, respectively. The primary outcome measures were the cumulative delivery rate (DR) per oocyte retrieval cycle and multiple DR. PARTICIPANTS/MATERIALS, SETTING, METHODS Surplus oocytes and/or embryos were vitrified during the entire study period. In the post-intervention period, all couples with good quality embryos and less than two previous implantation failures were offered eSET. Embryo selection was enhanced by blastocyst culture and PGS (blastocyst stage biopsy and 24-chromosomal screening). Elective SET was also

  17. Observation of a single spin by transferring its coherence to a high level macroscopic pure state

    SciTech Connect

    Kawamura, Minaru

    2014-12-04

    We discuss about quantum measurement of a single spin in a superconducting RF resonator, where amplification of coherence of the spin is enabled by transferring its coherence to the harmonic oscillator in an non-coherent state with high energy level. This quantum amplification allows that a single spin can induce macroscopic current to permits observation of a single spin state in the number and phase uncertainty relation.

  18. p38 MAPK Regulates Cavitation and Tight Junction Function in the Mouse Blastocyst

    PubMed Central

    Bell, Christine E.; Watson, Andrew J.

    2013-01-01

    Blastocyst formation is essential for implantation and maintenance of pregnancy and is dependent on the expression and coordinated function of a series of proteins involved in establishing and maintaining the trans-trophectoderm ion gradient that enables blastocyst expansion. These consist of Na/K-ATPase, adherens junctions, tight junctions (TJ) and aquaporins (AQP). While their role in supporting blastocyst formation is established, the intracellular signaling pathways that coordinate their function is unclear. The p38 MAPK pathway plays a role in regulating these proteins in other cell types and is required for embryo development at the 8–16 cell stage, but its role has not been investigated in the blastocyst. Hypothesis p38 MAPK regulates blastocyst formation by regulating blastocyst formation gene expression and function. Methods Embryos were cultured from the early blastocyst stage for 12 h or 24 h in the presence of a potent and specific p38 MAPK inhibitor, SB 220025. Blastocyst expansion, hatching, gene family expression and localization, TJ function and apoptosis levels were analyzed. Results Inhibition of the p38 MAPK pathway reduced blastocyst expansion and hatching, increased tight junction permeability, affected TJP1 localization, reduced Aqp3 expression, and induced a significant increase in apoptosis. Conclusion The p38 MAPK pathway coordinates the overall events that regulate blastocyst formation. PMID:23593143

  19. 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

  20. The single electron transfer chemistry of coals. Final report

    SciTech Connect

    Larsen, J.W.; Flowers, R.A. II

    1994-12-31

    This research addressed electron donar properties and radical reactions in coal. Solid residues from pyridine Soxhlet extractions of Pocahontas No. 3, Upper Freeport, Pittsburgh No. 8, Illinois No. 6 and Wyodak coals were exposed to 4-vinylpyridine vapors and swelled. All of the 4-vinylpyridine could not be removed under vacuum at 100{degree}C. Diffuse reflectance FTIR revealed the presence of poly-(4-vinylpyridine) in the Illinois No. 6 and Wyodak coals. EPR spectra displayed the loss of inertinite radicals in Upper Freeport, Illinois No. 6 and Wyodak residues after exposure to 4-vinylpyridine. There was little change in the vitrinite radical density or environment. The molecule N,N{prime}-Diphenyl-p-phenylene diamine (DPPD) was exposed to the solid residues from pyridine Soxhlet extractions of the above coals. Diffuse reflectance FTIR failed to detect the imine product from radical reaction with DPPD. EPR spectra displayed the loss of inertinite radicals in Upper Freeport and Wyodak residues. 7,7,8,8-Tetracyanoquinodimethane (TCNQ) and Tetracyanoethylene (TCNE) were deposited into coals in pyridine. FTIR indicated complete conversion of TCNQ to a material with a singly occupied LUMO. In TCNE the LUMO is about 30% occupied. TCNQ and TCNE were deposited into the pyridine extracts and residues of Illinois No. 6 and Pittsburgh No. 8 coals. Only a small amount of the TCNQ and TCNE displayed nitrile shifts in the IR spectrum of a material with an occupied LUMO. It has been concluded that TCNQ must be part of the aromatic stacks in coal and the TCNQ LUMO is part of an extended band.

  1. Diameter selective electron transfer from encapsulated ferrocenes to single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Iizumi, Yoko; Suzuki, Hironori; Tange, Masayoshi; Okazaki, Toshiya

    2014-10-01

    The diameter selective photoluminescence quenching of single-walled carbon nanotubes (SWCNTs) is observed upon ferrocene encapsulation, which can be attributed to electron transfer from the encapsulated ferrocenes to the SWCNTs. Interestingly, the dependence of the electron transfer process on the nanotube diameter is governed by the molecular orientation of the ferrocenes in the SWCNT rather than the reduction potentials of the SWCNT.The diameter selective photoluminescence quenching of single-walled carbon nanotubes (SWCNTs) is observed upon ferrocene encapsulation, which can be attributed to electron transfer from the encapsulated ferrocenes to the SWCNTs. Interestingly, the dependence of the electron transfer process on the nanotube diameter is governed by the molecular orientation of the ferrocenes in the SWCNT rather than the reduction potentials of the SWCNT. Electronic supplementary information (ESI) available: Calculated binding energies of FeCp2@SWCNTs and additional spectroscopic characterization are described in ESI. See DOI: 10.1039/c4nr04398g

  2. Probe-based measurement of lateral single-electron transfer between individual molecules

    PubMed Central

    Steurer, Wolfram; Fatayer, Shadi; Gross, Leo; Meyer, Gerhard

    2015-01-01

    The field of molecular electronics aims at using single molecules as functional building blocks for electronics components, such as switches, rectifiers or transistors. A key challenge is to perform measurements with atomistic control over the alignment of the molecule and its contacting electrodes. Here we use atomic force microscopy to examine charge transfer between weakly coupled pentacene molecules on insulating films with single-electron sensitivity and control over the atomistic details. We show that, in addition to the imaging capability, the probe tip can be used to control the charge state of individual molecules and to detect charge transfers to/from the tip, as well as between individual molecules. Our approach represents a novel route for molecular charge transfer studies with a host of opportunities, especially in combination with single atom/molecule manipulation and nanopatterning techniques. PMID:26387533

  3. Charge transfer at junctions of a single layer of graphene and a metallic single walled carbon nanotube.

    PubMed

    Paulus, Geraldine L C; Wang, Qing Hua; Ulissi, Zachary W; McNicholas, Thomas P; Vijayaraghavan, Aravind; Shih, Chih-Jen; Jin, Zhong; Strano, Michael S

    2013-06-10

    Junctions between a single walled carbon nanotube (SWNT) and a monolayer of graphene are fabricated and studied for the first time. A single layer graphene (SLG) sheet grown by chemical vapor deposition (CVD) is transferred onto a SiO₂/Si wafer with aligned CVD-grown SWNTs. Raman spectroscopy is used to identify metallic-SWNT/SLG junctions, and a method for spectroscopic deconvolution of the overlapping G peaks of the SWNT and the SLG is reported, making use of the polarization dependence of the SWNT. A comparison of the Raman peak positions and intensities of the individual SWNT and graphene to those of the SWNT-graphene junction indicates an electron transfer of 1.12 × 10¹³ cm⁻² from the SWNT to the graphene. This direction of charge transfer is in agreement with the work functions of the SWNT and graphene. The compression of the SWNT by the graphene increases the broadening of the radial breathing mode (RBM) peak from 3.6 ± 0.3 to 4.6 ± 0.5 cm⁻¹ and of the G peak from 13 ± 1 to 18 ± 1 cm⁻¹, in reasonable agreement with molecular dynamics simulations. However, the RBM and G peak position shifts are primarily due to charge transfer with minimal contributions from strain. With this method, the ability to dope graphene with nanometer resolution is demonstrated. PMID:23281165

  4. Monitoring Conformational Dynamics with Single-Molecule Fluorescence Energy Transfer: Applications in Nucleosome Remodeling

    PubMed Central

    Deindl, Sebastian; Zhuang, Xiaowei

    2016-01-01

    Due to its ability to track distance changes within individual molecules or molecular complexes on the nanometer scale and in real time, single-molecule fluorescence resonance energy transfer (single-molecule FRET) is a powerful tool to tackle a wide range of important biological questions. Using our recently developed single-molecule FRET assay to monitor nucleosome translocation as an illustrative example, we describe here in detail how to set up, carry out, and analyze single-molecule FRET experiments that provide time-dependent information on biomolecular processes. PMID:22929765

  5. How was the proton transfer process in bis-3, 6-(2- benzoxazolyl)-pyrocatechol, single or double proton transfer?

    PubMed Central

    Zhang, Yongjia; Sun, Mengtao; Li, Yongqing

    2016-01-01

    A theoretical analysis of proton transfer process for the symmetric systems with two intramolecular hydrogen bonds, bis-3,6-(2-benzoxazolyl)-pyrocatechol(BBPC) in hexane solvent, has been researched. In this study, we utilized ωB97X-D/ 6-311 + g (d,p) and B3LYP/6-31 + G(d) two procedures calculating the foremost bond length and bond angle, respectively. Our calculations demonstrate the two intramolecular hydrogen bonds were strengthened in S1 state, thus the proton transfer reaction can be facilitated. Furthermore, the calculated IR vibrational spectra confirmed hydrogen bonds were enhanced in S1 state. We found three local minima A B and C from the potential energy surfaces (PESs) on the S1 state, and the energy of B point and C point are identical. A new ESIPT mechanism has been proposed that was not equal to the previous conclusions. The new ESIPT mechanism elucidates that single proton transfer more likely occurs in the symmetric BBPC molecule in comparison with the double proton transfer reaction. And the frontier molecular orbitals(MOs) further illustrate the trend of ESIPT reaction. PMID:27157994

  6. 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

  7. Evidence that Additions of Grignard Reagents to Aliphatic Aldehydes Do Not Involve Single-Electron-Transfer Processes.

    PubMed

    Otte, Douglas A L; Woerpel, K A

    2015-08-01

    Addition of allylmagnesium reagents to an aliphatic aldehyde bearing a radical clock gave only addition products and no evidence of ring-opened products that would suggest single-electron-transfer reactions. The analogous Barbier reaction also did not provide evidence for a single-electron-transfer mechanism in the addition step. Other Grignard reagents (methyl-, vinyl-, t-Bu-, and triphenylmethylmagnesium halides) also do not appear to add to an alkyl aldehyde by a single-electron-transfer mechanism. PMID:26214553

  8. Mathematical equivalence between time-dependent single-rate and multirate mass transfer models

    NASA Astrophysics Data System (ADS)

    Fernández-Garcia, D.; Sanchez-Vila, X.

    2015-05-01

    The often observed tailing of tracer breakthrough curves is caused by a multitude of mass transfer processes taking place over multiple scales. Yet, in some cases, it is convenient to fit a transport model with a single-rate mass transfer coefficient that lumps all the non-Fickian observed behavior. Since mass transfer processes take place at all characteristic times, the single-rate mass transfer coefficient derived from measurements in the laboratory or in the field vary with time ω>(t>). The literature review and tracer experiments compiled by Haggerty et al. (2004) from a number of sites worldwide suggest that the characteristic mass transfer time, which is proportional to ω>(t>)-1, scales as a power law of the advective and experiment duration. This paper studies the mathematical equivalence between the multirate mass transfer model (MRMT) and a time-dependent single-rate mass transfer model (t-SRMT). In doing this, we provide new insights into the previously observed scale-dependence of mass transfer coefficients. The memory function, g(t), which is the most salient feature of the MRMT model, determines the influence of the past values of concentrations on its present state. We found that the t-SRMT model can also be expressed by means of a memory function φ>(t,τ>). In this case, though the memory function is nonstationary, meaning that in general it cannot be written as φ>(t-τ>). Nevertheless, the full behavior of the concentrations using a single time-dependent rate ω>(t>) is approximately analogous to that of the MRMT model provided that the equality ω>(t>)=-dln⁡g>(t>)/dt holds and the field capacity is properly chosen. This relationship suggests that when the memory function is a power law, g>(t>)˜t1-k, the equivalent mass transfer coefficient scales as ω>(t>)˜t-1, nicely fitting without calibration the estimated mass transfer coefficients compiled by Haggerty et al. (2004).

  9. Phthalimides as exceptionally efficient single electron transfer acceptors in reductive coupling reactions promoted by samarium diiodide.

    PubMed

    Vacas, Tatiana; Alvarez, Eleuterio; Chiara, Jose Luis

    2007-12-20

    Experimental and theoretical evidence shows that phthalimides are highly efficient single electron transfer acceptors in reactions promoted by samarium diiodide, affording ketyl radical anion intermediates, which participate in high-yielding inter- and intramolecular reductive coupling processes with different radicophiles including imides, oxime ethers, nitrones, and Michael acceptors.

  10. 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.

  11. Polarization Transfer in Wide-Angle Compton Scattering and Single-Pion Photoproduction from the Proton

    NASA Astrophysics Data System (ADS)

    Fanelli, C.; Cisbani, E.; Hamilton, D. J.; Salmé, G.; Wojtsekhowski, B.; Ahmidouch, A.; Annand, J. R. M.; Baghdasaryan, H.; Beaufait, J.; Bosted, P.; Brash, E. J.; Butuceanu, C.; Carter, P.; Christy, E.; Chudakov, E.; Danagoulian, S.; Day, D.; Degtyarenko, P.; Ent, R.; Fenker, H.; Fowler, M.; Frlez, E.; Gaskell, D.; Gilman, R.; Horn, T.; Huber, G. M.; de Jager, C. W.; Jensen, E.; Jones, M. K.; Kelleher, A.; Keppel, C.; Khandaker, M.; Kohl, M.; Kumbartzki, G.; Lassiter, S.; Li, Y.; Lindgren, R.; Lovelace, H.; Luo, W.; Mack, D.; Mamyan, V.; Margaziotis, D. J.; Markowitz, P.; Maxwell, J.; Mbianda, G.; Meekins, D.; Meziane, M.; Miller, J.; Mkrtchyan, A.; Mkrtchyan, H.; Mulholland, J.; Nelyubin, V.; Pentchev, L.; Perdrisat, C. F.; Piasetzky, E.; Prok, Y.; Puckett, A. J. R.; Punjabi, V.; Shabestari, M.; Shahinyan, A.; Slifer, K.; Smith, G.; Solvignon, P.; Subedi, R.; Wesselmann, F. R.; Wood, S.; Ye, Z.; Zheng, X.

    2015-10-01

    Wide-angle exclusive Compton scattering and single-pion photoproduction from the proton have been investigated via measurement of the polarization transfer from a circularly polarized photon beam to the recoil proton. The wide-angle Compton scattering polarization transfer was analyzed at an incident photon energy of 3.7 GeV at a proton scattering angle of θcmp=70 ° . The longitudinal transfer KLL, measured to be 0.645 ±0.059 ±0.048 , where the first error is statistical and the second is systematic, has the same sign as predicted for the reaction mechanism in which the photon interacts with a single quark carrying the spin of the proton. However, the observed value is ˜3 times larger than predicted by the generalized-parton-distribution-based calculations, which indicates a significant unknown contribution to the scattering amplitude.

  12. Polarization transfer in wide-angle Compton scattering and single-pion photoproduction from the proton

    DOE PAGES

    Fanelli, Cristiano V.

    2015-10-06

    Wide-angle exclusive Compton scattering and single-pion photoproduction from the proton have been investigated via measurement of the polarization transfer from a circularly polarized photon beam to the recoil proton. The WACS polarization transfer was analyzed at an incident photon energy of 3.7 GeV at a proton scattering angle of θPcm = 70°. The longitudinal transfer KLL, measured to be 0.645 ± 0.059 ± 0.048, where the first error is statistical and the second is systematic, has the same sign as predicted for the reaction mechanism in which the photon interacts with a single quark carrying the spin of the proton.more » However, the observed value is ~3 times larger than predicted by the GPD-based calculations, which indicates a significant unknown contribution to the scattering amplitude.« less

  13. Polarization transfer in wide-angle Compton scattering and single-pion photoproduction from the proton

    SciTech Connect

    Fanelli, Cristiano V.

    2015-10-06

    Wide-angle exclusive Compton scattering and single-pion photoproduction from the proton have been investigated via measurement of the polarization transfer from a circularly polarized photon beam to the recoil proton. The WACS polarization transfer was analyzed at an incident photon energy of 3.7 GeV at a proton scattering angle of θPcm = 70°. The longitudinal transfer KLL, measured to be 0.645 ± 0.059 ± 0.048, where the first error is statistical and the second is systematic, has the same sign as predicted for the reaction mechanism in which the photon interacts with a single quark carrying the spin of the proton. However, the observed value is ~3 times larger than predicted by the GPD-based calculations, which indicates a significant unknown contribution to the scattering amplitude.

  14. Research on urban public traffic network with multi-weights based on single bus transfer junction

    NASA Astrophysics Data System (ADS)

    An, Xin-lei; Zhang, Li; Zhang, Jian-gang

    2015-10-01

    Regarding single bus transfer junction as a research object, this paper constructs the urban traffic network models with multi-weights taking different bus lines in bus transfer junction as the network nodes, that is, the urban traffic network with multi-weights is given different properties weights at every edge. According to the method of network split, the complex network with multi-weights is split into several different single weighted complex networks. Then, we study the global synchronization of the new network model by changing congestion degrees, transfers coefficient and passenger flow density between different bus lines. Finally, analytical and simulated results are given to show the impact of different properties weights to the public traffic network balance.

  15. X chromosome regulation of autosomal gene expression in bovine blastocysts.

    PubMed

    Itoh, Yuichiro; Arnold, Arthur P

    2014-10-01

    Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.

  16. Hybrid Graphene and Single-Walled Carbon Nanotube Films for Enhanced Phase-Change Heat Transfer.

    PubMed

    Seo, Han; Yun, Hyung Duk; Kwon, Soon-Yong; Bang, In Cheol

    2016-02-10

    Nucleate boiling is an effective heat transfer method in power generation systems and cooling devices. In this letter, hybrid graphene/single-walled carbon nanotube (SWCNT), graphene, and SWCNT films deposited on indium tin oxide (ITO) surfaces were fabricated to investigate the enhancement of nucleate boiling phenomena described by the critical heat flux and heat transfer coefficient. The graphene films were grown on Cu foils and transferred to ITO surfaces. Furthermore, SWCNTs were deposited on the graphene layer to fabricate hybrid graphene/SWCNT films. We determined that the hybrid graphene/SWCNT film deposited on an ITO surface is the most effective heat transfer surface in pool boiling because of the interconnected network of carbon structures.

  17. Single and Multi-Nucleon Transfer Reactions for Nuclear Moment Studies Toward Radioactive-Ion Beams

    SciTech Connect

    Lozeva, R. L.; Georgiev, G. P.; Audi, G.; Cabaret, S.; Fiori, E.; Gaulard, C.; Hauschilda, K.; Lopez-Martens, A.; Risegari, L.; Blazhev, A.; Jolie, J.; Moschner, K.; Zell, K.-O.; Daugas, J.-M.; Faul, T.; Morel, P.; Roig, O.; Ferraton, M.; Ibrahim, F.

    2010-04-30

    This study is a part of an experimental program to measure nuclear moments in transfer reactions. It aims to probe for a first time the nuclear -spin orientation in multi-nucleon transfer. Fist experiments were performed to measure the quadrupole moment of an isomeric state in {sup 66}Cu (I{sup p}i 6{sup -}, E{sub x} = 1154 keV, T{sub 1/2} = 595(20) ns) in single nucleon transfer and the population of mus isomers in {sup 66}Cu and {sup 63}Ni in multi-nucleon transfer. The experimentally tested methodology allows broad applications toward more exotic species and feasibility of these reactions to produce species away from stability.

  18. Electric Field Induced Fluorescence Modulation of Single Molecules in PMMA Based on Electron Transfer

    PubMed Central

    Chen, Ruiyun; Gao, Yan; Zhang, Guofeng; Wu, Ruixiang; Xiao, Liantuan; Jia, Suotang

    2012-01-01

    We present a method to modulate the fluorescence of non-polar single squaraine-derived rotaxanes molecules embedded in a polar poly(methyl methacrylate) (PMMA) matrix under an external electric field. The electron transfer between single molecules and the electron acceptors in a PMMA matrix contributes to the diverse responses of fluorescence intensities to the electric field. The observed instantaneous and non-instantaneous electric field dependence of single-molecule fluorescence reflects the redistribution of electron acceptors in PMMA induced by electronic polarization and orientation polarization of polar polymer chains in an electric field. PMID:23109842

  19. Photoluminescence enhancement of aligned arrays of single-walled carbon nanotubes by polymer transfer

    NASA Astrophysics Data System (ADS)

    Schweiger, Manuel; Zakharko, Yuriy; Gannott, Florentina; Grimm, Stefan B.; Zaumseil, Jana

    2015-10-01

    The photoluminescence of as-grown, aligned single-walled carbon nanotubes (SWNTs) on quartz is strongly quenched and barely detectable. Here we show that transferring these SWNTs to another substrate such as clean quartz or glass increases their emission efficiency by up to two orders of magnitude. By statistical analysis of large nanotube arrays we show at what point of the transfer process the emission enhancement occurs and how it depends on the receiving substrate and the employed transfer polymer. We find that hydrophobic polystyrene (PS) as the transfer polymer results in higher photoluminescence enhancement than the more hydrophilic poly(methyl methacrylate) (PMMA). Possible mechanisms for this enhancement such as strain relief, disruption of the strong interaction of SWNTs with the substrate and localized emissive states are discussed.The photoluminescence of as-grown, aligned single-walled carbon nanotubes (SWNTs) on quartz is strongly quenched and barely detectable. Here we show that transferring these SWNTs to another substrate such as clean quartz or glass increases their emission efficiency by up to two orders of magnitude. By statistical analysis of large nanotube arrays we show at what point of the transfer process the emission enhancement occurs and how it depends on the receiving substrate and the employed transfer polymer. We find that hydrophobic polystyrene (PS) as the transfer polymer results in higher photoluminescence enhancement than the more hydrophilic poly(methyl methacrylate) (PMMA). Possible mechanisms for this enhancement such as strain relief, disruption of the strong interaction of SWNTs with the substrate and localized emissive states are discussed. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05163k

  20. Proton-transfer mechanism for dispersed decay kinetics of single molecules isolated in potassium hydrogen phthalate.

    PubMed

    Bott, Eric D; Riley, Erin A; Kahr, Bart; Reid, Philip J

    2009-08-25

    The excited-state decay kinetics of single 2',7'-dichlorofluorescein (DCF) molecules oriented and overgrown within crystals of potassium acid phthalate (KAP) are reported. Time-correlated single-photon counting measurements (TCSPC) of 56 DCF molecules in KAP reveal that single-exponential decay is exhibited by roughly half of the molecules. The remainder demonstrates complex excited-state decay kinetics that are well fit by a stretched exponential function consistent with dispersed kinetics. Histograms of single-molecule luminescence energies revealed environmental fluctuations and distinct chemical species. The TCSPC results are compared to Monte Carlo simulations employing a first-passage model for excited-state decay. Agreement between experiment and theory, on both bulk and single-molecule levels, suggests that a subset of the DCF molecules in KAP experience fluctuations in the surrounding environment that modify the energy barrier to proton transfer leading to dispersed kinetics.

  1. Effect of microinjection of a single IVF-derived blastomere on the development of cloned embryos at the eight-cell stage in bovine.

    PubMed

    Chen, Xiuping; Wang, Jianwu; Li, Rong; Ding, Fangrong; Li, Song; Zhang, Lei; Dai, Yunping; Li, Ning

    2010-12-01

    This study was conducted to determine the effect of microinjection of a single blastomere from in vitro fertilization (IVF)-derived eight-cell embryo into eight-cell cloned embryos harboring the gene encoding recombinant human lactoferrin (rhLF), GFP, and NEO markers in bovine. The reconstructed chimeric embryos were assessed for their development to blastocyst, or to term after transfer, and tissues of offspring were evaluated by polymerase chain reaction (PCR) for the presence of nuclear transfer (NT)-derived transgenic cells, and the cloned embryos without microinjection were used as controls. The chimeric embryos showed slightly higher blastocyst rate than that for controls. The single IVF-derived blastomere appeared to preferential contribute to inner cell mass (ICM) in the chimeric blastocysts. After transfer, the rates of development of chimeric embryos to day 60, to term, and to weaning were significantly higher than those of controls. Sixty-three chimeric blastocysts were transferred and 11 calves were born: 7 calves of them were dead, and the remaining 4 calves are apparently normal and healthy. Most of the tissues collected from dead fetus were transgenic, whereas NT-derived transgenic cells were not detected in some tissues of the living calves. Our results indicated that a single blastomere from IVF-derived eight-cell embryo improves the in vivo developmental potential of transgenic cloned eight-cell embryos in bovine; however, the single IVF-derived blastomere appears to be better able to populate the ICM and many tissues of offspring than NT-derived blastomeres.

  2. Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

    PubMed Central

    Egashira, Mahiro; Bai, Rulan; Nomura, Nana; Nomura, Shintaro; Hirota, Yasushi; Sakurai, Toshihiro; Imakawa, Kazuhiko

    2012-01-01

    Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity. PMID:23152823

  3. Near-infrared fluorescent single walled carbon nanotube-chitosan composite: Interfacial strain transfer efficiency assessment

    NASA Astrophysics Data System (ADS)

    Mol Menamparambath, Mini; Arabale, Girish; Nikolaev, Pavel; Baik, Seunghyun; Arepalli, Sivaram

    2013-04-01

    Effective load transfer at the single walled carbon nanotube (SWCNT)-polymer interface is most desirable for mechanically reinforced polymer composites. Versatile layer-by-layer assembly technique achieved dispersion and uniform distribution of sodium carboxymethylcellulose (CMC)-solubilized SWCNTs within the polymer matrix. Electrostatic interaction between positively charged chitosan and negatively charged CMC facilitates design of an optically active biocompatible nanocomposite. Interfacial strain transfer efficiency of SWCNT-chitosan nanocomposite was assessed via SWCNT Raman and photoluminescence band shifts under uniaxial strain. Photoluminescence peak shift rates of individual semiconducting SWCNTs were investigated and compared with tight binding model calculations.

  4. Expression of HSG is essential for mouse blastocyst formation

    SciTech Connect

    Jiang Guangjian; Pan Lei; Huang Xiuying; Han Mei; Wen Jinkun . E-mail: wjk@hebmu.edu.cn; Sun Fangzhen . E-mail: fzsun@genetics.ac.cn

    2005-09-23

    It has been shown recently that hyperplasia suppressor gene (HSG) is a powerful regulator for cell proliferation and has a critical role in mitochondrial fusion in many cells. However, little is known about its expression, localization, and function during oocyte maturation and early embryogenesis. In this study, with indirect immunofluorescent staining and Western blotting, we found that HSG was expressed in mouse oocytes and preimplantation embryos which primarily exhibited a submembrane distribution pattern in the cytoplasm. Moreover, HSG mainly associated with {beta}-tubulin during oocyte maturation and early embryonic development. When mouse zygotes were injected with HSG antisense plasmid and cultured in vitro, their capacity to form blastocysts was severely impaired. Our results indicate that HSG plays an essential role in mouse preimplantation development.

  5. Single electron transfer between selectfluor and chloride: A mass spectrometric and theoretical study

    NASA Astrophysics Data System (ADS)

    Zhang, Xiang

    2013-10-01

    The reaction between 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis-tetrafluoroborate (selectfluor) and chloride has been studied experimentally and modeled computationally at the ab initio levels. Based on the interception experiments by electrospray ionization-mass spectrometry (ESI-MS), it is found that only 5,5-dimethyl-l-pyrroline N-oxide (DMPO) succeeds in trapping the chlorine free radical. This result indicates that the single electron transfer (SET) is likely to occur between selectfluor and chloride. According to the Marcus' theory, the activation and reaction free energies for this electron transfer have been calculated. The theoretical study shows that the electron transfer reaction is both thermodynamically and kinetically beneficial, which is consistent with the experiment.

  6. Multi-Color Single Molecule Fluorescence Resonance Energy Transfer (smFret)

    NASA Astrophysics Data System (ADS)

    Anderson, Trevor; Weninger, Keith

    2008-10-01

    The assembly of multi-protein complexes is a vital part of intracellular biology. High resolution methods for characterizing such multi-protein complexes are required to understand functions of these complexes at the mechanistic level. Single molecule Fluorescence Resonance Energy Transfer is a promising method for both characterizing protein conformations and co-localizing different members of such multi-protein complexes. We present our progress towards developing an instrument for three and four color FRET studies at the single molecule level. This method will be useful for characterizing multi-protien complexes.

  7. Electron Transfer-Based Single Molecule Fluorescence as a Probe for Nano-Environment Dynamics

    PubMed Central

    Chen, Ruiyun; Wu, Ruixiang; Zhang, Guofeng; Gao, Yan; Xiao, Liantuan; Jia, Suotang

    2014-01-01

    Electron transfer (ET) is one of the most important elementary processes that takes place in fundamental aspects of biology, chemistry, and physics. In this review, we discuss recent research on single molecule probes based on ET. We review some applications, including the dynamics of glass-forming systems, surface binding events, interfacial ET on semiconductors, and the external field-induced dynamics of polymers. All these examples show that the ET-induced changes of fluorescence trajectory and lifetime of single molecules can be used to sensitively probe the surrounding nano-environments. PMID:24496314

  8. Single Neutron Transfer Experiments Close to the r-Process Path

    SciTech Connect

    Grzywacz-Jones, Kate L; Adekola, Aderemi S; Bardayan, Daniel W; Blackmon, Jeff C; Chae, Kyung Yuk; Chipps, K.; Cizewski, Jolie; Dean, David Jarvis; Erikson, Luke; Fitzgerald, R. P.; Gaddis, A. L.; Greife, U.; Harlin, Christopher W; Hatarik, Robert; Howard, Joshua A; Johnson, Micah; Kozub, R. L.; Liang, J Felix; Livesay, Jake; Ma, Zhanwen; Moazen, Brian; O'Malley, Patrick; Nesaraja, Caroline D; Pain, S. D.; Patterson, N. P.; Paulauskas, Stanley V; Shapira, Dan; ShrinerJr., J. F.; Sissom, D. J.; Smith, Michael Scott; Swan, T. P.; Thomas, J. S.

    2007-01-01

    The first measurements using the (d, p) transfer reaction to study single- particle states in nuclei on the expected r-process path have been made at the Holifield Radioactive Ion Beam Facility. The shell closure at N = 50 has been crossed using the 82Ge(d, p) and 84Se(d, p) reactions. The prop- erties of the lowest-lying states have been determined. Furthermore, the 132Sn(d, p) reaction has been used for the first time to populate single- particle states in 133Sn.

  9. Method of calculating gas dynamics and heat transfer in single stage refrigeration units

    NASA Technical Reports Server (NTRS)

    Zhitomirskiy, I. S.; Popolskiy, A. B.

    1974-01-01

    A generalized mathematical model of gas-dynamic and heat transfer processes in single-stage regenerative installations operating in Stirling, MacMahon, Gifford-MacMahon, and pulsating tube cycles is proposed. A numerical method os solving initial equations on a digital computer is given. This makes it possible to calculate the change in the thermodynamic parameters in the working cycle in different machine components, as well as the dependence of cold productivity on the temperature level in the steady regime.

  10. Single Molecule Spectroelectrochemistry of Interfacial Charge Transfer Dynamics In Hybrid Organic Solar Cell

    SciTech Connect

    Pan, Shanlin

    2014-11-16

    Our research under support of this DOE grant is focused on applied and fundamental aspects of model organic solar cell systems. Major accomplishments are: 1) we developed a spectroelectorchemistry technique of single molecule single nanoparticle method to study charge transfer between conjugated polymers and semiconductor at the single molecule level. The fluorescence of individual fluorescent polymers at semiconductor surfaces was shown to exhibit blinking behavior compared to molecules on glass substrates. Single molecule fluorescence excitation anisotropy measurements showed the conformation of the polymer molecules did not differ appreciably between glass and semiconductor substrates. The similarities in molecular conformation suggest that the observed differences in blinking activity are due to charge transfer between fluorescent polymer and semiconductor, which provides additional pathways between states of high and low fluorescence quantum efficiency. Similar spectroelectrochemistry work has been done for small organic dyes for understand their charge transfer dynamics on various substrates and electrochemical environments; 2) We developed a method of transferring semiconductor nanoparticles (NPs) and graphene oxide (GO) nanosheets into organic solvent for a potential electron acceptor in bulk heterojunction organic solar cells which employed polymer semiconductor as the electron donor. Electron transfer from the polymer semiconductor to semiconductor and GO in solutions and thin films was established through fluorescence spectroscopy and electroluminescence measurements. Solar cells containing these materials were constructed and evaluated using transient absorption spectroscopy and dynamic fluorescence techniques to understand the charge carrier generation and recombination events; 3) We invented a spectroelectorchemistry technique using light scattering and electroluminescence for rapid size determination and studying electrochemistry of single NPs in an

  11. Electron Transport, Energy Transfer, and Optical Response in Single Molecule Junctions

    NASA Astrophysics Data System (ADS)

    White, Alexander

    2015-03-01

    The field of molecular electronics has grown significantly since the first measurements of single molecule conductance. The single molecule junction, a device in which two conducting leads are spanned by a single molecule, has become a powerful tool for studying charge transfer at the molecular level. While early experiments were focused on elastic electron conductance, today measurements of vibronic effects, molecular optical response, spintronics, thermal conductance, and quantum interference and decoherence effects are prominent areas of research. These new experimental advancements demand improved theoretical treatments which properly account for the interactions between different degrees of freedom: charge, electronic, vibrational, spin, etc.; all in physically relevant parameter ranges. This talk focuses on using a many-body states based approach to investigate the regime of strong interaction between these degrees of freedom, with relatively weak coupling between the molecule and the electric reservoirs created by the conducting leads. We focused on three related processes, electron transfer, electronic energy transfer and molecular excitation. In collaboraton with Boris Fainberg, Faculty of Sciences, Holon Institute of Technology; Sergei Tretiak, Theoretical Division, Center for Integrated Nanotechnologies, Los Alamos National Laboratory; and Michael Galperin, Department of Chemistry and Biochemistry, University of California San Diego.

  12. Single-phase ambient and cryogenic temperature heat transfer coefficients in microchannels

    NASA Astrophysics Data System (ADS)

    Baek, S.; Bradley, P. E.

    2015-12-01

    Micro-scaling cryogenic refrigerators, in particular the Joule-Thomson (JT) variety require very good information about heat transfer characteristics of the refrigerants flowing in the microchannels for optimal design and performance. The extremely low Reynolds flow is present in a micro JT cryocooler, the heat transfer characteristics at these conditions require investigation. There are numerous studies regarding heat transfer coefficient measurements of liquid flow in microchannels at/near ambient temperature and high Reynolds flow (Re>2000), that agree well with the conventional correlations. However, results from previous studies of gaseous flow in microchannels at low Reynolds flow (Re<1000) disagree with conventional theory. Moreover, the studies performed at cryogenic temperatures are quite limited in number. In this paper, the single-phase heat transfer coefficients and friction factors for nitrogen are measured at ambient and cryogenic temperatures. The hydraulic diameters for this study are 60, 110 and 180 μm for circular microchannels. The Reynolds numbers varied from a very low value of 10 to 3000. The measured friction factors are comparable to those in macro-scale tubes. The experimental results of the heat transfer indicate that Nusselt numbers derived from measurements are significantly affected by axial conduction at low Reynolds flow (Re<500). The Nusselt numbers at high Reynolds flow (Re>1000) follow conventional theory. The detailed experiment, procedure, and measured results are presented in this paper and discussed regarding deviation from ideal theory at low Reynolds flow.

  13. Distance dependence of the energy transfer rate from a single semiconductor nanostructure to graphene.

    PubMed

    Federspiel, François; Froehlicher, Guillaume; Nasilowski, Michel; Pedetti, Silvia; Mahmood, Ather; Doudin, Bernard; Park, Serin; Lee, Jeong-O; Halley, David; Dubertret, Benoît; Gilliot, Pierre; Berciaud, Stéphane

    2015-02-11

    The near-field Coulomb interaction between a nanoemitter and a graphene monolayer results in strong Förster-type resonant energy transfer and subsequent fluorescence quenching. Here, we investigate the distance dependence of the energy transfer rate from individual, (i) zero-dimensional CdSe/CdS nanocrystals and (ii) two-dimensional CdSe/CdS/ZnS nanoplatelets to a graphene monolayer. For increasing distances d, the energy transfer rate from individual nanocrystals to graphene decays as 1/d(4). In contrast, the distance dependence of the energy transfer rate from a two-dimensional nanoplatelet to graphene deviates from a simple power law but is well described by a theoretical model, which considers a thermal distribution of free excitons in a two-dimensional quantum well. Our results show that accurate distance measurements can be performed at the single particle level using graphene-based molecular rulers and that energy transfer allows probing dimensionality effects at the nanoscale. PMID:25607231

  14. Interfacial electrochemical electron transfer in biology - towards the level of the single molecule.

    PubMed

    Zhang, Jingdong; Chi, Qijin; Hansen, Allan G; Jensen, Palle S; Salvatore, Princia; Ulstrup, Jens

    2012-03-01

    Physical electrochemistry has undergone a remarkable evolution over the last few decades, integrating advanced techniques and theory from solid state and surface physics. Single-crystal electrode surfaces have been a core notion, opening for scanning tunnelling microscopy directly in aqueous electrolyte (in situ STM). Interfacial electrochemistry of metalloproteins is presently going through a similar transition. Electrochemical surfaces with thiol-based promoter molecular monolayers (SAMs) as biomolecular electrochemical environments and the biomolecules themselves have been mapped with unprecedented resolution, opening a new area of single-molecule bioelectrochemistry. We consider first in situ STM of small redox molecules, followed by in situ STM of thiol-based SAMs as molecular views of bioelectrochemical environments. We then address electron transfer metalloproteins, and multi-centre metalloenzymes including applied single-biomolecular perspectives based on metalloprotein/metallic nanoparticle hybrids.

  15. Biophysical Insights from Temperature-Dependent Single-Molecule Förster Resonance Energy Transfer

    NASA Astrophysics Data System (ADS)

    Holmstrom, Erik D.; Nesbitt, David J.

    2016-05-01

    Single-molecule fluorescence microscopy techniques can be used in combination with micrometer length-scale temperature control and Förster resonance energy transfer (FRET) in order to gain detailed information about fundamental biophysical phenomena. In particular, this combination of techniques has helped foster the development of remarkable quantitative tools for studying both time- and temperature-dependent structural kinetics of biopolymers. Over the past decade, multiple research efforts have successfully incorporated precise spatial and temporal control of temperature into single-molecule FRET (smFRET)-based experiments, which have uncovered critical thermodynamic information on a wide range of biological systems such as conformational dynamics of nucleic acids. This review provides an overview of various temperature-dependent smFRET approaches from our laboratory and others, highlighting efforts in which such methods have been successfully applied to studies of single-molecule nucleic acid folding.

  16. Probing the charge-transfer dynamics in DNA at the single-molecule level.

    PubMed

    Kawai, Kiyohiko; Matsutani, Eri; Maruyama, Atsushi; Majima, Tetsuro

    2011-10-01

    Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.

  17. Heat transfer enhancement due to a longitudinal vortex produced by a single winglet in a pipe

    SciTech Connect

    Oyakawa, Kenyu; Senaha, Izuru; Ishikawa, Shuji; Hokama, Mashashige

    1999-07-01

    Longitudinal vortices were artificially generated by a single winglet vortex generator in a pipe. The purpose of this study is to analyze the motion of longitudinal vortices and their effects on heat transfer enhancement. The flow pattern was visualized by means of both fluorescein and rhodamine B as traces in a water flow. The main vortex was moved spirally along the circumference and the behavior of the other vortices was observed. Streamwise and circumferential heat transfer coefficients on the wall, wall static pressure, and velocity distribution in an overall cross section were also measured for the air flow in a range of Reynolds numbers from 18,800 to 62,400. The distributions of the streamwise heat transfer coefficient had a periodic pattern, and the peaks in the distribution were circumferentially moved due to the spiral motion of the main vortex. Lastly, the relationships between the iso-velocity distribution, wall static pressure, and heat transfer characteristics was shown. In the process of forming the vortex behind the winglet vortex generator, behaviors of both the main vortex and the corner vortex were observed as streak lines. The vortex being raised along the end of the winglet, and the vortex ring being rolled up to the main vortex were newly observed. Both patterns of the streamwise velocity on a cross-section and the static pressure on the wall show good correspondences to phenomena of the main vortex spirally flowing downstream. The increased ratio of the heat transfer is similar to that of the friction factor based on the shear stress on the wall surface of the pipe. The quantitative analogy between the heat transfer and the shear stress is confirmed except for some regions, where the effects of the down-wash or blow-away of the secondary flows is caused due to the main vortex.

  18. Charge transfer structure-reactivity dependence of fullerene-single-walled carbon nanotube heterojunctions.

    PubMed

    Hilmer, Andrew J; Tvrdy, Kevin; Zhang, Jingqing; Strano, Michael S

    2013-08-14

    Charge transfer at the interface between single-walled carbon nanotubes (SWCNTs) of distinct chiral vectors and fullerenes of various molecular weights is of interest both fundamentally and because of its importance in emerging photovoltaic and optoelectronic devices. One approach for generating isolated, discretized fullerene-SWCNT heterojunctions for spectroscopic investigation is to form an amphiphile, which is able to disperse the latter at the single-SWCNT level in aqueous solution. Herein, we synthesize a series of methanofullerene amphiphiles, including derivatives of C60, C70, and C84, and investigated their electron transfer with SWCNT of specific chirality, generating a structure-reactivity relationship. In the cases of two fullerene derivatives, lipid-C61-polyethylene glycol (PEG) and lipid-C71-PEG, band gap dependent, incomplete quenching was observed across all SWCNT species, indicating that the driving force for electron transfer is small. This is further supported by a variant of Marcus theory, which predicts that the energy offsets between the nanotube conduction bands and the C61 and C71 LUMO levels are less than the exciton binding energy in SWCNT. In contrast, upon interfacing nanotubes with C85 methanofullerene, a complete quenching of all semiconducting SWCNT is observed. This enhancement in quenching efficiency is consistent with the deeper LUMO level of C85 methanofullerene in comparison with the smaller fullerene adducts, and suggests its promise as for SWCNT-fullerene heterojunctions.

  19. 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

  20. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

    PubMed Central

    2011-01-01

    Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses) via horizontal gene transfer (HGT). It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA) viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep)-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts. PMID:21943216

  1. 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.

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

    PubMed

    Mead, R A; Rourke, A W

    1985-07-01

    The in vivo incorporation of 3H-uridine into RNA was studied in delayed implanting and activated blastocysts obtained from 33 western spotted skunks. 3H-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 3H-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 (43.9% vs. 27.5%). Our 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.

  3. Differential regulation of receptivity in two uterine horns of a recipient mouse following asynchronous embryo transfer

    PubMed Central

    Li, Shi-Jie; Wang, Tong-Song; Qin, Fu-Niu; Huang, Zhu; Liang, Xiao-Huan; Gao, Fei; Song, Zhuo; Yang, Zeng-Ming

    2015-01-01

    Receptivity is a limited time in which uterine endometrium can establish a successful dialogue with blastocyst. This study was to investigate the effect of asynchronous embryo transfer on uterine receptivity in mice. Embryos under different stages were transferred into two oviduct sides of a recipient mouse on day 1 of pseudopregnancy. Our results showed the asynchronously transferred embryos can implant in all groups. Compared to zygote-transfer group, the length of implanted embryos is longer in 8-cell embryo- or blastocyst-transfer group. The levels of Snail and COX-2 immunostaining in blastocyst-transfer group are significantly stronger than that in zygote-transfer group. Embryos in blastocyst-transfer group migrate faster than that in zygote-transfer group within uterus. Blastocysts are in a state of developmental delay after they are transferred into oviducts, and they are reactivated and implanted rapidly in uterus. The developmental rate to newborn in zygote-transfer group is obviously higher than that in blastocyst-transfer group, suggesting that a delay in embryo development and implantation will lead to a decrease of litter size. These results indicated that the window of implantation is differentially regulated in two uterine horns of a recipient by embryos at different stages. PMID:26531680

  4. High-fidelity transfer and storage of photon states in a single nuclear spin

    NASA Astrophysics Data System (ADS)

    Yang, Sen; Wang, Ya; Rao, D. D. Bhaktavatsala; Hien Tran, Thai; Momenzadeh, Ali S.; Markham, M.; Twitchen, D. J.; Wang, Ping; Yang, Wen; Stöhr, Rainer; Neumann, Philipp; Kosaka, Hideo; Wrachtrup, Jörg

    2016-08-01

    Long-distance quantum communication requires photons and quantum nodes that comprise qubits for interaction with light and good memory capabilities, as well as processing qubits for the storage and manipulation of photons. Owing to the unavoidable photon losses, robust quantum communication over lossy transmission channels requires quantum repeater networks. A necessary and highly demanding prerequisite for these networks is the existence of quantum memories with long coherence times to reliably store the incident photon states. Here we demonstrate the high-fidelity (˜98%) coherent transfer of a photon polarization state to a single solid-state nuclear spin that has a coherence time of over 10 s. The storage process is achieved by coherently transferring the polarization state of a photon to an entangled electron-nuclear spin state of a nitrogen-vacancy centre in diamond. The nuclear spin-based optical quantum memory demonstrated here paves the way towards an absorption-based quantum repeater network.

  5. High-fidelity transfer and storage of photon states in a single nuclear spin

    NASA Astrophysics Data System (ADS)

    Yang, Sen; Wang, Ya; Rao, D. D. Bhaktavatsala; Hien Tran, Thai; Momenzadeh, Ali S.; Markham, M.; Twitchen, D. J.; Wang, Ping; Yang, Wen; Stöhr, Rainer; Neumann, Philipp; Kosaka, Hideo; Wrachtrup, Jörg

    2016-08-01

    Long-distance quantum communication requires photons and quantum nodes that comprise qubits for interaction with light and good memory capabilities, as well as processing qubits for the storage and manipulation of photons. Owing to the unavoidable photon losses, robust quantum communication over lossy transmission channels requires quantum repeater networks. A necessary and highly demanding prerequisite for these networks is the existence of quantum memories with long coherence times to reliably store the incident photon states. Here we demonstrate the high-fidelity (∼98%) coherent transfer of a photon polarization state to a single solid-state nuclear spin that has a coherence time of over 10 s. The storage process is achieved by coherently transferring the polarization state of a photon to an entangled electron–nuclear spin state of a nitrogen–vacancy centre in diamond. The nuclear spin-based optical quantum memory demonstrated here paves the way towards an absorption-based quantum repeater network.

  6. Charge transfer processes and ultraviolet induced absorption in Yb:YAG single crystal laser materials

    NASA Astrophysics Data System (ADS)

    Rydberg, S.; Engholm, M.

    2013-06-01

    Charge transfer (CT) transitions and UV induced color centers in Yb:YAG single crystals have been investigated. A simultaneous pair formation of a stable Yb2+ ion and a hole related (O-) color center (hole polaron) are observed through a CT-process. Slightly different types of hole related color centers are formed in Yb:YAG crystals containing small levels of iron impurities. Furthermore, excitation spectroscopy on the UV irradiated Yb:YAG samples could confirm an energy transfer process between Yb3+ and Yb2+ ions. The findings are important for an increased knowledge of the physical loss mechanisms observed in Yb-doped laser materials, such as the nonlinear decay process in Yb:YAG crystals as well as the photodarkening phenomenon in Yb-doped fiber lasers.

  7. Bovine blastocysts obtained from reconstructed cytoplast and karyoplasts using a simple portable CO2 incubator.

    PubMed

    Varisanga, M D; Dong, Y J; Mtango, N R; Fahrudin, M; Ni Wayan, K K; Suzuki, T

    2000-01-01

    To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO(2) incubator. CO(2) and O(2) gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO(2) (2-5%) and O(2) (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5 degrees C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O(2), CO(2), and air pressure.

  8. 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

  9. On the charge transfer between single-walled carbon nanotubes and graphene

    SciTech Connect

    Rao, Rahul Pierce, Neal; Dasgupta, Archi

    2014-08-18

    It is important to understand the electronic interaction between single-walled carbon nanotubes (SWNTs) and graphene in order to use them efficiently in multifunctional hybrid devices. Here, we deposited SWNT bundles on graphene-covered copper and SiO{sub 2} substrates by chemical vapor deposition and investigated the charge transfer between them by Raman spectroscopy. Our results revealed that, on both copper and SiO{sub 2} substrates, graphene donates electrons to the SWNTs, resulting in p-type doped graphene and n-type doped SWNTs.

  10. Observation of Frenkel and charge transfer excitons in pentacene single crystals using spectroscopic generalized ellipsometry

    NASA Astrophysics Data System (ADS)

    Qi, Dongchen; Su, Haibin; Bastjan, M.; Jurchescu, O. D.; Palstra, T. M.; Wee, Andrew T. S.; Rübhausen, M.; Rusydi, A.

    2013-09-01

    We report on the emerging and admixture of Frenkel and charge transfer (CT) excitons near the absorption onset in pentacene single crystals. Using high energy-resolution spectroscopic generalized ellipsometry with in-plane polarization dependence, the excitonic nature of three lowest lying excitations is discussed. Their distinct polarization dependence strongly indicates the presence of both Frenkel and CT types of excitons near the excitation onset. In particular, the peculiar polarization behavior of the second excitation can only be rationalized by taking into account the inherent CT transition dipole moment. This observation has important implications for the pentacene-based optoelectronic devices.

  11. Electron transfer behaviour of biological macromolecules towards the single-molecule level

    NASA Astrophysics Data System (ADS)

    Zhang, Jingdong; Grubb, Mikala; Hansen, Allan G.; Kuznetsov, Alexander M.; Boisen, Anja; Wackerbarth, Hainer; Ulstrup, Jens

    2003-05-01

    Redox metalloproteins immobilized on metallic surfaces in contact with aqueous biological media are important in many areas of pure and applied sciences. Redox metalloprotein films are currently being addressed by new approaches where biotechnology including modified and synthetic proteins is combined with state-of-the-art physical electrochemistry with emphasis on single-crystal, atomically planar electrode surfaces, in situ scanning tunnelling microscopy (STM) and other surface techniques. These approaches have brought bioelectrochemistry important steps forward towards the nanoscale and single-molecule levels. We discuss here these advances with reference to two specific redox metalloproteins, the blue single-copper protein Pseudomonas aeruginosa azurin and the single-haem protein Saccharomyces cerevisiae yeast cytochrome c, and a short oligonucleotide. Both proteins can be immobilized on Au(111) by chemisorption via exposed sulfur-containing residues. Voltammetric, interfacial capacitance, x-ray photoelectron spectroscopy and microcantilever sensor data, together with in situ STM with single-molecule resolution, all point to a coherent view of monolayer organization with protein electron transfer (ET) function retained. In situ STM can also address the microscopic mechanisms for electron tunnelling through the biomolecules and offers novel notions such as coherent multi-ET between the substrate and tip via the molecular redox levels. This differs in important respects from electrochemical ET at a single metal/electrolyte interface. Similar data for a short oligonucleotide immobilized on Au(111) show that oligonucleotides can be characterized with comparable detail, with novel perspectives for addressing DNA electronic conduction mechanisms and for biological screening towards the single-molecule level.

  12. Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells.

    PubMed

    Madan, M L; Chauhan, M S; Singla, S K; Manik, R S

    1994-09-01

    Buffalo ovaries were collected immediately after slaughter and were transported to laboratory in sterile saline at 37 degrees C. Follicular oocytes with the cumulus mass aspirated from 2 to 6 mm in diameter follicles were cultured in TCM-199 medium supplemented with 10% buffalo estrus serum (BES) in 5% CO(2) at 38.5 degrees C. After 20 to 24 h of incubation, the oocytes were inseminated with precapacitated frozen thawed spermatozoa for 6 h. The fertilization rate was 78.15% of the matured oocytes. Over an in vitro culture period of 3 to 9 d, 4.02% of the inseminated oocytes developed to the morula stage when cultured with cumulus cells alone and 17.83% when cumulus cells plus oviductal epithelial cells were used. The percentage of developed blastocysts was very low (0.57%) when the oocytes were co-cultured with cumulus cells from the original oocytes. However, 8% of the inseminated oocytes that were denuded 3 d after insemination developed to the blastocyst stage when they were co-cultured with cumulus and oviductal epithelial cells. Sixteen early/expanded blastocysts were transferred non-surgically to 16 recipients. Four of the 16 recipients became pregnant, of which 2 delivered normal buffalo male calves.

  13. Higher β-HCG concentrations and higher birthweights ensue from single vitrified embryo transfers.

    PubMed

    Keane, Kevin N; Mustafa, Kamarul Bahyah; Hinchliffe, Peter; Conceicao, Jason; Yovich, John L

    2016-08-01

    To examine the effect of cryopreservation on developmental potential of human embryos, this study compared quantitative β-HCG concentrations at pregnancy test after IVF-fresh embryo transfer (IVF-ET) with those arising after frozen embryo transfer (FET). It also tracked outcomes of singleton pregnancies resulting from single-embryo transfers that resulted in singleton live births (n = 869; with 417 derived from IVF-ET and 452 from FET). The initial serum β-HCG concentration indicating successful implantation was measured along with the birthweight of the ensuing infants. With testing at equivalent luteal phase lengths, the median pregnancy test β-HCG was significantly higher following FET compared with fresh IVF-ET (844.5 IU/l versus 369 IU/l; P < 0.001). Despite no significant difference in the average period of gestation (38 weeks 5 days for both groups), the mean birthweight of infants born following FET was significantly heavier by 161 g (3370 g versus 3209 g; P < 0.001). Furthermore, more infants exceeded 4000 g (P < 0.001) for FET although there was no significant difference for the macrosomic category (≥4500 g). We concluded that FET programme embryos lead to infants with equivalent (if not better) developmental potential compared with IVF-ET, demonstrated by higher pregnancy β-HCG concentrations and ensuing birthweights. PMID:27209497

  14. What about the remaining twins since single-embryo transfer? How far can (should) we go?

    PubMed

    De Neubourg, D; Gerris, J

    2006-04-01

    Single-embryo transfer (SET) and more specifically elective SET (eSET) have taken their place in good clinical IVF/ICSI practice. After the initial cautious search for the characteristics of the twin-prone patient and of the selection of the embryo with the highest implantation potential many centres have embarked on the (progressive) implementation of SET, either by conviction or forced by legislation or both. It was only because the ongoing pregnancy rates remained largely unaffected that SET was accepted. Generally speaking, it can be said that the twinning rate after IVF/ICSI has dropped by at least 50% simply by transferring only one good-quality embryo in the first and second fresh IVF/ICSI cycles in young women, without decrease in the overall pregnancy rate. Preventing 'the second half' of IVF/ICSI twins constitutes another and probably tougher challenge because the target group is a heterogeneous mix consisting of patients in very different clinical situations. Can we expand our experience for further twin prevention to women of older age and to cycles of higher rank without a significant drop in pregnancy rates? Can we extend it to more cryopreservation cycles? To have an idea of future target groups for increased application of SET, we analysed the remaining twins after double-embryo transfer (DET), and from these data we suggest expanding the eSET policy to women <38 years of age until the third cycle and to cryopreservation cycles.

  15. Photoinduced charge transfer from vacuum-deposited molecules to single-layer transition metal dichalcogenides

    NASA Astrophysics Data System (ADS)

    Osada, Kazuki; Tanaka, Masatoshi; Ohno, Shinya; Suzuki, Takanori

    2016-06-01

    Variations of photoluminescence (PL) and Raman spectra of single-layer MoS2, MoSe2, WS2, and WSe2 due to the vacuum deposition of C60 or copper phthalocyanine (CuPc) molecules have been investigated. PL spectra are decomposed into two competitive components, an exciton and a charged exciton (trion), depending on carrier density. The variation of PL spectra is interpreted in terms of charge transfer across the interfaces between transition metal dichalcogenides (TMDs) and dopant molecules. We find that deposited C60 molecules inject photoexcited electrons into MoS2, MoSe2, and WS2 or holes into WSe2. CuPc molecules also inject electrons into MoS2, MoSe2, and WS2, while holes are depleted from WSe2 to CuPc. We then propose a band alignment between TMDs and dopant molecules. Peak shifts of Raman spectra and doped carrier density estimated using a three-level model also support the band alignment. We thus demonstrate photoinduced charge transfer from dopant molecules to single-layer TMDs.

  16. Analysis of Convective Heat Transfer Enhancement by Nanofluids: Single-Phase and Two-Phase Treatments

    NASA Astrophysics Data System (ADS)

    Kakaç, S.; Pramuanjaroenkij, A.

    2016-05-01

    Nanofluids have been investigated regarding their advantages and potentialities for the purpose of increasing convective heat transfer rates inside thermal systems where they are used as working fluids. Researchers in thermophysics have investigated these fluids experimentally and numerically. This review provides extensive theoretical information concerning nanofluids in the single-phase and two-phase treatments. Important published works on nanofluid properties and correlations are summarized and reviewed in detail. Heat transfer enhancement by nanofluids is a challenging problem due to the difficulties inherent in the model of the physical mechanism of interaction between the paricles. Here the interaction between the phases is modeled by several two-phase models, and the results are given in graphical and tabular forms. Despite the advantages of the mixture model, such as imlementation of physical properties and less computational power requirements, some studies showed that the results of the single-phase and two-phase models are very similar. The main difference consists in the effect of the drift velocities of the phases relative to each other.

  17. 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

    Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid held for 1.5 h at ambient temperature (26°C-33°C) yielded 32% blastocysts; however, in experiment 1, fluid held at 32 °C for 2 h after aspiration yielded 16% blastocysts versus 23% for aspirates processed immediately. Performing TVA/ICSI throughout the year would increase production from valuable mares, but efficiency during the nonbreeding season is unknown. In addition, to reduce the possibility of infection after TVA, administration of antibiotics to the mare before TVA is indicated; however, these could affect oocyte quality. In experiment 2, follicle numbers at the time of TVA were significantly higher in December to January than for the same mares during the breeding season. Oocyte recovery rates on TVA were 60% to 66% and the blastocyst rate was 18%. An equivalent blastocyst rate (18%) was achieved after administration of ampicillin and gentamicin to mares before TVA. In experiment 3, we verified that stallion differences exist in rates of cleavage after ICSI with motile sperm. In sperm from a low-performing stallion, density-gradient centrifugation followed by swim-up was associated with significantly higher rates of cleavage (45% vs. 18%) and blastocyst development (14% vs. 0%) than those for density gradient alone. In experiment 4, parthenogenetic activation with ionomycin and 6-dimethylaminopurine yielded 40% blastocysts. Frozen-thawed sperm that were immotile

  18. Shell thickness dependent photoinduced hole transfer in hybrid conjugated polymer/quantum dot nanocomposites: from ensemble to single hybrid level.

    PubMed

    Xu, Zhihua; Hine, Corey R; Maye, Mathew M; Meng, Qingping; Cotlet, Mircea

    2012-06-26

    Photoinduced hole transfer is investigated in inorganic/organic hybrid nanocomposites of colloidal CdSe/ZnS quantum dots and a cationic conjugated polymer, poly(9,9'-bis(6-N,N,N-trimethylammoniumhexyl)fluorene-alt-phenylene, in solution and in solid thin film, and down to the single hybrid level and is assessed to be a dynamic quenching process. We demonstrate control of hole transfer rate in these quantum dot/conjugated polymer hybrids by using a series of core/shell quantum dots with varying shell thickness, for which a clear exponential dependency of the hole transfer rate vs shell thickness is observed, for both solution and thin-film situations. Furthermore, we observe an increase of hole-transfer rate from solution to film and correlate this with changes in quantum dot/polymer interfacial morphology affecting the hole transfer rate, namely, the donor-acceptor distance. Single particle spectroscopy experiments reveal fluctuating dynamics of hole transfer at the single conjugated polymer/quantum dot interface and an increased heterogeneity in the hole-transfer rate with the increase of the quantum dot's shell thickness. Although hole transfer quenches the photoluminescence intensity of quantum dots, it causes little or no effect on their blinking behavior over the time scales probed here.

  19. Elective single-embryo transfer: persuasive communication strategies can affect choice in a young British population.

    PubMed

    van den Akker, O B A; Purewal, S

    2011-12-01

    This study tested the effectiveness of the framing effect and fear appeals to inform young people about the risks of multiple births and the option of selecting elective single-embryo transfer (eSET). A non-patient student sample (age (mean±SD) 23±5.5 years; n=321) were randomly allocated to one of seven groups: (1) framing effect: (1a) gain and (1b) loss frame; (2) fear appeal: (2a) high, (2b) medium and (2c) low fear; or (3) a control group: (3a) education and (3b) non-education. The primary outcome measure was the Attitudes towards Single Embryo Transfer questionnaire, before exposure to the messages (time 1) and immediately afterwards (time 2). Results revealed participants in the high fear, medium fear and gain condition demonstrated the most positive and significant differences (P<0.001 to P<0.05) in their knowledge, hypothetical intentions and modest changes in attitudes towards eSET than the low fear, loss frame and education and non-education messages. The results demonstrate that the use of complex persuasive communication techniques on a student population to promote immediate and hypothetical eSET preferences is more successful at promoting eSET than merely reporting educational content. Future research should investigate its application in a clinical population. A multiple pregnancy is a health risk to both infant and mother following IVF treatment. The aims of this study were to test the effectiveness of two persuasive communication techniques (the framing effect and fear appeals) to inform young people about the risks of multiple births and the hypothetical option of selecting elective single-embryo transfer (eSET) (i.e., only one embryo is transferred to the uterus using IVF treatment). A total of 321 non-patient student sample (mean age 23) were randomly allocated to read a message from one of seven groups: (1) framing effect: (1a) gain and (1b) loss frame; (2) fear appeal: (2a) high, (2b) medium and (2c) low fear; or (3) a control group

  20. Enhanced single seed trait predictions in soybean (Glycine max) and robust calibration model transfer with near infrared reflectance spectroscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single seed near infrared reflectance (NIR) spectroscopy predicts soybean (Glycine max) seed quality traits of moisture, oil, and protein. We tested the accuracy of transferring calibrations between different single seed NIR analyzers of the same design by collecting NIR spectra and analytical trait...

  1. Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population

    PubMed Central

    Labonté, Jessica M.; Field, Erin K.; Lau, Maggie; Chivian, Dylan; Van Heerden, Esta; Wommack, K. Eric; Kieft, Thomas L.; Onstott, Tullis C.; Stepanauskas, Ramunas

    2015-01-01

    A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment. PMID:25954269

  2. Single Molecule Nanospectroscopy Visualizes Proton-Transfer Processes within a Zeolite Crystal

    PubMed Central

    2016-01-01

    Visualizing proton-transfer processes at the nanoscale is essential for understanding the reactivity of zeolite-based catalyst materials. In this work, the Brønsted-acid-catalyzed oligomerization of styrene derivatives was used for the first time as a single molecule probe reaction to study the reactivity of individual zeolite H-ZSM-5 crystals in different zeolite framework, reactant and solvent environments. This was accomplished via the formation of distinct dimeric and trimeric fluorescent carbocations, characterized by their different photostability, as detected by single molecule fluorescence microscopy. The oligomerization kinetics turned out to be very sensitive to the reaction conditions and the presence of the local structural defects in zeolite H-ZSM-5 crystals. The remarkably photostable trimeric carbocations were found to be formed predominantly near defect-rich crystalline regions. This spectroscopic marker offers clear prospects for nanoscale quality control of zeolite-based materials. Interestingly, replacing n-heptane with 1-butanol as a solvent led to a reactivity decrease of several orders and shorter survival times of fluorescent products due to the strong chemisorption of 1-butanol onto the Brønsted acid sites. A similar effect was achieved by changing the electrophilic character of the para-substituent of the styrene moiety. Based on the measured turnover rates we have established a quantitative, single turnover approach to evaluate substituent and solvent effects on the reactivity of individual zeolite H-ZSM-5 crystals. PMID:27709925

  3. Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population.

    PubMed

    Labonté, Jessica M; Field, Erin K; Lau, Maggie; Chivian, Dylan; Van Heerden, Esta; Wommack, K Eric; Kieft, Thomas L; Onstott, Tullis C; Stepanauskas, Ramunas

    2015-01-01

    A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

  4. Single step high-speed printing of continuous silver lines by laser-induced forward transfer

    NASA Astrophysics Data System (ADS)

    Puerto, D.; Biver, E.; Alloncle, A.-P.; Delaporte, Ph.

    2016-06-01

    The development of high-speed ink printing process by Laser-Induced Forward Transfer (LIFT) is of great interest for the printing community. To address the problems and the limitations of this process that have been previously identified, we have performed an experimental study on laser micro-printing of silver nanoparticle inks by LIFT and demonstrated for the first time the printing of continuous conductive lines in a single pass at velocities of 17 m/s using a 1 MHz repetition rate laser. We investigated the printing process by means of a time-resolved imaging technique to visualize the ejection dynamics of single and adjacent jets. The control of the donor film properties is of prime importance to achieve single step printing of continuous lines at high velocities. We use a 30 ps pulse duration laser with a wavelength of 343 nm and a repetition rate from 0.2 to 1 MHz. A galvanometric mirror head controls the distance between two consecutives jets by scanning the focused beam along an ink-coated donor substrate at different velocities. Droplets and lines of silver inks are laser-printed on glass and PET flexible substrates and we characterized their morphological quality by atomic force microscope (AFM) and optical microscope.

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

    PubMed

    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.

  6. Transcriptomic signature to oxidative stress exposure at the time of embryonic genome activation in bovine blastocysts.

    PubMed

    Cagnone, Gael L M; Sirard, Marc-André

    2013-04-01

    In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions. Two pro-oxidant agents, one that acts extracellularly by promoting reactive oxygen species (ROS) production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) or another that acts intracellularly by inhibiting glutathione synthesis (0.4 mM buthionine sulfoximine [BSO]) were added separately to in vitro culture media from Day 3 (8-16-cell stage) onward. Transcriptomic analysis was then performed on resulting Day-7 blastocysts. In the literature, these two pro-oxidant conditions were shown to induce delayed degeneration in a proportion of Day-8 blastocysts. In our experiment, no morphological difference was visible, but AAPH tended to decrease the blastocyst rate while BSO significantly reduced it, indicating a differential impact on the surviving population. At the transcriptomic level, blastocysts that survived either pro-oxidant exposure showed oxidative stress and an inflammatory response (ARRB2), although AAPH induced higher disturbances in cellular homeostasis (SERPINE1). Functional genomics of the BSO profile, however, identified differential expression of genes related to glycine metabolism and energy metabolism (TPI1). These differential features might be indicative of pre-degenerative blastocysts (IGFBP7) in the AAPH population whereas BSO exposure would select the most viable individuals (TKDP1). Together, these results illustrate how oxidative disruption of pre-attachment development is associated with systematic up-regulation of several metabolic markers. Moreover, it indicates that a better capacity to survive anti-oxidant depletion may allow for the survival of blastocysts with a quieter metabolism after compaction.

  7. 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

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

    PubMed

    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

  9. Conjugate Heat Transfer Computation for Evaluation of Single-Blow Method for Compact Fin-Tube Heat Exchangers

    NASA Astrophysics Data System (ADS)

    Song, Gil-Dal; Nishino, Koichi

    Computations have been carried out to evaluate heat transfer coefficients given by the single-blow method that is characterized by a transient and conjugate heat transfer problem between the fluid and the solid. Both heat conduction and convection equations are solved numerically to obtain the transient fluid and fin temperature distributions. Finite volume solutions indicate that the fin temperature distribution of the single-blow method varies with time and position and that the fluid temperature distribution of the single-blow method is close to that observed in the steady-state computation specified with the constant wall temperature condition. It is found that the local heat transfer coefficient resulting from the single-blow method is almost identical to that from the steady-state constant wall temperature computation and is nearly time-independent.

  10. Proposal for probing energy transfer pathway by single-molecule pump-dump experiment

    PubMed Central

    Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

    2016-01-01

    The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices. PMID:27277702

  11. Elective Single Embryo Transfer: an update to UK Best Practice Guidelines.

    PubMed

    Harbottle, Stephen; Hughes, Ciara; Cutting, Rachel; Roberts, Steve; Brison, Daniel

    2015-09-01

    A significant number of multiple pregnancies and births worldwide continue to occur following treatment with Assisted Reproductive Technologies (ARTs). Whilst efforts have been made to increase the proportion of elective single embryo transfer (eSET) cycles, the multiple pregnancy rate or MPR remains at a level that most consider unacceptable given the associated clinical risks to mothers and babies, and the additional costs associated with neonatal care of premature and low birth weight babies. Northern Europe, Australia and Japan have continued to lead the way in the adoption of eSET. Randomised controlled trials or RCTs, meta-analyses and economic analyses support the implementation of an eSET policy, particularly in light of recent advances in ARTs. This paper provides a review of current evidence and an update to the eSET guidelines first published by Cutting et al. (2008) intended to assist ART clinics in the implementation of an effective eSET policy.

  12. Elective Single Embryo Transfer: an update to UK Best Practice Guidelines.

    PubMed

    Harbottle, Stephen; Hughes, Ciara; Cutting, Rachel; Roberts, Steve; Brison, Daniel

    2015-09-01

    A significant number of multiple pregnancies and births worldwide continue to occur following treatment with Assisted Reproductive Technologies (ARTs). Whilst efforts have been made to increase the proportion of elective single embryo transfer (eSET) cycles, the multiple pregnancy rate or MPR remains at a level that most consider unacceptable given the associated clinical risks to mothers and babies, and the additional costs associated with neonatal care of premature and low birth weight babies. Northern Europe, Australia and Japan have continued to lead the way in the adoption of eSET. Randomised controlled trials or RCTs, meta-analyses and economic analyses support the implementation of an eSET policy, particularly in light of recent advances in ARTs. This paper provides a review of current evidence and an update to the eSET guidelines first published by Cutting et al. (2008) intended to assist ART clinics in the implementation of an effective eSET policy. PMID:26391438

  13. Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence.

    PubMed

    Branchini, Bruce R; Behney, Curran E; Southworth, Tara L; Fontaine, Danielle M; Gulick, Andrew M; Vinyard, David J; Brudvig, Gary W

    2015-06-24

    Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.

  14. Proposal for probing energy transfer pathway by single-molecule pump-dump experiment.

    PubMed

    Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

    2016-01-01

    The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices. PMID:27277702

  15. Proposal for probing energy transfer pathway by single-molecule pump-dump experiment

    NASA Astrophysics Data System (ADS)

    Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

    2016-06-01

    The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices.

  16. Multiple-layer SOI based on Single-Crystal Si Nanomembrane Transfer

    NASA Astrophysics Data System (ADS)

    Peng, Weina; Roberts, Michelle; Nordberg, Eric; Flack, Frank; Colavita, Paula; Hamers, Robert; Savage, Donald; Lagally, Max; Eriksson, Mark

    2007-03-01

    Silicon-on-insulator (SOI) has many advantages over bulk Si including the reduction of parasitic resistance and increased device speed. Multiple-layer SOI, having more device layers per unit area, enables 3D process integration as well as applications in optics. However, it is impossible to achieve such a system by growth techniques (one can grow only non-crystalline Si on SiO2), and multiple Smart Cut transfers used to create single layer SOI may be prohibitively expensive. We present here a novel method to fabricate such a multiple SOI system using transferred Si nanomembranes^ and subsequent oxidation. The surface roughness and interface quality are examined respectively by AFM and cross-sectional SEM. Low surface roughness (0.176nm) and smooth interfaces are achieved. As an example optical application, we apply the multilayer system to fabricate a Si-based Bragg reflector. The specular reflectivity of one, two, and three-membrane mirrors is measured using FTIR. High specular reflectivity, above 99%, is achieved for three stacked membranes. Comparison of the measured reflectivity with theoretical calculations shows good agreement.

  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. Endometrial glands are essential for blastocyst implantation and decidualization in the mouse uterus.

    PubMed

    Filant, Justyna; Spencer, Thomas E

    2013-04-01

    Uterine glands and their secretions are hypothesized to be essential for blastocyst implantation and decidualization in the uterus of rodents and humans. One factor solely expressed by uterine glands in mice is leukemia inhibitory factor (LIF), and Lif null mice are infertile because of defective blastocyst attachment to the uterine luminal epithelium (LE). Progesterone treatment of neonatal mice permanently ablates differentiation of uterine glands, resulting in an aglandular uterus in the adult. Progesterone-induced uterine gland knockout (PUGKO) mice were used to investigate the biological role of uterine glands in blastocyst implantation and stromal cell decidualization. As compared to controls, PUGKO mice cycled normally but were infertile. Histological assessment of PUGKO uteri on Days 5.5 and 8.5 postmating found a hatched blastocyst apposed to an intact LE without evidence of implantation or stromal cell decidualization. Expression of several implantation-related factors, including Lif and PTGS2, were altered in the PUGKO uterus, whereas expression of steroid hormone receptors and their regulated genes was not different. Artificial decidualization was observed in the uteri of control but not PUGKO mice. Further, intrauterine administration of LIF failed to promote artificial decidualization in the uterus of PUGKO mice. Thus, uterine glands and their secretions have important biological roles in blastocyst implantation and stromal cell decidualization in the uterus.

  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. FTIR microspectroscopic imaging as a new tool to distinguish chemical composition of mouse blastocyst

    NASA Astrophysics Data System (ADS)

    Thumanu, Kanjana; Tanthanuch, Waraporn; Lorthongpanich, Chanchao; Heraud, Philip; Parnpai, Rangsun

    2009-09-01

    Synchrotron-Infrared (SR-IR) mapping and Focal plane array (FPA) imaging have been applied for discrimination of the three biochemical components of the mouse blastocyst. The mouse blastocyst consists of two clusters of cells known as the inner cell mass (ICM) formed within the blastocoel cavity and the thin layer of outer cells called the trophectoderm. Using Hierachical Cluster Analysis (HCA) and Principle Component Analysis (PCA), it can be shown that the composition and distribution of biochemical components within the blastocyst show differences in the protein secondary structure and the lipid content. It is worth noting that the secondary structure of the outer layer cells indicates more distinctive β-type secondary structure. The blastocoel cavity was observed to be predominantly α-helix. Significantly, the ICM region showed the predominant high absorption intensities of lipid content (CH 2, CH 3 symmetric and asymmetric stretching around 3000-2800 cm -1). The results show agreement between both SR-IR mapping and FPA-IR imaging. We propose that the biochemical difference within the blastocyst, especially the high lipid content in the ICM region, could be involved in the process of lipid signaling during pre-implantation. The use of both techniques is shown to be significant approach for revealing the biochemical components within the blastocyst.

  1. Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Ernst, S.; Verhalen, B.; Zarrabi, N.; Wilkens, S.; Börsch, M.

    2011-03-01

    In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.

  2. Group transfer theory of single molecule imaging experiments in the F-ATPase biomolecular motor

    NASA Astrophysics Data System (ADS)

    Volkan-Kacso, Sandor; Marcus, Rudolph

    I describe a chemo-mechanical theory to treat single molecule imaging and ``stalling'' experiments on the F-ATPase enzyme. This enzyme is an effective stepping biomolecular rotary motor with a rotor shaft and a stator ring. Using group transfer theoretical approach the proposed structure-based theory couples the binding transition of nucleotides in the stator subunits and the physics of torsional elasticity in the rotor. The twisting of the elastic rotor domain acts as a perturbation upon the driving potential, the Gibbs free energy. In the theory, without the use of adjustastable parameters, we predict the rate and equilibrium constant dependence of steps such as ATP binding and phosphate release as a function of manipulated rotor angle. Then we compare these predictions to available data from stalling experiments. Besides treating experiments, the theory can provide guides for atomistic simulations, which could calculate the reorganization parameter and the torsional spring constant. The framework is generic and I discuss its application to other single molecule experiments, such as controlled rotation and other biomolecular motors, including motor-DNA complexes and linear motors.[PNAS, Early Edition, Oct. 19, 2015, doi: 10.1073/pnas.1518489112

  3. Group transfer theory of single molecule imaging experiments in the F-ATPase biomolecular motor

    NASA Astrophysics Data System (ADS)

    Volkan-Kacso, Sandor; Marcus, Rudolph

    I describe a chemo-mechanical theory to treat single molecule imaging and ``stalling'' experiments on the F-ATPase enzyme. This enzyme is an effective stepping biomolecular rotary motor with a rotor shaft and a stator ring. Using group transfer theoretical approach the proposed structure-based theory couples the binding transition of nucleotides in the stator subunits and the physics of torsional elasticity in the rotor. The twisting of the elastic rotor domain acts as a perturbation upon the driving potential, the Gibbs free energy. In the theory, without the use of adjustastable parameters, we predict the rate and equilibrium constant dependence of steps such as ATP binding and phosphate release as a function of manipulated rotor angle. Then we compare these predictions to available data from stalling experiments. Besides treating experiments, the theory can provide guides for atomistic simulations, which could calculate the reorganization parameter and the torsional spring constant. The framework is generic and I discuss its application to other single molecule experiments, such as controlled rotation and other biomolecular motors, including motor-DNA complexes and linear motors.[PNAS, Early Edition, Oct. 19, 2015, doi: 10.1073/pnas.1518489112] The authors would like to acknowledge support from the Office of the Naval Research, the Army Research Office, and the James W. Glanville Foundation.

  4. Toward automated denoising of single molecular Förster resonance energy transfer data.

    PubMed

    Lee, Hao-Chih; Lin, Bo-Lin; Chang, Wei-Hau; Tu, I-Ping

    2012-01-01

    A wide-field two-channel fluorescence microscope is a powerful tool as it allows for the study of conformation dynamics of hundreds to thousands of immobilized single molecules by Förster resonance energy transfer (FRET) signals. To date, the data reduction from a movie to a final set containing meaningful single-molecule FRET (smFRET) traces involves human inspection and intervention at several critical steps, greatly hampering the efficiency at the post-imaging stage. To facilitate the data reduction from smFRET movies to smFRET traces and to address the noise-limited issues, we developed a statistical denoising system toward fully automated processing. This data reduction system has embedded several novel approaches. First, as to background subtraction, high-order singular value decomposition (HOSVD) method is employed to extract spatial and temporal features. Second, to register and map the two color channels, the spots representing bleeding through the donor channel to the acceptor channel are used. Finally, correlation analysis and likelihood ratio statistic for the change point detection (CPD) are developed to study the two channels simultaneously, resolve FRET states, and report the dwelling time of each state. The performance of our method has been checked using both simulation and real data.

  5. Clinical outcomes resulting from the transfer of vitrified human embryos using a new device for cryopreservation (plastic blade)

    PubMed Central

    Sugiyama, Rikikazu; Shirai, Asako; Sugiyama, Rie; Nishi, Yayoi; Kuribayashi, Yasushi; Inoue, Masato

    2010-01-01

    Purpose We attempted clinical application of a plastic blade, which is a novel cryopreservation device, for vitrification of human embryos and blastocysts. Methods Between February 2003 and December 2007, a total of 4,430 Day 3 embryos from 898 patients (Day 3 group) and 55 blastocysts from 29 patients (blastocyst group) were vitrified and cryopreserved with a plastic device, and subsequently thawed for embryo transfer. Clinical outcomes after thawing and transfer of vitrified embryos and blastocysts were evaluated. Results In the Day 3 group, all embryos resulting from 1,441 oocyte retrieval cycles were recovered, and the thawed embryo survival rate was 98.4%. In the blastocyst group, the survival rate after thawing was 100%. A total of 3,026 day 3 embryos and 46 blastocysts were transferred. The pregnancy and implantation rates in the Day 3 group were 25.0% and 15.5%, respectively, and in the blastocyst group the rates were 24.2% and 26.1%, respectively. The miscarriage rates in the Day 3 and blastocyst groups were 18.3% and 50.0%, respectively. Conclusions A plastic blade is a useful novel device in cryopreservation of vitrified human embryos. PMID:20127161

  6. Ultrafast single-electron transfer in coupled quantum dots driven by a few-cycle chirped pulse

    SciTech Connect

    Yang, Wen-Xing; Chen, Ai-Xi; Bai, Yanfeng; Lee, Ray-Kuang

    2014-04-14

    We theoretically study the ultrafast transfer of a single electron between the ground states of a coupled double quantum dot (QD) structure driven by a nonlinear chirped few-cycle laser pulse. A time-dependent Schrödinger equation without the rotating wave approximation is solved numerically. We demonstrate numerically the possibility to have a complete transfer of a single electron by choosing appropriate values of chirped rate parameters and the intensity of the pulse. Even in the presence of the spontaneous emission and dephasing processes of the QD system, high-efficiency coherent transfer of a single electron can be obtained in a wide range of the pulse parameters. Our results illustrate the potential to utilize few-cycle pulses for the excitation in coupled quantum dot systems through the nonlinear chirp parameter control, as well as a guidance in the design of experimental implementation.

  7. Conditional deletion of Msx homeobox genes in the uterus inhibits blastocyst implantation by altering uterine receptivity.

    PubMed

    Daikoku, Takiko; Cha, Jeeyeon; Sun, Xiaofei; Tranguch, Susanne; Xie, Huirong; Fujita, Tomoko; Hirota, Yasushi; Lydon, John; DeMayo, Francesco; Maxson, Robert; Dey, Sudhansu K

    2011-12-13

    An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs.

  8. Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations.

    PubMed

    Choi, Young-Ho; Ross, Pablo; Velez, Isabel C; Macías-García, B; Riera, Fernando L; Hinrichs, Katrin

    2015-07-01

    Equine embryos develop in vitro in the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31-46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively for POU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did. GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation in in vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

  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.

  10. Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation.

    PubMed Central

    Bhatt, H; Brunet, L J; Stewart, C L

    1991-01-01

    We have analyzed the expression of the cytokine leukemia inhibitory factor (LIF) during embryogenesis and in tissues of neonatal and adult mice. The site of the most abundant LIF expression is the uterine endometrial glands, specifically on day 4 of pregnancy. Analysis of LIF expression in pseudopregnant mice and in females undergoing delayed implantation showed that it is under maternal control and that its expression coincides with blastocyst formation and always precedes implantation. These results suggest that a principal function of LIF in vivo may be to regulate the growth and to initiate implantation of blastocysts. Images PMID:1722331

  11. Electron Transport, Energy Transfer, and Optical Response in Single Molecule Junctions

    NASA Astrophysics Data System (ADS)

    White, Alexander James

    The last decade has seen incredible growth in the quality of experiments being done on single molecule junctions. Contemporary experimental measurements have expanded far beyond simple electron transport. Measurement of vibronic eects, quantum interference and decoherence eects, molecular optical response (Raman spectroscopy), and molecular spintronics are just some of the continuing areas of research in single molecule junctions. Experimental advancements demand advanced theoretical treatments, which can be used accurately within appropriate physical regimes, in order to understand measured phenomena and predict interesting directions for future study. In this dissertation we will study systems with strong intra-system interactions using a many-body states based approach. We will be focused on three related processes in molecular junctions: electron transport, electronic energy transfer, and molecular excitation. Inelastic electron transport in the regime of strong and nonlinear electron-vibration coupling within and outside of the Born-Oppenheimer regime will be investigated. To understand their appropriateness, we will compare simple semi-classical approximations in molecular redox junctions and electron-counting devices to fully quantum calculations based on many-body system states. The role of coherence and quantum interference in energy and electron transfer in molecular junctions is explored. Experiments that simultaneously measure surface enhanced Raman scattering and electron conduction have revealed a strong interaction between conducting electrons and molecular excitation. We investigate the role of the molecular response to a classical surface plasmon enhanced electric eld considering the back action of the oscillating molecular dipole. Raman scattering is quantum mechanical by nature and involves strong interaction between surface plasmons in the contacts and the molecular excitation. We develop a scheme for treating strong plasmon-molecular excitation

  12. Repeated growth and bubbling transfer of graphene with millimetre-size single-crystal grains using platinum.

    PubMed

    Gao, Libo; Ren, Wencai; Xu, Huilong; Jin, Li; Wang, Zhenxing; Ma, Teng; Ma, Lai-Peng; Zhang, Zhiyong; Fu, Qiang; Peng, Lian-Mao; Bao, Xinhe; Cheng, Hui-Ming

    2012-02-28

    Large single-crystal graphene is highly desired and important for the applications of graphene in electronics, as grain boundaries between graphene grains markedly degrade its quality and properties. Here we report the growth of millimetre-sized hexagonal single-crystal graphene and graphene films joined from such grains on Pt by ambient-pressure chemical vapour deposition. We report a bubbling method to transfer these single graphene grains and graphene films to arbitrary substrate, which is nondestructive not only to graphene, but also to the Pt substrates. The Pt substrates can be repeatedly used for graphene growth. The graphene shows high crystal quality with the reported lowest wrinkle height of 0.8 nm and a carrier mobility of greater than 7,100 cm(2) V(-1) s(-1) under ambient conditions. The repeatable growth of graphene with large single-crystal grains on Pt and its nondestructive transfer may enable various applications.

  13. 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.

  14. 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

  15. Laser-induced forward transfer for flip-chip packaging of single dies.

    PubMed

    Kaur, Kamal S; Van Steenberge, Geert

    2015-01-01

    Flip-chip (FC) packaging is a key technology for realizing high performance, ultra-miniaturized and high-density circuits in the micro-electronics industry. In this technique the chip and/or the substrate is bumped and the two are bonded via these conductive bumps. Many bumping techniques have been developed and intensively investigated since the introduction of the FC technology in 1960(1) such as stencil printing, stud bumping, evaporation and electroless/electroplating2. Despite the progress that these methods have made they all suffer from one or more than one drawbacks that need to be addressed such as cost, complex processing steps, high processing temperatures, manufacturing time and most importantly the lack of flexibility. In this paper, we demonstrate a simple and cost-effective laser-based bump forming technique known as Laser-induced Forward Transfer (LIFT)3. Using the LIFT technique a wide range of bump materials can be printed in a single-step with great flexibility, high speed and accuracy at RT. In addition, LIFT enables the bumping and bonding down to chip-scale, which is critical for fabricating ultra-miniature circuitry. PMID:25867627

  16. Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets

    SciTech Connect

    Rahmanseresht, Sheema; Ramos, Kieran P.; Gamari, Ben D.; Goldner, Lori S.; Milas, Peker

    2015-05-11

    Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.

  17. Laser-induced forward transfer for flip-chip packaging of single dies.

    PubMed

    Kaur, Kamal S; Van Steenberge, Geert

    2015-03-20

    Flip-chip (FC) packaging is a key technology for realizing high performance, ultra-miniaturized and high-density circuits in the micro-electronics industry. In this technique the chip and/or the substrate is bumped and the two are bonded via these conductive bumps. Many bumping techniques have been developed and intensively investigated since the introduction of the FC technology in 1960(1) such as stencil printing, stud bumping, evaporation and electroless/electroplating2. Despite the progress that these methods have made they all suffer from one or more than one drawbacks that need to be addressed such as cost, complex processing steps, high processing temperatures, manufacturing time and most importantly the lack of flexibility. In this paper, we demonstrate a simple and cost-effective laser-based bump forming technique known as Laser-induced Forward Transfer (LIFT)3. Using the LIFT technique a wide range of bump materials can be printed in a single-step with great flexibility, high speed and accuracy at RT. In addition, LIFT enables the bumping and bonding down to chip-scale, which is critical for fabricating ultra-miniature circuitry.

  18. High-Content Analysis of Breast Cancer Using Single-Cell Deep Transfer Learning.

    PubMed

    Kandaswamy, Chetak; Silva, Luís M; Alexandre, Luís A; Santos, Jorge M

    2016-03-01

    High-content analysis has revolutionized cancer drug discovery by identifying substances that alter the phenotype of a cell, which prevents tumor growth and metastasis. The high-resolution biofluorescence images from assays allow precise quantitative measures enabling the distinction of small molecules of a host cell from a tumor. In this work, we are particularly interested in the application of deep neural networks (DNNs), a cutting-edge machine learning method, to the classification of compounds in chemical mechanisms of action (MOAs). Compound classification has been performed using image-based profiling methods sometimes combined with feature reduction methods such as principal component analysis or factor analysis. In this article, we map the input features of each cell to a particular MOA class without using any treatment-level profiles or feature reduction methods. To the best of our knowledge, this is the first application of DNN in this domain, leveraging single-cell information. Furthermore, we use deep transfer learning (DTL) to alleviate the intensive and computational demanding effort of searching the huge parameter's space of a DNN. Results show that using this approach, we obtain a 30% speedup and a 2% accuracy improvement. PMID:26746583

  19. High-Content Analysis of Breast Cancer Using Single-Cell Deep Transfer Learning.

    PubMed

    Kandaswamy, Chetak; Silva, Luís M; Alexandre, Luís A; Santos, Jorge M

    2016-03-01

    High-content analysis has revolutionized cancer drug discovery by identifying substances that alter the phenotype of a cell, which prevents tumor growth and metastasis. The high-resolution biofluorescence images from assays allow precise quantitative measures enabling the distinction of small molecules of a host cell from a tumor. In this work, we are particularly interested in the application of deep neural networks (DNNs), a cutting-edge machine learning method, to the classification of compounds in chemical mechanisms of action (MOAs). Compound classification has been performed using image-based profiling methods sometimes combined with feature reduction methods such as principal component analysis or factor analysis. In this article, we map the input features of each cell to a particular MOA class without using any treatment-level profiles or feature reduction methods. To the best of our knowledge, this is the first application of DNN in this domain, leveraging single-cell information. Furthermore, we use deep transfer learning (DTL) to alleviate the intensive and computational demanding effort of searching the huge parameter's space of a DNN. Results show that using this approach, we obtain a 30% speedup and a 2% accuracy improvement.

  20. Single-crystal organic charge-transfer interfaces probed using Schottky-gated heterostructures.

    PubMed

    Gutiérrez Lezama, Ignacio; Nakano, Masaki; Minder, Nikolas A; Chen, Zhihua; Di Girolamo, Flavia V; Facchetti, Antonio; Morpurgo, Alberto F

    2012-09-01

    Organic semiconductors based on small conjugated molecules generally behave as insulators when undoped, but the heterointerfaces of two such materials can show electrical conductivity as large as in a metal. Although charge transfer is commonly invoked to explain the phenomenon, the details of the process and the nature of the interfacial charge carriers remain largely unexplored. Here we use Schottky-gated heterostructures to probe the conducting layer at the interface between rubrene and PDIF-CN(2) single crystals. Gate-modulated conductivity measurements demonstrate that interfacial transport is due to electrons, whose mobility exhibits band-like behaviour from room temperature to ~150 K, and remains as high as ~1 cm(2) V(-1) s(-1) at 30 K for the best devices. The electron density decreases linearly with decreasing temperature, an observation that can be explained quantitatively on the basis of the heterostructure band diagram. These results elucidate the electronic structure of rubrene/PDIF-CN(2) interfaces and show the potential of Schottky-gated organic heterostructures for the investigation of transport in molecular semiconductors. PMID:22820687

  1. Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning.

    PubMed

    Hayes, O; Rodríguez, L L; González, A; Falcón, V; Aguilar, A; Castro, F O

    2005-11-01

    The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect its efficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and the protocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect of two protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesis is that freezing affects cell membranes in a way that interferes with the fusion process upon cloning but without hampering normal cell development in vitro. We found that freezing cell lines without controlling the cooling rate gives lower yields in the fusion step and in the final development into blastocysts, compared with cells frozen with a controlled cooling rate of approximately 1 degrees C/min. Transmission electron microscopy of the cells subjected to different freezing procedures showed major damage to the cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning is a critical step in the procedure and should be monitored carefully using a method that allows for a step-wise, controlled cooling rate.

  2. Enhanced Single Seed Trait Predictions in Soybean (Glycine max) and Robust Calibration Model Transfer with Near-Infrared Reflectance Spectroscopy.

    PubMed

    Hacisalihoglu, Gokhan; Gustin, Jeffery L; Louisma, Jean; Armstrong, Paul; Peter, Gary F; Walker, Alejandro R; Settles, A Mark

    2016-02-10

    Single seed near-infrared reflectance (NIR) spectroscopy predicts soybean (Glycine max) seed quality traits of moisture, oil, and protein. We tested the accuracy of transferring calibrations between different single seed NIR analyzers of the same design by collecting NIR spectra and analytical trait data for globally diverse soybean germplasm. X-ray microcomputed tomography (μCT) was used to collect seed density and shape traits to enhance the number of soybean traits that can be predicted from single seed NIR. Partial least-squares (PLS) regression gave accurate predictive models for oil, weight, volume, protein, and maximal cross-sectional area of the seed. PLS models for width, length, and density were not predictive. Although principal component analysis (PCA) of the NIR spectra showed that black seed coat color had significant signal, excluding black seeds from the calibrations did not impact model accuracies. Calibrations for oil and protein developed in this study as well as earlier calibrations for a separate NIR analyzer of the same design were used to test the ability to transfer PLS regressions between platforms. PLS models built from data collected on one NIR analyzer had minimal differences in accuracy when applied to spectra collected from a sister device. Model transfer was more robust when spectra were trimmed from 910 to 1679 nm to 955-1635 nm due to divergence of edge wavelengths between the two devices. The ability to transfer calibrations between similar single seed NIR spectrometers facilitates broader adoption of this high-throughput, nondestructive, seed phenotyping technology.

  3. Detection of single-nucleotide variations by monitoring the blinking of fluorescence induced by charge transfer in DNA.

    PubMed

    Kawai, Kiyohiko; Majima, Tetsuro; Maruyama, Atsushi

    2013-08-19

    Charge transfer dynamics in DNA: Photo-induced charge separation and charge-recombination dynamics in DNA was assessed by monitoring the blinking of fluorescence. Single nucleotide variations, mismatch and one base deletion, were differentiated based on the length of the off-time of the blinking, which corresponds to the lifetime of the charge-separated state. PMID:23846860

  4. Development of a Novel Bidimensional Spectroelectrochemistry Cell Using Transfer Single-Walled Carbon Nanotubes Films as Optically Transparent Electrodes.

    PubMed

    Garoz-Ruiz, Jesus; Heras, Aranzazu; Palmero, Susana; Colina, Alvaro

    2015-06-16

    A really easy method to transfer commercial single-walled carbon nanotubes (SWCNTs) to different substrates is proposed. In this paper, a homogeneous transference of SWCNTs films to nonconductor and transparent supports, such as polyethylene terephthalate, glass, and quartz, and to conductor supports, such as indium tin oxide, aluminum, highly ordered pyrolytic graphite, and glassy carbon, was achieved using a very fast, reproducible, and clean methodology. In order to test these transferences, SWCNTs films transferred on quartz were used as working optically UV-vis transparent electrodes due to their optimal electrical and optical properties. A new easy-to-use, homemade optical fiber based cell for bidimensional spectroelectrochemistry was developed, offering the possibility to measure in normal and parallel configuration. The cell was tested with ferrocenemethanol, a compound widely used in electrochemistry but scarcely studied by spectroelectrochemistry, covering the UV-vis spectral region. PMID:25989247

  5. 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.

  6. Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts

    PubMed Central

    HWANG, Jae Yeon; CHOI, Kwang-Hwan; LEE, Dong-Kyung; KIM, Seung-Hun; KIM, Eun Bae; HYUN, Sang-Hwan; LEE, Chang-Kyu

    2015-01-01

    X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts. PMID:26255835

  7. Morphological changes in the blastocyst of the western spotted skunk during activation from delayed implantation.

    PubMed

    Enders, A C; Schlafke, S; Hubbard, N E; Mead, R A

    1986-03-01

    Blastocysts collected from the spotted skunk during delay of implantation, early activation and late activation demonstrate three-tiered growth and developmental changes. The slow-growing blastocyst from the several months of delay is small (less than 1.1 mm) with a rounded inner cell mass consisting of clusters of rounded, lipid-filled cells. During the several days of early activation, the lipid in both inner cell mass and trophoblast diminishes, polyribosomes increase in number, and the endodermal layer differentiates as the blastocyst grows (1.2-1.6 mm). At activation the inner cell mass flattens, becomes uncovered by polar trophoblast, and forms a disc of columnar epiblast cells. The blastocyst expands rapidly during the last 24-48 h prior to implantation to 1.7-2.0 mm, and the trophoblast becomes cuboidal with a marked endocytotic apparatus. The morphological evidence, together with previous studies of protein and RNA synthesis, suggests a tooling-up period during early activation with progressive increases in rates of growth and differentiation in the last hours as implantation approaches.

  8. FGF4-DEPENDENT STEM CELLS DERIVED FROM RAT BLASTOCYSTS DIFFERENTIATE ALONG THE TROPHOBLAST LINEAGE

    PubMed Central

    Asanoma, Kazuo; Rumi, M.A. Karim; Kent, Lindsey N.; Chakraborty, Damayanti; Renaud, Stephen J.; Wake, Norio; Lee, Dong-Soo; Kubota, Kaiyu; Soares, Michael J.

    2011-01-01

    Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation. PMID:21215265

  9. 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

  10. 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

    2016-06-03

    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.

  11. 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

  12. Preliminary investigation of transfer between single-word decoding ability and contextual reading comprehension by poor readers in grade six.

    PubMed

    Blanchard, J S

    1980-12-01

    The study sought to investigate transfer between single-word decoding skill and contextual reading comprehension using 60 sixth grade pupils classified as 30 poor and 30 very poor readers. There were two training groups, content-specific and non-content-specific. One group received single-word decoding training, which led to decoding mastery of all words to be read in narrative prose passages and the accompanying literal and inferential comprehension questions. The other group learned to identify and pronounce words, equal in difficulty and number to those of the content-specific group but never a part of the passages and questions for the study. Pupils given content-specific training answered significantly more literal and inferential questions correctly than did those given non-content-specific training. It is suggested that there is transfer between single-word decoding skill and contextual, literal and inferential comprehension.

  13. 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.

  14. 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

  15. Molecular adsorption on ZnO(1010) single-crystal surfaces: morphology and charge transfer.

    PubMed

    Chen, Jixin; Ruther, Rose E; Tan, Yizheng; Bishop, Lee M; Hamers, Robert J

    2012-07-17

    While ZnO has excellent electrical properties, it has not been widely used for dye-sensitized solar cells, in part because ZnO is chemically less stable than widely used TiO(2). The functional groups typically used for surface passivation and for attaching dye molecules either bind weakly or etch the ZnO surface. We have compared the formation of molecular layers from alkane molecules with terminal carboxylic acid, alcohol, amine, phosphonic acid, or thiol functional groups on single-crystal zinc oxide (1010) surfaces. Atomic force microscopy (AFM) images show that alkyl carboxylic acids etch the surface whereas alkyl amine and alkyl alcohols bind only weakly on the ZnO(1010) surface. Phosphonic acid-terminated molecules were found to bind to the surface in a heterogeneous manner, forming clusters of molecules. Alkanethiols were found to bind to the surface, forming highly uniform monolayers with some etching detected after long immersion times in an alkanethiol solution. Monolayers of hexadecylphosphonic acid and octadecanethiol were further analyzed by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and electrochemical measurements. AFM scratching shows that thiols were bound strongly to the ZnO surface, suggesting the formation of strong Zn-S covalent bonds. Surprisingly, the tridentate phosphonic acids adhered much more weakly than the monodentate thiol. The influence of organic grafting on the charge transfer to ZnO was studied by time-resolved surface photovoltage measurements and electrochemical impedance measurements. Our results show that the grafting of thiols to ZnO leads to robust surfaces and reduces the surface band bending due to midgap surface states.

  16. Single embryo transfer and IVF/ICSI outcome: a balanced appraisal.

    PubMed

    Gerris, Jan M R

    2005-01-01

    This review considers the value of single embryo transfer (SET) to prevent multiple pregnancies (MP) after IVF/ICSI. The incidence of MP (twins and higher order pregnancies) after IVF/ICSI is much higher (approximately 30%) than after natural conception (approximately 1%). Approximately half of all the neonates are multiples. The obstetric, neonatal and long-term consequences for the health of these children are enormous and costs incurred extremely high. Judicious SET is the only method to decrease this epidemic of iatrogenic multiple gestations. Clinical trials have shown that programmes with >50% of SET maintain high overall ongoing pregnancy rates ( approximately 30% per started cycle) while reducing the MP rate to <10%. Experience with SET remains largely European although the need to reduce MP is accepted worldwide. An important issue is how to select patients suitable for SET and embryos with a high putative implantation potential. The typical patient suitable for SET is young (aged <36 years) and in her first or second IVF/ICSI trial. Embryo selection is performed using one or a combination of embryo characteristics. Available evidence suggests that, for the overall population, day 3 and day 5 selection yield similar results but better than zygote selection results. Prospective studies correlating embryo characteristics with documented implantation potential, utilizing databases of individual embryos, are needed. The application of SET should be supported by other measures: reimbursement of IVF/ICSI (earned back by reducing costs), optimized cryopreservation to augment cumulative pregnancy rates per oocyte harvest and a standardized format for reporting results. To make SET the standard of care in the appropriate target group, there is a need for more clinical studies, for intensive counselling of patients, and for an increased sense of responsibility in patients, health care providers and health insurers.

  17. Mass transfer resistance in a liquid-phase microextraction employing a single hollow fiber under unsteady-state conditions.

    PubMed

    Kumrić, Ksenija R; Vladisavljević, Goran T; Đorđević, Jelena S; Jönsson, Jan Åke; Trtić-Petrović, Tatjana M

    2012-09-01

    In this study, the mass transport resistance in liquid-phase microextraction (LPME) in a single hollow fiber was investigated. A mathematical model has been developed for the determination of the overall mass transfer coefficient based on the acceptor phase in an unsteady state. The overall mass transfer coefficient in LPME in a single hollow fiber has been estimated from time-dependent concentration of extracted analyte in the acceptor phase while maintaining a constant analyte concentration in the donor phase. It can be achieved either using a high volume of donor to acceptor phase ratio or tuning the extraction conditions to obtain a low-enrichment factor, so that the analyte concentration in the sample is not significantly influenced by the mass transfer. Two extraction systems have been used to test experimentally the developed model: the extraction of Lu(III) from a buffer solution and the extraction of three local anesthetics from a buffer or plasma solution. The mass transfer resistance, defined as a reciprocal values of the mass transfer coefficient, was found to be 1.2 × 10(3) cm(-1) min for Lu(III) under optimal conditions and from 1.96 to 3.3 × 10(3) cm(-1) min for the local anesthetics depending on the acceptor pH and the hydrophobicity of the drug.

  18. Facile electrochemical transfer of large-area single crystal epitaxial graphene from Ir(1 1 1)

    NASA Astrophysics Data System (ADS)

    Koefoed, Line; Kongsfelt, Mikkel; Ulstrup, Søren; Grubišić Čabo, Antonija; Cassidy, Andrew; Whelan, Patrick R.; Bianchi, Marco; Dendzik, Maciej; Pizzocchero, Filippo; Jørgensen, Bjarke; Bøggild, Peter; Hornekær, Liv; Hofmann, Philip; Pedersen, Steen U.; Daasbjerg, Kim

    2015-03-01

    High-quality growth of graphene and subsequent reliable transfer to insulating substrates are needed for various technological applications, such as flexible screens and high speed electronics. In this paper, we present a new electrochemical method for the transfer of large-area, high-quality single crystalline graphene from Ir(1 1 1) to Si/SiO2 under ambient conditions. The method is based on intercalation of tetraoctylammonium ions between the graphene layer and the Ir surface. This simple technique allows transfer of graphene single crystals having the same size as the substrate they are grown on (diameter ≈7 mm). In addition, the substrate can be reused for further growth cycles. A detailed Raman map analysis of the transferred graphene reveals straight lines, in which the Raman peaks characteristic for graphene are shifted. These lines originate from scratches in the Ir(1 1 1) crystal introduced by the polishing procedure. Furthermore, areas with numerous wrinkles exist inbetween these lines, forming a network across the entire graphene crystal. Hence, the initial characteristics and imprints left on the sheet of graphene in terms of strain and wrinkles from the growth process remain after transfer.

  19. Single-Turnover Kinetics of Methyl Transfer to tRNA by Methyltransferases

    PubMed Central

    Hou, Ya-Ming

    2016-01-01

    Summary Methyl transfer from S-adenosyl methionine (abbreviated as AdoMet) to biologically active molecules such as mRNAs and tRNAs is one of the most fundamental and widespread reactions in nature, occurring in all three domains of life. The measurement of kinetic constants of AdoMet-dependent methyl transfer is therefore important for understanding the reaction mechanism in the context of biology. When kinetic constants of methyl transfer are measured in steady state over multiple rounds of turnover, the meaning of these constants is difficult to define and is often limited by non-chemical steps of the reaction, such as product release after each turnover. Here the measurement of kinetic constants of methyl transfer by tRNA methyltransferases in rapid equilibrium binding condition for one methyl transfer is described. The advantage of such a measurement is that the meaning of kinetic constants can be directly assigned to the steps associated with the chemistry of methyl transfer, including the substrate binding affinity to the methyl transferase, the pre-chemistry re-arrangement of the active site, and the chemical step of methyl transfer. An additional advantage is that kinetic constants measured for one methyl transfer can be correlated with structural information of the methyl transferase to gain direct insight into its reaction mechanism. PMID:26965259

  20. Effects of prenatal exposure to extremely low electro-magnetic field on in vivo derived blastocysts of mice

    PubMed Central

    Bayat, Parvin-Dokht; Darabi, Mohmmad Reza; Ghanbari, Ali; Amiri, Sara; Sohouli, Pardis

    2012-01-01

    Background: Indisputable population exposure to widespread electromagnetic fields, has grown concerns over the probable health effects of these fields. Objective: The present study was aimed to examine the possible effects of 50 Hz extremely low frequency electromagnetic field (ELF-EMF) exposure on the number and quality of mice blastocysts. Materials and Methods: In present study, 66 NMRI pregnant females divided into two treated and non-treated groups. The treated group exposed to ELF-EMF (50 Hz and 6×10-3 T). Subsequently, embryos were collected by flushing the uterine horn and Fallopian tubes on the day 3 of gestation. Number of trophoectoderm (TE) and Inner Cell Mass (ICM) cells in blastocysts were determined after differential nuclei staining using a modified method. Furthermore, number of all flushed blastocysts calculated in each group. Results: There was no significant difference in mean number of blastocysts in treated (6.64±1.34) and none treated (8.22±1.59) groups. In treated group, there were significant decreased in total cell number of blastocysts (p=0.000), number of ICM cells (p=0.000), and number of TE cells (p=0.001) whereas the ratio of ICM/TE cells increased (p=0.002). Conclusion: The data indicate that ELF-EMF is able to affect cellular composition of blastocysts, but it can't omit total volume of blastocysts. PMID:25246926

  1. Frequency-domain time-resolved four wave mixing spectroscopy of vibrational coherence transfer with single-color excitation.

    PubMed

    Pakoulev, Andrei V; Rickard, Mark A; Mathew, Nathan A; Kornau, Kathryn M; Wright, John C

    2008-07-17

    Triply vibrationally enhanced four-wave mixing spectroscopy is employed to observe vibrational coherence transfer between the asymmetric and symmetric CO-stretching modes of rhodium(I) dicarbonyl acetylacetonate (RDC). Coherence transfer is a nonradiative transition of a coherent superposition of quantum states to a different coherent superposition due to coupling of the vibrational modes through the bath. All three excitation pulses in the experiment are resonant with a single quantum coherence, but coherence transfer results in new coherences with different frequencies. The new output frequency is observed with a monochromator that resolves it from the stronger peak at the original excitation frequency. This technique spectrally resolves pathways that include coherence transfer, discriminates against spectral features created solely by radiative transitions, and temporally resolves modulations created by interference between different coherence transfer pathways. Redfield theory simulates the temporal modulations in the impulsive limit, but it is also clear that coherence transfer violates the secular approximation invoked in most Redfield theories. Instead, it requires non-Markovian and bath memory effects. RDC may provide a simple model for the development of theories that incorporate these effects.

  2. Refuting a misguided campaign against the goal of single-embryo transfer and singleton birth in assisted reproduction.

    PubMed

    Stillman, Robert J; Richter, Kevin S; Jones, Howard W

    2013-10-01

    Much recent progress has been made by assisted reproductive technology (ART) professionals toward minimizing the incidence of multiple pregnancy following ART treatment. While a healthy singleton birth is widely considered to be the ideal outcome of such treatment, a vocal minority continues a campaign to advocate the benefits of multiple embryo transfer as treatment and twin pregnancy as outcome for most ART patients. Proponents of twinning argue four points: that patients prefer twins, that multiple embryo transfer maximizes success rates, that the costs per infant are lower with twins and that one twin pregnancy and birth is associated with no higher risk than two consecutive singleton pregnancies and births. We find fault with the reasoning and data behind each of these tenets. First, we respect the principle of patient autonomy to choose the number of embryos for transfer but counter that it has been shown that better patient education reduces their desire for twins. In addition, reasonable and evidentially supported limits may be placed on autonomy in exchange for public or private insurance coverage for ART treatment, and counterbalancing ethical principles to autonomy exist, especially beneficence (doing good) and non-maleficence (doing no harm). Second, comparisons between success rates following single-embryo transfer (SET) and double-embryo transfers favor double-embryo transfers only when embryo utilization is not comparable; cumulative pregnancy and birth rates that take into account utilization of cryopreserved embryos (and the additional cryopreserved embryo available with single fresh embryo transfer) consistently demonstrate no advantage to double-embryo transfer. Third, while comparisons of costs are system dependent and not easy to assess, several independent studies all suggest that short-term costs per child (through the neonatal period alone) are lower with transfers of one rather than two embryos. And, finally, abundant evidence conclusively

  3. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts.

    PubMed

    Wen, Duancheng; Saiz, Nestor; Rosenwaks, Zev; Hadjantonakis, Anna-Katerina; Rafii, Shahin

    2014-01-01

    Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  4. Plasmon-induced spatial electron transfer between single Au nanorods and ALD-coated TiO2: dependence on TiO2 thickness.

    PubMed

    Zheng, Zhaoke; Tachikawa, Takashi; Majima, Tetsuro

    2015-10-01

    We employed single-particle photoluminescence (PL) measurements to investigate the interfacial electron transfer between single Au nanorods (NRs) and TiO2 coated by ALD. Analyzing the energy relaxation path of plasmon-generated hot electrons as well as the PL intensities allowed for the detection and study of the interfacial electron transfer process spatially.

  5. Friction and metal transfer for single-crystal silicon carbide in contact with various metals in vacuum

    NASA Technical Reports Server (NTRS)

    Miyoshi, K.; Buckley, D. H.

    1978-01-01

    Sliding friction experiments were conducted with single-crystal silicon carbide in contact with transition metals (tungsten, iron, rhodium, nickel, titanium, and cobalt), copper, and aluminum. Results indicate the coefficient of friction for a silicon carbide-metal system is related to the d bond character and relative chemical activity of the metal. The more active the metal, the higher the coefficient of friction. All the metals examined transferred to the surface of silicon carbide in sliding. The chemical activity of metal to silicon and carbon and shear modulus of the metal may play important roles in metal transfer and the form of the wear debris. The less active and greater resistance to shear the metal has, with the exception of rhodium and tungsten, the less transfer to silicon carbide.

  6. Ideal dipole approximation fails to predict electronic coupling and energy transfer between semiconducting single-wall carbon nanotubes.

    PubMed

    Wong, Cathy Y; Curutchet, Carles; Tretiak, Sergei; Scholes, Gregory D

    2009-02-28

    The electronic coupling values and approximate energy transfer rates between semiconductor single-wall carbon nanotubes are calculated using two different approximations, the point dipole approximation and the distributed transition monopole approximation, and the results are compared. It is shown that the point dipole approximation fails dramatically at tube separations typically found in nanotube bundles ( approximately 12-16 A) and that the disagreement persists at large tube separations (>100 A, over ten nanotube diameters). When used in Forster resonance energy transfer theory, the coupling between two point transition dipoles is found to overestimate energy transfer rates. It is concluded that the point dipole approximation is inappropriate for use with elongated systems such as carbon nanotubes and that methods which can account for the shape of the particle are more suitable. PMID:19256589

  7. Transfer-printing-based integration of single-mode waveguide-coupled III-V-on-silicon broadband light emitters.

    PubMed

    De Groote, Andreas; Cardile, Paolo; Subramanian, Ananth Z; Fecioru, Alin M; Bower, Christopher; Delbeke, Danae; Baets, Roel; Roelkens, Günther

    2016-06-27

    We present the first III-V opto-electronic components transfer printed on and coupled to a silicon photonic integrated circuit. Thin InP-based membranes are transferred to an SOI waveguide circuit, after which a single-spatial-mode broadband light source is fabricated. The process flow to create transfer print-ready coupons is discussed. Aqueous FeCl3 at 5°C was found to be the best release agent in combination with the photoresist anchoring structures that were used. A thin DVS-BCB layer provides a strong bond, accommodating the post-processing of the membranes. The resulting optically pumped LED has a 3 dB bandwidth of 130 nm, comparable to devices realized using a traditional die-to-wafer bonding method. PMID:27410539

  8. Single-pair fluorescence resonance energy transfer of nucleosomes in free diffusion: optimizing stability and resolution of subpopulations.

    PubMed

    Gansen, Alexander; Hauger, Florian; Tóth, Katalin; Langowski, Jörg

    2007-09-15

    We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosomes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nucleosomes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning.

  9. Embryo transfer in water buffalo (Bubalus bubalis).

    PubMed

    Drost, M; Wright, J M; Cripe, W S; Richter, A R

    1983-11-01

    A normal, live 35-kg water buffalo bull calf was born 300 days after it was nonsurgically collected as a 7-day blastocyst from a water buffalo donor and transferred nonsurgically to an unrelated water buffalo recipient. The development of estrus synchronization, superovulation and estrus detection methods in water buffalo are described.

  10. Telomere elongation during morula-to-blastocyst transition in cloned porcine embryos.

    PubMed

    Dang-Nguyen, Thanh Quang; Haraguchi, Seiki; Akagi, Satoshi; Somfai, Tamas; Kaneda, Masahiro; Watanabe, Shinya; Kikuchi, Kazuhiro; Tajima, Atsushi; Nagai, Takashi

    2012-12-01

    Although telomeres are elongated during morula-to-blastocyst transition in cloned embryos, it is still unknown whether donor cell types have any effect on this elongation. In the present study, we examined the changes of telomere length during morula-to-blastocyst transition in cloned porcine embryos using different types of donor cells. Porcine embryonic stem-like cells (pESLCs), porcine cumulus cells (PCs), and porcine embryonic fibroblasts at passages 7 and 10 (PEF7s and PEF10s, respectively) were used as donor cells. Telomere lengths of pESLCs (35.8±1.5 kb), PCs (24.4±0.5 kb), PEF7s (18.7±0.6 kb), and PEF10s (17.2±0.1 kb) were significantly different. In contrast, telomere length in morulae derived from pESLCs (18.2±0.3 kb), PC (17.8±0.7 kb), PEF7 (18.5±0.3 kb), and PEF10 (18.4±0.4 kb) did not differ significantly. Likewise, telomeres in blastocysts derived from pESLCs (22.3±1.5 kb), PCs (23.5±2.6 kb), PEF7s (20.2±1.0 kb), and PEF10s (20.9±1.0 kb) had similar lengths. However, telomeres in blastocysts were significant longer (p<0.05) compared with morulae in each group. Relative telomerase activities of morulae derived from pESLCs (4.2±0.4), PCs (4.0±0.5), PEF7s (5.1±0.4), and PEF10s (4.9±0.4) were significantly lower (p<0.01) than those of blastocysts derived from pESLCs (8.2±1.1), PCs (8.6±0.6), PEF7s (12.5±2.9), and PEF10s (8.3±1.1). In conclusion, the telomere elongation in cloned pig embryos that occurred during morula-to-blastocyst transition may be related to the rise of telomerase activity. The telomere elongation may also be independent of the type and telomere length of the donor cell.

  11. Monitoring single coffee bean roasting by direct volatile compound analysis with proton transfer reaction time-of-flight mass spectrometry.

    PubMed

    Yener, Sine; Navarini, Luciano; Lonzarich, Valentina; Cappellin, Luca; Märk, Tilmann D; Bonn, Günther K; Biasioli, Franco

    2016-09-01

    This study applies proton transfer reaction time-of-flight mass spectrometry for the rapid analysis of volatile compounds released from single coffee beans. The headspace volatile profiles of single coffee beans (Coffeea arabica) from different geographical origins (Brazil, Guatemala and Ethiopia) were analyzed via offline profiling at different stages of roasting. The effect of coffee geographical origin was reflected on volatile compound formation that was supported by one-way ANOVA. Clear origin signatures were observed in the formation of different coffee odorants. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27476633

  12. Mothers, Workers and Students: Examining the Experiences of Single Mothers Transferring from Community Colleges into Universities

    ERIC Educational Resources Information Center

    Robinson, Emily Erin Peterson

    2010-01-01

    Single parent households are on the rise, and female headed households are more likely to live in poverty than other single parent households (Holyfield, 2002). Many single mothers who do not have an undergraduate degree see education as a way out of poverty (Holyfield, 2002; Heller & Bjorklund, 2004). This research was undertaken to highlight…

  13. Electronic Coupling Dependence of Ultrafast Interfacial Electron Transfer on Nanocrystalline Thin Films and Single Crystal

    SciTech Connect

    Lian, Tianquan

    2014-04-22

    The long-term goal of the proposed research is to understand electron transfer dynamics in nanoparticle/liquid interface. This knowledge is essential to many semiconductor nanoparticle based devices, including photocatalytic waste degradation and dye sensitized solar cells.

  14. Probing Ion Transfer across Liquid-Liquid Interfaces by Monitoring Collisions of Single Femtoliter Oil Droplets on Ultramicroelectrodes.

    PubMed

    Deng, Haiqiang; Dick, Jeffrey E; Kummer, Sina; Kragl, Udo; Strauss, Steven H; Bard, Allen J

    2016-08-01

    We describe a method of observing collisions of single femtoliter (fL) oil (i.e., toluene) droplets that are dispersed in water on an ultramicroelectrode (UME) to probe the ion transfer across the oil/water interface. The oil-in-water emulsion was stabilized by an ionic liquid, in which the oil droplet trapped a highly hydrophobic redox probe, rubrene. The ionic liquid also functions as the supporting electrolyte in toluene. When the potential of the UME was biased such that rubrene oxidation would be possible when a droplet collided with the electrode, no current spikes were observed. This implies that the rubrene radical cation is not hydrophilic enough to transfer into the aqueous phase. We show that current spikes are observed when tetrabutylammonium trifluoromethanesulfonate or tetrahexylammonium hexafluorophosphate are introduced into the toluene phase and when tetrabutylammonium perchlorate is introduced into the water phase, implying that the ion transfer facilitates electron transfer in the droplet collisions. The current (i)-time (t) behavior was evaluated quantitatively, which indicated the ion transfer is fast and reversible. Furthermore, the size of these emulsion droplets can also be calculated from the electrochemical collision. We further investigated the potential dependence on the electrochemical collision response in the presence of tetrabutylammonium trifluoromethanesulfonate in toluene to obtain the formal ion transfer potential of tetrabutylammonium across the toluene/water interface, which was determined to be 0.754 V in the inner potential scale. The results yield new physical insights into the charge balance mechanism in emulsion droplet collisions and indicate that the electrochemical collision technique can be used to probe formal ion transfer potentials between water and solvents with very low (ε < 5) dielectric constants. PMID:27387789

  15. Tutorial on Quantification of Differences between Single- and Two-Component Two-Phase Flow and Heat Transfer

    NASA Astrophysics Data System (ADS)

    Delil, A. A. M.

    2003-01-01

    Single-component two-phase systems are envisaged for aerospace thermal control applications: Mechanically Pumped Loops, Vapour Pressure Driven Loops, Capillary Pumped Loops and Loop Heat Pipes. Thermal control applications are foreseen in different gravity environments: Micro-g, reduced-g for Mars or Moon bases, 1-g during terrestrial testing, and hyper-g in rotating spacecraft, during combat aircraft manoeuvres and in systems for outer planets. In the evaporator, adiabatic line and condenser sections of such single-component two-phase systems, the fluid is a mixture of the working liquid (for example ammonia, carbon dioxide, ethanol, or other refrigerants, etc.) and its saturated vapour. Results of two-phase two-component flow and heat transfer research (pertaining to liquid-gas mixtures, e.g. water/air, or argon or helium) are often applied to support research on flow and heat transfer in two-phase single-component systems. The first part of the tutorial updates the contents of two earlier tutorials, discussing various aerospace-related two-phase flow and heat transfer research. It deals with the different pressure gradient constituents of the total pressure gradient, with flow regime mapping (including evaporating and condensing flow trajectories in the flow pattern maps), with adiabatic flow and flashing, and with thermal-gravitational scaling issues. The remaining part of the tutorial qualitatively and quantitatively determines the differences between single- and two-component systems: Two systems that physically look similar and close, but in essence are fully different. It was already elucidated earlier that, though there is a certain degree of commonality, the differences will be anything but negligible, in many cases. These differences (quantified by some examples) illustrates how careful one shall be in interpreting data resulting from two-phase two-component simulations or experiments, for the development of single-component two-phase thermal control

  16. 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.

  17. [Production of porcine blastocysts expressed EGFP by handmade cloning].

    PubMed

    Zhang, Peng; Yang, Zhen-Zhen; Dou, Hong-Wei; Li, Wei-Hang; Lv, Bo; Bolund, Lars; DU, Yu-Tao; Tan, Ping-Ping; Ma, Run-Lin

    2011-05-01

    Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production. PMID:21586400

  18. Innate immunity in an in vitro murine blastocyst model using embryonic and trophoblast stem cells.

    PubMed

    Aikawa, Hiroaki; Tamai, Miho; Mitamura, Keisuke; Itmainati, Fakhria; Barber, Glen N; Tagawa, Yoh-ichi

    2014-03-01

    The immune system has two broad components-innate and adaptive immunity. Adaptive immunity becomes established only after the onset of hematopoiesis, whereas the innate immune system may be actively protecting organisms from microbial invasion much earlier in development. Here, we address the question of whether the innate immune system functions in the early-stage embryo, i.e., the blastocyst. The innate immune system was studied by using in vitro blastocyst models, e.g., embryonic stem (ES) and trophoblast stem (TS) cell cultures. The expression of Toll-like receptors (TLR)-2, -3, and -5 could be detected in both ES and TS cells. The expression of interferon (IFN)-β was induced by the addition of polyinosinic:polycytidylic acid [poly(I:C)] in TS cells, but not ES cells, although TLR-3 was expressed at the same level in both cell types. In turn, ES cells responded to IFN-β exposure by expressing IFN-induced anti-viral genes, e.g., RNA-dependent protein kinase and 2', 5'-oligoadenylate synthetase (OAS). Neither a reduction in ES cell proliferation nor cell death in these cultures was observed after IFN-β stimulation. Furthermore, OAS1a expression was induced in ES/TS co-cultures after poly(I:C) stimulation, but was not induced when either cell type was cultured alone. In conclusion, TS cells react to poly(I:C) stimulation by producing IFN-β, which induces IFN-inducible genes in ES cells. This observation suggests that the trophectoderm, the outer layer of the blastocyst, may respond to viral infection, and then induce anti-viral gene expression via IFN-β signaling to the blastocyst inner cell mass.

  19. 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

  20. Resveratrol protects against 2-bromopropane-induced apoptosis and disruption of embryonic development in blastocysts.

    PubMed

    Chan, Wen-Hsiung

    2011-01-01

    2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. In the present work, we show that 2-BP induces apoptosis in the inner cell mass of mouse blastocysts, and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties. 2-BP-treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro, and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented 2-BP-induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that 2-BP directly promotes ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-3, whereas resveratrol effectively blocks 2-BP-induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that 2-BP triggers the mitochondrion-dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents 2-BP-induced toxicity. PMID:21954340

  1. Resveratrol Protects against 2-Bromopropane-Induced Apoptosis and Disruption of Embryonic Development in Blastocysts

    PubMed Central

    Chan, Wen-Hsiung

    2011-01-01

    2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. In the present work, we show that 2-BP induces apoptosis in the inner cell mass of mouse blastocysts, and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties. 2-BP-treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro, and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented 2-BP-induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that 2-BP directly promotes ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-3, whereas resveratrol effectively blocks 2-BP-induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that 2-BP triggers the mitochondrion-dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents 2-BP-induced toxicity. PMID:21954340

  2. Quantum coherent energy transfer over varying pathways in single light-harvesting complexes.

    PubMed

    Hildner, Richard; Brinks, Daan; Nieder, Jana B; Cogdell, Richard J; van Hulst, Niek F

    2013-06-21

    The initial steps of photosynthesis comprise the absorption of sunlight by pigment-protein antenna complexes followed by rapid and highly efficient funneling of excitation energy to a reaction center. In these transport processes, signatures of unexpectedly long-lived coherences have emerged in two-dimensional ensemble spectra of various light-harvesting complexes. Here, we demonstrate ultrafast quantum coherent energy transfer within individual antenna complexes of a purple bacterium under physiological conditions. We find that quantum coherences between electronically coupled energy eigenstates persist at least 400 femtoseconds and that distinct energy-transfer pathways that change with time can be identified in each complex. Our data suggest that long-lived quantum coherence renders energy transfer in photosynthetic systems robust in the presence of disorder, which is a prerequisite for efficient light harvesting.

  3. Occurrence of amitotic division of trophoblast cell nuclei in blastocysts of the western spotted skunk (Spilogale putorius latifrons).

    PubMed

    Isakova, Galina K; Mead, Rodney A

    2004-01-01

    A cytogenetic examination of spreaded cells of diapausing and early activated blastocysts obtained from 7 female western spotted skunks was performed. Mitosis was not observed in 1626 cells obtained from 9 diapausing blastocysts; however, 12 (1.5%) figures of diploid mitosis were seen in 851 cells from 5 early activated embryos. Diameter of the cell nuclei varied from 4 to 29 microm during diapause, and from 5 to 40 microm in activated blastocyst, and the heterogeneity in nuclear size was significantly different between diapausing and activated embryos (P<0.01). About 80% of nuclei from diapausing blastocysts measured 9 to 16 microm, whereas a similar percentage of nuclei from activated blastocysts ranged from 15 to 27 microm. Many enlarged nuclei exhibited morphological features characteristic of mammalian polytene (i.e. endopolyploid with polytenic organization of chromosomes) trophoblast cells. The number of silver stained nucleoli in all the nuclei did not exceed 2, which corresponds to the number of nucleolus organizers in the diploid karyotype in this species of skunk and suggests the polytene organization of chromosomes in enlarged nuclei. About 10% of large interphase nuclei were observed to undergo amitosis, i.e. direct division by constriction. The resulting nuclear fragments in diapausing blastocysts usually had normal morphology and active nucleoli. In activated embryos, nearly 15% of amitotically divided nuclei appeared to be dividing into fragments of unequal size, one of which had normal cell nuclear morphology and extremely large silver positive nucleoli, and the other fragment exhibited signs of cell death. We interpret these data as indicating that 1) amitotic division of trophoblast endopolyploid cell nuclei in the skunk blastocysts may generate new trophoblast cells which contribute to increased cell number during both diapause and activation stages, and 2) activation of blastocysts after diapause is related to the production of trophoblast

  4. [How can we nowadays select the best embryo to transfer?].

    PubMed

    Alter, L; Boitrelle, F; Sifer, C

    2014-01-01

    Multiple pregnancies stand as the most common adverse outcome of assisted reproduction technologies (ART) and the dangers associated with those pregnancies have been reduced by doing elective single embryo transfers (e-SET). Many studies have shown that e-SET is compatible with a continuously high pregnancy rate per embryo transfer. Yet, it still becomes necessary to improve the selection process in order to define the quality of individual embryos - so that the ones we choose for transfer are more likely to implant. First, analysis of embryo morphology has greatly helped in this identification and remains the most relevant criterion for choosing the embryo. The introduction of time-lapse imaging provides new criteria predictive of implantation potential, but the real contribution of this system - including the benefit/cost ratio - seems to be not yet properly established. In this context, extended culture until blastocyst stage is an essential practice but it appears wise to keep it for a population showing a good prognosis. Then, the failure of aneuploid embryos to implant properly led to achieve preimplantation genetic screening (PGS) in order to increase pregnancy and delivery rates after ART. However, PGS by fluorescence in situ hybridization (FISH) at day 3 is a useless process - and may even be harmful. Another solution involves using comparative genomic hybridisation (CGH) and moving to blastocyst biopsy. Finally, it is envisaged that morphology will also be significantly aided by non-invasive analysis of biomarkers in the culture media that give a better reflection of whole-embryo physiology and function. PMID:24951187

  5. The effect of the site of laser zona opening on the complete hatching of mouse blastocysts and their cell numbers

    PubMed Central

    Sanmee, Usanee; Piromlertamorn, Waraporn

    2016-01-01

    Objective We studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts. Methods Mouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125). Results The rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group (69±19.3, 74±15.7, and 71±16.8, respectively; p=0.680). Conclusion These findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers. PMID:27689037

  6. Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts.

    PubMed

    Zhao, Xue-Ming; Du, Wei-Hua; Hao, Hai-Sheng; Wang, Dong; Qin, Tong; Liu, Yan; Zhu, Hua-Bin

    2012-07-01

    The present study was designed to determine the effects of vitrification on promoter methylation and the expression levels of pluripotency and differentiation genes in mouse blastocysts. Promoter region CpG methylation patterns and the expression levels of octamer-binding transcription factor (Oct4), Nanog homeobox (Nanog), caudal-type homeobox 2 (Cdx2), and heart and neural crest derivatives-expressed transcript 1 (Hand1) were analyzed in fresh and vitrified mouse blastocysts. Methylation was measured by bisulphate mutagenesis and sequencing; gene expression was determined by real-time reverse transcription-PCR. The results showed that vitrification significantly reduced the methylation levels of the Oct4 (85% vs. 62.5%), Nanog (77.5% vs. 55%), and Cdx2 promoters (4.6% vs. 1.4%; P < 0.05) in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog in vitrified blastocysts. Hand1 promoter methylation was not significantly different in the fresh (17.9%) versus vitrification group (21.4%; P > 0.05). The expression levels of Cdx2 and Hand1 were not significantly different in fresh and vitrified blastocysts. In conclusion, vitrification significantly decreased Oct4, Nanog, and Cdx2 promoter methylation in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog.

  7. The effect of the site of laser zona opening on the complete hatching of mouse blastocysts and their cell numbers

    PubMed Central

    Sanmee, Usanee; Piromlertamorn, Waraporn

    2016-01-01

    Objective We studied the effect of the site of laser zona opening on the complete hatching of mouse blastocysts and the cell numbers of the completely hatched blastocysts. Methods Mouse blastocysts were randomly allocated to the inner cell mass (ICM) group (zona opening performed at the site of the ICM, n=125), the trophectoderm (TE) group (zona opening performed opposite to the ICM, n=125) and the control group (no zona opening, n=125). Results The rate of complete hatching of the blastocysts was not significantly different in the ICM and the TE group (84.8% vs 80.8%, respectively; p=0.402), but was significantly lower in the control group (51.2%, p<0.001). The cell numbers in the completely hatched blastocysts were comparable in the control group, the ICM group, and the TE group (69±19.3, 74±15.7, and 71±16.8, respectively; p=0.680). Conclusion These findings indicate that the site of laser zona opening did not influence the rate of complete hatching of mouse blastocysts or their cell numbers.

  8. Reduction of high order multiples in frozen embryo transfers.

    PubMed

    Anderson, Anthony R; Wilkinson, Shan S; Price, Sandy; Crain, Jack L

    2005-03-01

    The aim of this study was to determine if blastocyst frozen embryo transfers are able to reduce the potential for the rate of high order multiples (HOM) (>2 fetal sacs) without affecting the overall pregnancy and implantation potential. Group A included all frozen blastocyst transfers prior to 1 January 2002. Group B included all frozen blastocyst transfers between 1 January 2002 and 12 December 2003. There was no significant difference for survival for the two time periods (79 versus 80%). A significantly (P<0.05) lower number of embryos were transferred in group B (1.9) compared with group A (2.8). There was a significant (P<0.05) reduction of (HOM) from 31 to 3% for groups A and B respectively. Ongoing pregnancy rates for group A resulted in 52% of 25 embryo transfers and 41% of 75 embryo transfers in group B (not significant). Reducing the number of embryos transferred between groups A and B did not significantly impact implantation rates. It is concluded that blastocyst freezing is effective for overall survival, pregnancy and implantation while reducing the rate of high order multiples.

  9. Experimental study of single-phase pressure drop and heat transfer in a micro-fin tube

    SciTech Connect

    Li, Xiao-Wei; Meng, Ji-An; Li, Zhi-Xin

    2007-11-15

    The single-phase pressure drop and heat transfer in a micro-fin tube were measured using oil and water as the working fluids. The Prandtl number varied from 3.2 to 220 and the Reynolds number ranged from 2500 to 90,000. The results show that there is a critical Reynolds number, Re{sub cr}, for heat transfer enhancement. For Retransfer in the micro-fin tube is the same as that in a smooth tube, but for Reynolds numbers higher than Re{sub cr}, the heat transfer in the micro-fin tube is gradually enhanced compared with a smooth tube. It reaches more than twice that in a smooth tube for Reynolds numbers greater than 30,000 with water as the working fluid. The Nusselt number is proportional to Pr {sup 0.56} in the enhanced region and is proportional to Pr {sup 0.3} in the non-enhanced region. For the high Prandtl number working fluid (oil, 80< Pr <220), the critical Reynolds number for heat transfer enhancement is about 6000, while for the low Prandtl number working fluid (water, 3.2< Pr <5.8), the critical Reynolds number for heat transfer enhancement is about 10,000. The friction factors in the micro-fin tube are almost the same as for a smooth tube for Reynolds numbers below 10,000. For Reynolds numbers higher than 30,000, the friction factor is about 40-50% higher than for a smooth tube. (author)

  10. (Analysis of proteins essential for Agrobacterium mediated DNA transfer to plant cells). [Single-stranded DNA binding proteins

    SciTech Connect

    Not Available

    1989-12-14

    The tumor inducing (Ti) plasmid of Agrobacterium contains two regions important for infection and transformation of plant cells. One region, the T-DNA, is transferred as a single strand into the plant cell, while the virulence (vir) region is responsible for recognition of susceptible cells, synthesis of the T-DNA single strand (T-strand), formation of a T-strand protein complex and transfer of this complex into susceptible cells. A DNA binding protein, VirE2, was identified as a product of the vir region. Sequencing of the 9000 kilobase pair virB region has been completed. Expression of 10 of the predicted 11 open reading frames (ORFs) was demonstrated in Escherichia coli. Translational coupling was demonstrated for 5 ORFs. Hydropathy analysis indicates that 9 of 11 ORFs have hydrophobic regions that could permit membrane channel formation. In related work, analysis of protons that potentiate movement of plant viruses was discussed, with indications that the tobacco mosaic virus (TMV) protein P30 may mediate transfer of TMV RNA through plasmadesmata. Also, using the T-DNA element as a marker, genes responsible for abnormal flower development are being cloned and isolated. 3 refs. (MHB)

  11. Pressure drop and heat transfer characteristics for single-phase developing flow of water in rectangular microchannels

    NASA Astrophysics Data System (ADS)

    Mirmanto; Kenning, D. B. R.; Lewis, J. S.; Karayiannis, T. G.

    2012-11-01

    Experiments were conducted to investigate the pressure drop and heat transfer characteristics of single-phase flow of de-ionized water in single copper microchannels of hydraulic diameters 0.438 mm, 0.561 mm and 0.635 mm. The channel length was 62 mm. The experimental conditions covered a range of mass flux from 500 to 5000 kg/m2 s in the laminar, transitional and low Reynolds number turbulent regimes. Pressure drop was measured for adiabatic flows with fluid inlet temperatures of 30°C, 60°C and 90°C. In the heat transfer tests, the heat flux ranged from 256 kW/m2 to 519 kW/m2. Friction factors and Nusselt numbers determined from the measurements were higher than for fully-developed conditions, but in reasonable agreement with predictions made using published solutions for hydrodynamically and thermally developing flow. When entrance effects, experimental uncertainties, heat losses, inlet and exit losses, thermal boundary conditions and departure from laminar flow were considered, the results indicate that equations developed for flow and heat transfer in conventional size channels are applicable for water flows in microchannels of these sizes.

  12. Naphthalenedicarboxamides as fluorescent probes of inter- and intramolecular electron transfer in single strand, hairpin, and duplex DNA

    SciTech Connect

    Lewis, F.D.; Zhang, Y.; Liu, X.; Xu, N.; Letsinger, R.L.

    1999-04-01

    The 2,6-naphthalenedicarboxamide chromophore has been investigated as a fluorescent probe for DNA hairpin and duplex formation and DNA electron transfer. The high fluorescence quantum yield and long singlet lifetime of this chromophore make it an attractive candidate for these studies. The kinetics of intermolecular quenching of a naphthalenedicarboxamide by nucleosides is dependent upon the nucleoside oxidation potential and solvent. Bis(oligonucleotide) conjugates containing naphthalene linkers have been prepared by means of conventional phosphoramidite chemistry. The base-sequence dependence of the naphthalene fluorescence intensity and decay times in both single-strand and hairpin conjugates indicates that singlet naphthalene is quenched by neighboring dA more efficiently than by dT, in accord with an electron-transfer quenching mechanism. These data are analyzed by means of a three-state model which includes a nonemissive dark state. Duplexes formed between complementary naphthalene-linked oligonucleotides display naphthalene excimer emission. The base-sequence dependence of the excimer emission quantum yields indicates that the excimer is not quenched by neighboring dA but that distance-dependent electron-transfer quenching by dG may occur. Quenching serves to protect the naphthalene chromophore from photobleaching in both single strand and duplex structures.

  13. Origin of the red sites and energy transfer rates in single MEH-PPV chains at low temperature.

    PubMed

    Feist, Florian A; Zickler, Martin F; Basché, Thomas

    2011-06-01

    Single poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) molecules dispersed in thin poly(methylmethacrylate) films have been investigated by fluorescence emission, excitation and time-resolved spectroscopy at 1.2 K. For the molecular weight studied (∼200 kDa) a bimodal distribution of emission maxima is observed. Based on a comparison of the spectroscopic properties of blue and red sites and on polarisation-resolved measurements, we argue in agreement with recent quantum-chemical calculations that the red subpopulation most probably does not arise from interchromophoric excitation delocalisation but is to be attributed to longer chromophoric units originating from ordered regions of a polymer chain, where due to constraints on the chain conformation larger conjugation lengths can be realised. In excitation spectra within the red spectral region we can identify multiple chromophoric units, among them chromophores without correspondence in the emission spectrum-donors of the intramolecular energy transfer. Zero-phonon lines of donor chromophores proved to be significantly broadened, indicating fast excited-state population decay due to energy transfer. Thus, a distribution of energy transfer times within MEH-PPV chains could be determined from donor zero-phonon line widths, with an average value of 3.9 ps. Our study represents the first direct measurement of energy transfer times in conjugated polymers, parameters that are crucial for the performance of many technical applications based on this class of material. PMID:21472962

  14. Single-phase heat transfer improved by helical inserts in tubes

    NASA Technical Reports Server (NTRS)

    Gutsein, M. U.

    1970-01-01

    Helical-vane insert creates a single, well defined helical flow passage resulting in more reliable predictions and extrapolations of the thermal and hydraulic performance. Insert core houses instrumentation which does not disturb the flow.

  15. Magnetic Dynamics of Single-Domain Planar Spin-Transfer Devices

    NASA Astrophysics Data System (ADS)

    Bazaliy, Ya. B.

    2008-03-01

    We study spin-transfer devices with dynamic magnets characterized by large easy-plane anisotropy. This situation is standard for planar devices where it arises due to the shape anisotropy. Dominating easy-plane anisotropy keeps the motion of the magnetic moment close to the easy plane, with small out-of-plane deviations. As a result, it is possible to approximately describe magnetization vector by the in-plane angle and derive an effective one dimensional equation for that angle in the absence [1] and in the presence [2] of spin-transfer torques. Effective description maps a spin-transfer device problem onto a problem of an ``effective particle'' moving in external potential with variable friction coefficient. The advantage of such a description is that the motion of the effective particle can be qualitatively understood by applying the usual energy conservation and dissipation arguments. We show how the effective description produces analytic results for current induced precession states [3] and predicts unconventional ``stabilization by repulsion'' of static states [2]. // [1] C. J. Garcia-Cervera, Weinan E, J. Appl. Phys. 90, 370 (2001). [2] Ya. B. Bazaliy, Phys. Rev. B 76, 140402(R) (2007). [3] Ya. B. Bazaliy, arXiv:0705.0508, to be published in Appl. Phys. Lett. (2007).

  16. Single-stage dynamic reanimation of the smile in irreversible facial paralysis by free functional muscle transfer.

    PubMed

    Thiele, Jan; Bannasch, Holger; Stark, G Bjoern; Eisenhardt, Steffen U

    2015-03-01

    Unilateral facial paralysis is a common disease that is associated with significant functional, aesthetic and psychological issues. Though idiopathic facial paralysis (Bell's palsy) is the most common diagnosis, patients can also present with a history of physical trauma, infectious disease, tumor, or iatrogenic facial paralysis. Early repair within one year of injury can be achieved by direct nerve repair, cross-face nerve grafting or regional nerve transfer. It is due to muscle atrophy that in long lasting facial paralysis complex reconstructive methods have to be applied. Instead of one single procedure, different surgical approaches have to be considered to alleviate the various components of the paralysis. The reconstruction of a spontaneous dynamic smile with a symmetric resting tone is a crucial factor to overcome the functional deficits and the social handicap that are associated with facial paralysis. Although numerous surgical techniques have been described, a two-stage approach with an initial cross-facial nerve grafting followed by a free functional muscle transfer is most frequently applied. In selected patients however, a single-stage reconstruction using the motor nerve to the masseter as donor nerve is superior to a two-stage repair. The gracilis muscle is most commonly used for reconstruction, as it presents with a constant anatomy, a simple dissection and minimal donor site morbidity. Here we demonstrate the pre-operative work-up, the post-operative management, and precisely describe the surgical procedure of single-stage microsurgical reconstruction of the smile by free functional gracilis muscle transfer in a step by step protocol. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly comprehend the procedure. We further discuss indications and limitations of the technique and demonstrate representative results.

  17. Effects of synchronous and asynchronous embryo transfer on postnatal development, adult health, and behavior in mice.

    PubMed

    López-Cardona, Angela P; Fernández-González, Raúl; Pérez-Crespo, Miriam; Alén, Francisco; de Fonseca, Fernando Rodriguez; Orio, Laura; Gutierrez-Adan, Alfonso

    2015-10-01

    Asynchronous embryo transfer (ET) is a common assisted reproduction technique used in several species, but its biological effects on postnatal and early development remain unknown. The aim of this study was to determine whether asynchronous ET produces long-term effects in mice. Postnatal development, animal weight, systolic blood pressure (SBP), relative organ weight (liver, spleen, kidneys, heart, lungs, brain, and testicles), and behavior (assessed in open-field and elevated plus maze tests) were assessed in CD1 mice produced by different ET procedures: 1) the transfer of Day 3.5 (D3.5) blastocysts to the uterus (BL-UT); 2) the transfer of D3.5 blastocysts to the oviduct (BL-OV); or 3) the transfer of D0.5 zygotes to the oviduct (Z-OV). In vivo conceived animals served as controls (CT). The transfer of blastocysts to the uterus or zygotes to the oviduct was defined as synchronous, and transfer of blastocysts to the oviduct was defined as asynchronous. Both synchronous and asynchronous ET resulted in increased weight at birth that normalized thereafter with the exception of asynchronous ET females. In this group, female BL-OV, a clear lower body weight was recorded along postnatal life when compared with controls (P < 0.05). No effects on animal weight were produced during postnatal development in the synchronous ET groups (BL-UT, Z-OV, and CT). Both synchronous and asynchronous ET had impacts on adult (Wk 30) organ weight. SBP was modified in animals derived from blastocyst but not zygote ET. Effects on behavior (anxiety in the plus maze) were only detected in the BL-UT group (P < 0.05). Our findings indicate that zygotes are less sensitive than blastocysts to ET and that both synchronous and asynchronous blastocyst ET may have long-term consequences on health, with possible impacts on weight, arterial pressure, relative organ weight, and behavior.

  18. Kinetic energy release in thermal ion--molecule reactions: The Nb sup 2+ --(benzene) single charge--transfer reaction

    SciTech Connect

    Gord, J.R.; Freiser, B.S. ); Buckner, S.W. )

    1991-03-15

    We have adapted the techniques originally developed to measure ion kinetic energies in ion cyclotron resonance (ICR) spectrometry to study the single charge--transfer reaction of Nb{sup 2+} with benzene under thermal conditions in a Fourier transform ion cyclotron resonance mass spectrometer (FTICRMS). The partitioning of reaction exothermicity among the internal and translational modes available is consistent with a long-distance electron-transfer mechanism, in which the reactants approach on an ion-induced dipole attractive potential and cross to a repulsive potential at a critical separation of {similar to}7.5 A when electron transfer occurs. The reaction exothermicity, 5.08 eV, is partitioned to translation of Nb{sup +} , 0.81{plus minus}0.25 eV, translation of C{sub 6} H{sub 6}{sup +}, 1.22{plus minus}0.25 eV, and internal excitation of C{sub 6} H{sub 6}{sup +} to produce the la{sub 2{ital u}} electronic state, which is {similar to}3 eV above the ground state of the ion. We have also studied the kinetics of the reaction of Nb{sup 2+} with benzene and determined the rate constant, {ital k} = 1.4{times}10{sup {minus}9} cm{sup 3} molecule{sup {minus}1} s{sup {minus}1}, and the efficiency, 0.60, of the process. These also support the proposed charge--transfer mechanism. In addition to the charge--transfer pathway, which accounts for 95% of the reaction products, Nb{sup 2+} is observed to dehydrogenate benzene to form Nb{sup 2+} (benzyne). This process implies {ital D}(Nb{sup 2+} --benzyne){ge}79 kcal/mol.

  19. Oxidative catalysis using the stoichiometric oxidant as a reagent: an efficient strategy for single-electron-transfer-induced tandem anion-radical reactions.

    PubMed

    Kafka, František; Holan, Martin; Hidasová, Denisa; Pohl, Radek; Císařová, Ivana; Klepetářová, Blanka; Jahn, Ullrich

    2014-09-01

    Oxidative single-electron transfer-catalyzed tandem reactions consisting of a conjugate addition and a radical cyclization are reported, which incorporate the mandatory terminal oxidant as a functionality into the product.

  20. Optical and Electrical Characteristics of Graphene Double Layer Formed by a Double Transfer of Graphene Single Layers.

    PubMed

    Kim, Young Jun; Bae, Gi Yoon; Chun, Sungwoo; Park, Wanjun

    2016-03-01

    We demonstrate formation of double layer graphene by means of a double transfer using two single graphene layers grown by a chemical vapor deposition method. It is observed that shiftiness and broadness in the double-resonance of Raman scattering are much weaker than those of bilayer graphene formed naturally. Transport characteristics examined from transmission line measurements and field effect transistors show the similar behavior with those of single layer graphene. It indicates that interlayer separation, in electrical view, is large enough to avoid correlation between layers for the double layer structure. It is also observed from a transistor with the double layer graphene that molecules adsorpted on two inner graphene surfaces in the double layered structure are isolated and conserved from ambient environment. PMID:27455706

  1. Optical and Electrical Characteristics of Graphene Double Layer Formed by a Double Transfer of Graphene Single Layers.

    PubMed

    Kim, Young Jun; Bae, Gi Yoon; Chun, Sungwoo; Park, Wanjun

    2016-03-01

    We demonstrate formation of double layer graphene by means of a double transfer using two single graphene layers grown by a chemical vapor deposition method. It is observed that shiftiness and broadness in the double-resonance of Raman scattering are much weaker than those of bilayer graphene formed naturally. Transport characteristics examined from transmission line measurements and field effect transistors show the similar behavior with those of single layer graphene. It indicates that interlayer separation, in electrical view, is large enough to avoid correlation between layers for the double layer structure. It is also observed from a transistor with the double layer graphene that molecules adsorpted on two inner graphene surfaces in the double layered structure are isolated and conserved from ambient environment.

  2. 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.

  3. Single-Phase, Turbulent Heat-Transfer Friction-Factor Data Base Flow Enhanced Tb

    1994-01-21

    Heat-exchanger designers need to know what type of performance improvement can be obtained before they will consider enhanced tubes. In particular, they need access to the heat-transfer coefficients and friction-factor values of enhanced tube types that are commercially available. To compile these data from the numerous publications and reports in the open literature is a formidable task that can discourage the designer from using them. A computer program that contains a comprehensive data base withmore » a search feature would be a handy tool for the designer to obtain an estimate of the performance improvement that can be obtained with a particular enhanced tube geometry. In addition, it would be a valuable tool for researchers who are developing and/or validating new prediction methods. This computer program can be used to obtain friction-factor and/or heat-transfer data for a broad range of internally enhanced tube geometries with forced-convective turbulent flow. The program has search features; that is the user can select data for tubes with a particular enhancement geometry range or data obtained from a particular source or publication. The friction factor data base contains nearly 5,000 points and the heat-transfer data base contains more than 4,700 points. About 360 different tube geometries are included from the 36 different sources. Data for tubes with similar geometries and the same and/or different types can be easily extracted with the sort feature of this data base and compared. Users of the program are heat-exchanger designers, enhanced tubing suppliers, and research organizations or academia who are developing or validating prediction methods.« less

  4. Properties of sizeable [n]cycloparaphenylenes as molecular models of single-wall carbon nanotubes elucidated by Raman spectroscopy: structural and electron-transfer responses under mechanical stress.

    PubMed

    Peña Alvarez, Miriam; Mayorga Burrezo, Paula; Kertesz, Miklos; Iwamoto, Takahiro; Yamago, Shigeru; Xia, Jianlong; Jasti, Ramesh; López Navarrete, Juan T; Taravillo, Mercedes; Baonza, Valentín G; Casado, Juan

    2014-07-01

    [n]Cycloparaphenylenes behave as molecular templates of "perfectly chemically defined" single-wall carbon nanotubes. These [n]CPP molecules have electronic, mechanical, and chemical properties in size correspondence with their giant congeners. Under mechanical stress, they form charge-transfer salts, or complexes with fullerene, by one-electron concave-convex electron transfer. PMID:24838669

  5. Structural and EPR characterisation of single electron and alkyl transfer products from reaction of dimethyl magnesium with bulky alpha-diimine ligands.

    PubMed

    Bailey, Philip J; Coxall, Robert A; Dick, Caroline M; Fabre, Sylvie; Parsons, Simon; Yellowlees, Lesley J

    2005-09-28

    Treatment of dimethylmagnesium with bulky alpha-diimine ligands provides either the biradical methyl-bridged complexes [(alpha-diimine-.)Mg+(mu-CH3)]2 via single electron transfer (SET), or the product of methyl transfer to an imine carbon atom depending upon conditions.

  6. Studies of Nuclei Close to 132Sn Using Single-Neutron Transfer Reactions

    SciTech Connect

    Jones, K. L.; Pain, S. D.; Kozub, R. L.; Adekola, Aderemi S; Bardayan, Daniel W; Blackmon, Jeff C; Catford, Wilton N; Chae, K. Y.; Chipps, K.; Cizewski, J. A.; Erikson, Luke; Gaddis, A. L.; Greife, U.; Grzywacz, R. K.; Harlin, Christopher W; Hatarik, Robert; Howard, Joshua A; James, J.; Kapler, R.; Krolas, W.; Liang, J Felix; Ma, Zhanwen; Matei, Catalin; Moazen, Brian; Nesaraja, Caroline D; O'Malley, Patrick; Patterson, N. P.; Paulauskas, Stanley; Shapira, Dan; ShrinerJr., J. F.; Sikora, M.; Sissom, D. J.; Smith, Michael Scott; Swan, T. P.; Thomas, J. S.; Wilson, Gemma L

    2009-01-01

    Neutron transfer reactions were performed in inverse kinematics using radioactive ion beams of 132Sn, 130Sn, and 134Te and deuterated polyethylene targets. Preliminary results are presented. The Q-value spectra for 133Sn, 131Sn and 135Te reveal a number of previously unobserved peaks. The angular distributions are compatible with the expected lf7/2 nature of the ground state of 133Sn, and 2p3/2 for the 3.4 MeV state in 131Sn.

  7. Studies of nuclei close to {sup 132}Sn using single-neutron transfer reactions

    SciTech Connect

    Jones, K. J.; Pain, S. D.; Kozub, R. L.; Howard, J. A.; O'Malley, P. D.; Paulauskas, S. V.; Shriner, J. F.; Sissom, D. J.; Adekola, A. S.; Bardayan, D. W.; Blackmon, J. C.; Liang, J. F.; Nesaraja, C. D.; Shapira, D.; Smith, M. S.; Catford, W. N.; Harlin, C.; Patterson, N. P.; Swan, T. P.; Wilson, G. L.

    2009-03-04

    Neutron transfer reactions were performed in inverse kinematics using radioactive ion beams of {sup 132}Sn, {sup 130}Sn, and {sup 134}Te and deuterated polyethylene targets. Preliminary results are presented. The Q-value spectra for {sup 133}Sn, {sup 131}Sn and {sup 135}Te reveal a number of previously unobserved peaks. The angular distributions are compatible with the expected lf{sub 7/2} nature of the ground state of {sup 133}Sn, and 2p{sub 3/2} for the 3.4 MeV state in {sup 131}Sn.

  8. Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA REPLICATION FACTOR A.

    PubMed

    Dafny-Yelin, Mery; Levy, Avner; Dafny, Raz; Tzfira, Tzvi

    2015-01-01

    Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process. PMID:25424309

  9. Single-drop reactive extraction/extractive reaction with forced convective diffusion and interphase mass transfer

    NASA Technical Reports Server (NTRS)

    Kleinman, Leonid S.; Red, X. B., Jr.

    1995-01-01

    An algorithm has been developed for time-dependent forced convective diffusion-reaction having convection by a recirculating flow field within the drop that is hydrodynamically coupled at the interface with a convective external flow field that at infinity becomes a uniform free-streaming flow. The concentration field inside the droplet is likewise coupled with that outside by boundary conditions at the interface. A chemical reaction can take place either inside or outside the droplet, or reactions can take place in both phases. The algorithm has been implemented, and for comparison results are shown here for the case of no reaction in either phase and for the case of an external first order reaction, both for unsteady behavior. For pure interphase mass transfer, concentration isocontours, local and average Sherwood numbers, and average droplet concentrations have been obtained as a function of the physical properties and external flow field. For mass transfer enhanced by an external reaction, in addition to the above forms of results, we present the enhancement factor, with the results now also depending upon the (dimensionless) rate of reaction.

  10. Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Periasamy, Ammasi; Kay, Steve A.; Day, Richard N.

    1997-05-01

    Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.

  11. Single-drop reactive extraction/extractive reaction with forced convective diffusion and interphase mass transfer

    NASA Technical Reports Server (NTRS)

    Kleinman, Leonid S.; Reed, X. B., Jr.

    1995-01-01

    An algorithm has been developed for the forced convective diffusion-reaction problem for convection inside and outside a droplet by a recirculating flow field hydrodynamically coupled at the droplet interface with an external flow field that at infinity becomes a uniform streaming flow. The concentration field inside the droplet is likewise coupled with that outside by boundary conditions at the interface. A chemical reaction can take place either inside or outside the droplet or reactions can take place in both phases. The algorithm has been implemented and results are shown here for the case of no reaction and for the case of an external first order reaction, both for unsteady behavior. For pure interphase mass transfer, concentration isocontours, local and average Sherwood numbers, and average droplet concentrations have been obtained as a function of the physical properties and external flow field. For mass transfer enhanced by an external reaction, in addition to the above forms of results, we present the enhancement factor, with the results now also depending upon the (dimensionless) rate of reaction.

  12. Exciton Recombination, Energy-, and Charge Transfer in Single- and Multilayer Quantum-Dot Films on Silver Plasmonic Resonators

    PubMed Central

    Shin, Taeho; Cho, Kyung-Sang; Yun, Dong-Jin; Kim, Jinwoo; Li, Xiang-Shu; Moon, Eui-Seong; Baik, Chan-Wook; Il Kim, Sun; Kim, Miyoung; Choi, Jun Hee; Park, Gyeong-Su; Shin, Jai-Kwang; Hwang, Sungwoo; Jung, Tae-Sung

    2016-01-01

    We examine exciton recombination, energy-, and charge transfer in multilayer CdS/ZnS quantum dots (QDs) on silver plasmonic resonators using photoluminescence (PL) and excitation spectroscopy along with kinetic modeling and simulations. The exciton dynamics including all the processes are strongly affected by the separation distance between QDs and silver resonators, excitation wavelength, and QD film thickness. For a direct contact or very small distance, interfacial charge transfer and tunneling dominate over intrinsic radiative recombination and exciton energy transfer to surface plasmons (SPs), resulting in PL suppression. With increasing distance, however, tunneling diminishes dramatically, while long-range exciton-SP coupling takes place much faster (>6.5 ns) than intrinsic recombination (~200 ns) causing considerable PL enhancement. The exciton-SP coupling strength shows a strong dependence on excitation wavelengths, suggesting the state-specific dynamics of excitons and the down-conversion of surface plasmons involved. The overlayers as well as the bottom monolayer of QD multilayers exhibit significant PL enhancement mainly through long-range exciton-SP coupling. The overall emission behaviors from single- and multilayer QD films on silver resonators are described quantitatively by a photophysical kinetic model and simulations. The present experimental and simulation results provide important and useful design rules for QD-based light harvesting applications using the exciton-surface plasmon coupling. PMID:27184469

  13. Debating Elective Single Embryo Transfer after in vitro Fertilization: A Plea for a Context-Sensitive Approach

    PubMed Central

    Ezugwu, EC; Van der Burg, S

    2015-01-01

    The number of embryos transferred after in vitro fertilization (IVF) have been a topic of debate for over a decade now. Due to the risk associated with multiple pregnancy, there has been a global effort at reducing the multiple pregnancy rates to a minimum while maintaining an acceptable level of successful IVF pregnancy rate. Elective single embryo transfer (eSET) is advocated in most European countries. In Belgium and Sweden, eSET is mandatory for couples with a good prognosis. However, despite clinical recommendations and policy statements, patients in clinical practice frequently do request for the transfer of multiple embryos in order to have twins. Such requests conflict with policy guidelines and create an ethical dilemma for physicians: Should the physician do as the couple requests, and there with respect the autonomy of patients, or adhere to medical policy that takes the health of the mother and children at heart? This article provides an exploration of the arguments found in the literature that plays a role in the discussion on this topic and eventually argues that what a physician should do depends on the specificities of the context in which patients and physicians are implicated. These contextual issues can be taken into account in a shared decision-making procedure, which allows reflections and the responsibilities of both patients and physicians to be attended in decision about assisted reproduction. PMID:25745568

  14. Quantitative description of the lie-to-sit-to-stand-to-walk transfer by a single body-fixed sensor.

    PubMed

    Bagalà, Fabio; Klenk, Jochen; Cappello, Angelo; Chiari, Lorenzo; Becker, Clemens; Lindemann, Ulrich

    2013-07-01

    Sufficient capacity and quality of performance of complex movement patterns during daily activity, such as standing up from a bed, is a prerequisite for independent living and also may be an indicator of fall risk. Until now, the transfer from lying-to-sit-to-stand-to-walk (LSSW) was investigated by functional testing, subjective rating or for activity classification of subtasks. The aim of this study was to use a single body-fixed inertial sensor to describe the complex movement of the LSSW transfer. Fifteen older patients of a geriatric rehabilitation clinic (median age 81 years) and ten young, healthy persons (median age 37 years) were instructed to stand up from bed in a continuous movement and to start walking. Data acquisition was performed using an inertial measurement unit worn on the lower back. Parameters extracted from the sensor outputs were able to correctly classify the subjects into a correct group with sensitivity and specificity between 90% and 100%. ICCs 3,1 of the descriptive parameters ranged between 0.85 and 0.95 in the cohort of older patients. The different strategies adopted to transfer from lying to standing up were estimated through an extended Kalman filter. The results obtained in this study suggest the usability of the instrumented LSSW test in clinical settings.

  15. SCC-DFTB Energy Barriers for Single and Double Proton Transfer Processes in the Model Molecular Systems Malonaldehyde and Porphycene

    SciTech Connect

    Walewski, L.; Krachtus, D; Fischer, S.; Smith, Jeremy C; Bala, P.; Lesyng, B.

    2005-09-01

    Self-consistent charge-density functional tight-binding SCC-DFTB is a computationally efficient method applicable to large (bio)molecular systems in which (bio)chemical reactions may occur. Among these reactions are proton transfer processes. This method, along with more advanced ab initio techniques, is applied in this study to compute intramolecular barriers for single and double proton transfer processes in the model systems, malonaldehyde and porphycene, respectively. SCC-DFTB is compared with experimental data and higher-level ab initio calculations. For malonaldehyde, the SCC-DFTB barrier height is 3.1 kcal/mol in vacuo and 4.2 kcal/mol in water solution. In the case of porphycene, the minimum energy pathways for double intramolecular proton transfer were determined using the conjugate peak refinement (CPR) method. Six isomers of porphycene were ordered according to energy. The only energetically allowed pathway was found to connect two symmetrical trans states via an unstable cis-A isomer. The SCC-DFTB barrier heights are 11.1 kcal/mol for the trans-cis-A process, and 7.4 kcal/mol for the reverse cis-A-trans one with the energy difference of 3.7 kcal/mol between the trans- and cis-A states. The method provides satisfactory energy results when compared with reference ab initio and experimental data.

  16. Study of Sequential Dexter Energy Transfer in High Efficient Phosphorescent White Organic Light-Emitting Diodes with Single Emissive Layer

    NASA Astrophysics Data System (ADS)

    Kim, Jin Wook; You, Seung Il; Kim, Nam Ho; Yoon, Ju-An; Cheah, Kok Wai; Zhu, Fu Rong; Kim, Woo Young

    2014-11-01

    In this study, we report our effort to realize high performance single emissive layer three color white phosphorescent organic light emitting diodes (PHOLEDs) through sequential Dexter energy transfer of blue, green and red dopants. The PHOLEDs had a structure of; ITO(1500 Å)/NPB(700 Å)/mCP:Firpic-x%:Ir(ppy)3-0.5%:Ir(piq)3-y%(300 Å)/TPBi(300 Å)/Liq(20 Å)/Al(1200 Å). The dopant concentrations of FIrpic, Ir(ppy)3 and Ir(piq)3 were adjusted and optimized to facilitate the preferred energy transfer processes attaining both the best luminous efficiency and CIE color coordinates. The presence of a deep trapping center for charge carriers in the emissive layer was confirmed by the observed red shift in electroluminescent spectra. White PHOLEDs, with phosphorescent dopant concentrations of FIrpic-8.0%:Ir(ppy)3-0.5%:Ir(piq)3-0.5% in the mCP host of the single emissive layer, had a maximum luminescence of 37,810 cd/m2 at 11 V and a luminous efficiency of 48.10 cd/A at 5 V with CIE color coordinates of (0.35, 0.41).

  17. Study of sequential dexter energy transfer in high efficient phosphorescent white organic light-emitting diodes with single emissive layer.

    PubMed

    Kim, Jin Wook; You, Seung Il; Kim, Nam Ho; Yoon, Ju-An; Cheah, Kok Wai; Zhu, Fu Rong; Kim, Woo Young

    2014-11-12

    In this study, we report our effort to realize high performance single emissive layer three color white phosphorescent organic light emitting diodes (PHOLEDs) through sequential Dexter energy transfer of blue, green and red dopants. The PHOLEDs had a structure of; ITO(1500 Å)/NPB(700 Å)/mCP:Firpic-x%:Ir(ppy)3-0.5%:Ir(piq)3-y%(300 Å)/TPBi(300 Å)/Liq(20 Å)/Al(1200 Å). The dopant concentrations of FIrpic, Ir(ppy)3 and Ir(piq)3 were adjusted and optimized to facilitate the preferred energy transfer processes attaining both the best luminous efficiency and CIE color coordinates. The presence of a deep trapping center for charge carriers in the emissive layer was confirmed by the observed red shift in electroluminescent spectra. White PHOLEDs, with phosphorescent dopant concentrations of FIrpic-8.0%:Ir(ppy)3-0.5%:Ir(piq)3-0.5% in the mCP host of the single emissive layer, had a maximum luminescence of 37,810 cd/m(2) at 11 V and a luminous efficiency of 48.10 cd/A at 5 V with CIE color coordinates of (0.35, 0.41).

  18. 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

  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. 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

  1. Transfer RNA maturation in Chlamydomonas mitochondria, chloroplast and the nucleus by a single RNase P protein.

    PubMed

    Bonnard, Géraldine; Gobert, Anthony; Arrivé, Mathilde; Pinker, Franziska; Salinas-Giegé, Thalia; Giegé, Philippe

    2016-08-01

    The maturation of tRNA precursors involves the 5' cleavage of leader sequences by an essential endonuclease called RNase P. Beyond the ancestral ribonucleoprotein (RNP) RNase P, a second type of RNase P called PRORP (protein-only RNase P) evolved in eukaryotes. The current view on the distribution of RNase P in cells is that multiple RNPs, multiple PRORPs or a combination of both, perform specialised RNase P activities in the different compartments where gene expression occurs. Here, we identify a single gene encoding PRORP in the green alga Chlamydomonas reinhardtii while no RNP is found. We show that its product, CrPRORP, is triple-localised to mitochondria, the chloroplast and the nucleus. Its downregulation results in impaired tRNA biogenesis in both organelles and the nucleus. CrPRORP, as a single-subunit RNase P for an entire organism, makes up the most compact and versatile RNase P machinery described in either prokaryotes or eukaryotes.

  2. SU-E-QI-15: Single Point Dosimetry by Means of Cerenkov Radiation Energy Transfer (CRET)

    SciTech Connect

    Volotskova, O; Jenkins, C; Xing, L

    2014-06-15

    Purpose: Cerenkov light is generated when a charged particles with energy greater then 250 keV, moves faster than the speed of light in a given medium. Both x-ray photons and electrons produce optical Cerenkov photons during the static megavoltage linear accelerator (LINAC) operational mode. Recently, Cerenkov radiation gained considerable interest as possible candidate as a new imaging modality. Optical signals generated by Cerenkov radiation may act as a surrogate for the absorbed superficial radiation dose. We demonstrated a novel single point dosimetry method for megavoltage photon and electron therapy utilizing down conversion of Cerenkov photons. Methods: The custom build signal characterization system was used: a sample holder (probe) with adjacent light tight compartments was connected via fiber-optic cables to a photon counting photomultiplier tube (PMT). One compartment contains a medium only while the other contains medium and red-shifting nano-particles (Q-dots, nanoclusters). By taking the difference between the two signals (Cerenkov photons and CRET photons) we obtain a measure of the down-converted light, which we expect to be proportional to dose as measured with an adjacent ion chamber. Experimental results are compared to Monte Carlo simulations performed using the GEANT4 code. Results: The signal correlation between CR signal, CRET readings and dose produced by LINAC at a single point were investigated. The experimental results were compared with simulations. The dose linearity, signal to noise ratio and dose rate dependence were tested with custom build CRET based probe. Conclusion: Performance characteristics of the proposed single point CRET based probe were evaluated. The direct use of the induced Cerenkov emission and CRET in an irradiated single point volume as an indirect surrogate for the imparted dose was investigated. We conclude that CRET is a promising optical based dosimetry method that offers advantages over those already proposed.

  3. Single-Phase Active Boost Rectifier with Power Factor Correction for Wireless Power Transfer Applications

    SciTech Connect

    Chinthavali, Madhu Sudhan; Onar, Omer C; Miller, John M; Tang, Lixin

    2013-01-01

    Wireless Power Transfer (WPT) technology is a novel research area in the charging technology that bridges utility and the automotive industries. There are various solutions that are currently being evaluated by several research teams to find the most efficient way to manage the power flow from the grid to the vehicle energy storage system. There are different control parameters that can be utilized to compensate for the change in the impedance. To understand the power flow through the system this paper presents a novel approach to the system model and the impact of different control parameters on the load power. The implementation of an active front-end rectifier on the grid side for power factor control and voltage boost capability for load power regulation is also discussed.

  4. Electron Transfer between Cytochrome C and Cytochome C Peroxidase in Single Crystals

    SciTech Connect

    Kang, Seong A.; Marjavaara, Pieti J.; Crane, Brian R.

    2010-11-10

    Cytochrome c (Cc) and cytochrome c peroxidase (CcP) form an important redox pair for understanding interprotein electron transfer (ET). Measurements of ET rates from photoexcited CcP substituted with Zn porphyrin to either yeast Fe(III)Cc or horse Fe(III)Cc in crystals reveal that the molecular associations found in the respective crystal structures determine solution reactivity. Similar forward rates for yeast isozyme-1 Cc (yCc) and yCc homologue horse Cc (hCc), despite different orientations relative to CcP, suggest small-amplitude conformational gating of ET even in the crystalline state; faster back ET in the yCc compared to the hCc complex agrees with the relative coupling between redox sites predicted by the structures.

  5. Cross-plane heat transfer through single-layer carbon structures.

    PubMed

    Zhang, Huaichen; Nedea, Silvia V; Rindt, Camilo C M; Smeulders, David M J

    2016-02-21

    Graphene-based nano-structures have been recently proposed to function as additives to improve the conductivity of thermally sluggish phase change materials (PCMs). Based on the existing research studies, the improvement is dependent not only on the matrix material, but also on the geometry of the carbon structure. To gain more insight into the nano-scale thermal transport problem, we launched the current pilot research using water as the matrix material, to represent the hydroxyl-group-rich sugar alcohols as PCMs. We have found that the heat conduction across a graphene layer to water is much faster than the heat conduction to the graphene layer itself. Also, the high graphene-water thermal contact resistance fails to acknowledge the fast thermal kinetics of the low frequency phonons. In the investigation of the geometry effect, the cross-plane heat transfer coefficient is found to decrease with decreasing CNT diameter except CNT(9,9). PMID:26818392

  6. Radiant heat transfer from flames in a single tubular turbojet combustor / Leonard Topper

    NASA Technical Reports Server (NTRS)

    Topper, Leonard

    1952-01-01

    An experimental investigation of thermal radiation from the flame of a single tubular turbojet-engine combustor to the combustor liner is presented. The effects of combustor inlet-air pressure, air mass flow, and fuel-air ratio on the radiant intensity and the temperature and emissivity of the flame are reported. The total radiation of the "luminous" flames (containing incandescent soot particles) was much greater (4 to 21 times) than the "nonluminous" molecular radiation. The intensity of radiation from the flame increased rapidly with an increase in combustor inlet-air pressure; it was affected to a lesser degree by variations in fuel-air ratio and air mass flow.

  7. Single-electron Born approximations for charge transfer from multielectron atoms to protons

    NASA Technical Reports Server (NTRS)

    Omidvar, K.; Golden, J. E.; Mcguire, J. H.; Weaver, L.

    1976-01-01

    The Born approximation, including the internuclear interaction, is used to compute cross sections for the transfer of one electron from a multielectron atom to an incident proton. When the full internuclear interaction is included, the results lie far above high-energy experimental K-shell data for protons and argon. However, when only enough internuclear interaction is included so that the total projectile-target interaction goes to zero asymptotically in accordance with plane-wave functions actually used, fair agreement at high energies is obtained. The latter form of the Born approximation is compared with data on K-shell capture from helium as well as argon and with the simpler approximation of Oppenheimer and of Brinkman and Kramers (OBK), where no internuclear interaction is included. The OBK results typically lie a factor of 2 to 8 above the data, while the present Born results are within a factor of 2 of most of the existing high-velocity K-shell data.

  8. Selective on/off switching at room temperature of a magnetic bistable {Fe2Co2} complex with single crystal-to-single crystal transformation via intramolecular electron transfer.

    PubMed

    Cao, Li; Tao, Jun; Gao, Qian; Liu, Tao; Xia, Zhengcai; Li, Dongfeng

    2014-02-18

    A cyano-bridged {Fe2Co2} complex shows reversible single crystal-to-single crystal transformation between diamagnetic and paramagnetic states switched specifically by losing and absorbing methanol at room temperature in the solid state. And the solvent loss form presents temperature- and pressure-induced intramolecular electron transfer behaviour.

  9. Detection of single nucleotide variations by a hybridization proximity assay based on molecular beacons and luminescence resonance energy transfer.

    PubMed

    Root, Douglas D; Vaccaro, Carlos; Zhang, Zhiling; Castro, Miguel

    2004-09-01

    A powerful combination of molecular beacon and luminescence resonance energy transfer technology reveals alterations in nucleic acid structure by as little as a single nucleotide in a novel hybridization proximity assay. The assay measures the length of a single-stranded target when a terbium chelate-labeled molecular beacon hybridizes to one side of the nucleic acid segment to be measured and an acceptor probe carrying a convention fluorophore hybridizes to the opposite end of the target. Using a test sequence shortened incrementally by deleting single nucleotides, this assay reports a nearly linear relationship between sequence length and the distance separating acceptor and donor probes. Consequently, this assay can be used to detect alternative splicing, allele types, rearrangements, insertion, and deletion events by measuring separation distances within a predefined region. Furthermore, the use of terbium chelates in molecular beacons can produce exceptionally high signal-to-background ratios compared to the use of conventional fluorophores. Principles of optimal probe design are investigated experimentally and by computational simulations of plausible molecular beacon folding. Some molecular beacon designs form dimers that reduce their maximal response to target sequences. A simple assay to detect such dimers is reported as a tool to help improve the design of molecular beacons. Optimally designed molecular beacons with terbium chelates and hybridization proximity assays are expected to expand their applications in the analysis and screening of genetic diseases.

  10. 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

  11. Low-pressure airlift fermenter for single cell protein production. I. Design and oxygen transfer studies

    SciTech Connect

    Chen, N.Y.; Kondis, E.F.; Srinivasan, S.

    1987-03-01

    The energy consumption of a fermenter constitutes a major part of the operating expense of a single cell protein process. A low-pressure airlift fermenter was designed to reduce this cost. In this new design, the fermenter broth is kept below 120 cm in depth, and air alone is employed to fulfil the need of supplying oxygen, and cooling and agitating the broth. The use of low-pressure air from air blowers instead of air compressors lowers the capital cost of air delivery and reduces the energy consumption in the fermenter section to below 1 kWh/kg protein, a saving of over 70% as compared to a conventional stirred tank fermenter. It also eliminates the investment of mechanical agitators, heat exchangers, and air compressors. Sulfite oxidation studies confirmed the design concepts. 30 references.

  12. 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

  13. Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts

    PubMed Central

    Hajian, Mahdi; Hosseini, Seyed Morteza; Asgari, Vajiheh; Ostadhoosseini, Somayyeh; Forouzanfar, Mohsen; Nasr Esfahani, Mohammad Hossein

    2011-01-01

    Background This study investigated the effect of two in vitro embryo culture systems (co-culture system versus cell-free sequential-media) on developmental competence, cryosurvival and DNA- fragmentation of in vitro developed bovine blastocysts. Materials and Methods Bovine presumptive zygotes were cultured in Ménézo's B2 (B2) plus vero-cells or sequential synthetic oviductal fluid (SOF) for eight days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and post- warming embryos along with their corresponding non-vitrified embryos were cultured for two additional days in the same medium used before vitrification. Embryo development, cryosurvival and apoptosis were compared between the groups. Results For non-vitrified embryos, culture in SOF significantly promoted the potency of embryos to develop into blastocysts compared with the co-culture system. The difference in post vitrification survival rate of SOF blastocysts (83.3%) was insignificant compared with co-culture (84.3%). However, while total cell number of warmed blastocysts in the co-culture system was significantly higher in the co-culture versus the sequential system (215.4 vs. 170.4), the quality of survived embryos in terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF (65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05). Conclusion Although co-culture system may increase the viability of embryos following cryopreservation, the potency and dynamics of blastocyst formation significantly increased with sequential media compared to the co-culture system which can compensate for the lower efficiency of sequential media for vitrification/warming purposes. PMID:24917920

  14. Embryonic development in culture of two dasyurid marsupials, Sminthopsis crassicaudata (Gould) and Sminthopsis macroura (Spencer), during cleavage and blastocyst formation.

    PubMed

    Selwood, L

    1987-04-01

    Embryos of Sminthopsis crassicaudata and Sminthopsis macroura were cultured for up to 96 hours during cleavage and early expansion of the blastocyst in Dulbecco's modified Eagle's medium (DMEG), DMEG containing 2.76 gm/liter sodium lactate (DMEGL), DMEG containing 3.5 gm/liter galactose (DMEGAL), DMEG containing 15 ng/ml progesterone (DMEGP) or 150 ng/ml progesterone (DMEGP10), and DMEGL containing 15 ng/ml progesterone (DMEGLP). The disappearance of sperm was used to indicate the time of ovulation (day 0). Fertilized eggs were found in the uterus at the end of day 1, four-cell stages at the end of day 2, and embryos completing the fourth division by the end of day 3 in S. macroura and day 4 in S. crassicaudata. Estimated developmental times in culture were similar to those obtained in vivo. In both species, the first two divisions take about 24 hours, cleavage is arrested for 24 hours or longer at the rounded four-cell stage, and the third and fourth divisions take a further 24 hours. The blastocyst expands during the next 24 hours in which time the fifth and sixth divisions occur. It was possible to culture embryos from S. macroura but not S. crassicaudata over the four-cell stage to early expanding blastocysts. DMEGAL did not support cleavage in culture. DMEG, DMEGL, DMEGP, DMEGP10, and DMEGLP all supported culture during cleavage and early blastocyst expansion. Blastocyst expansion was slightly enhanced using media containing sodium lactate. More embryos completed the fifth division and formed expanding blastocysts in DMEG, DMEGL, and DMEGLP.

  15. SiC MOSFET Based Single Phase Active Boost Rectifier with Power Factor Correction for Wireless Power Transfer Applications

    SciTech Connect

    Onar, Omer C; Tang, Lixin; Chinthavali, Madhu Sudhan; Campbell, Steven L; Miller , John M.

    2014-01-01

    Wireless Power Transfer (WPT) technology is a novel research area in the charging technology that bridges the utility and the automotive industries. There are various solutions that are currently being evaluated by several research teams to find the most efficient way to manage the power flow from the grid to the vehicle energy storage system. There are different control parameters that can be utilized to compensate for the change in the impedance due to variable parameters such as battery state-of-charge, coupling factor, and coil misalignment. This paper presents the implementation of an active front-end rectifier on the grid side for power factor control and voltage boost capability for load power regulation. The proposed SiC MOSFET based single phase active front end rectifier with PFC resulted in >97% efficiency at 137mm air-gap and >95% efficiency at 160mm air-gap.

  16. Biological construction of single-walled carbon nanotube electron transfer pathways in dye-sensitized solar cells.

    PubMed

    Inoue, Ippei; Watanabe, Kiyoshi; Yamauchi, Hirofumi; Ishikawa, Yasuaki; Yasueda, Hisashi; Uraoka, Yukiharu; Yamashita, Ichiro

    2014-10-01

    We designed and mass-produced a versatile protein supramolecule that can be used to manufacture a highly efficient dye-sensitized solar cell (DSSC). Twelve single-walled carbon-nanotube (SWNT)-binding and titanium-mineralizing peptides were genetically integrated on a cage-shaped dodecamer protein (CDT1). A process involving simple mixing of highly conductive SWNTs with CDT1 followed by TiO2 biomineralization produces a high surface-area/weight TiO2 -(anatase)-coated intact SWNT nanocomposite under environmentally friendly conditions. A DSSC with a TiO2 photoelectrode containing 0.2 wt % of the SWNT-TiO2 nanocomposite shows a current density improvement by 80% and a doubling of the photoelectric conversion efficiency. The SWNT-TiO2 nanocomposite transfers photon-generated electrons from dye molecules adsorbed on the TiO2 to the anode electrode swiftly.

  17. Bond-length alternation and charge transfer in a linear carbon chain encapsulated within a single-walled carbon nanotube

    NASA Astrophysics Data System (ADS)

    Rusznyák, Á.; Zólyomi, V.; Kürti, J.; Yang, S.; Kertesz, M.

    2005-10-01

    The physical properties of a linear carbon chain encapsulated within single-walled carbon nanotubes are investigated with density-functional theory using periodic boundary conditions. The dominant feature of an isolated carbon chain is the Peierls dimerization and the opening of a Peierls gap. The two weakly interacting subsystems (infinite carbon chain and nanotube) establish a common Fermi level, resulting in charge transfer (CT) which leads to a metallic combined system with a high density of states at the Fermi level. The rigid band model provides useful insights. Unusual physics arises from the effects of CT and chain-tube orbital hybridization which both tend to suppress the Peierls dimerization. Implications for the observed Raman spectrum of the chain-nanotube system are discussed.

  18. Bipolar Latissimus Dorsi Transfer through a Single Incision: First Key–Step in Poland Syndrome Chest Deformity

    PubMed Central

    di Summa, Pietro G.; Raffoul, Wassim

    2016-01-01

    Summary: Poland syndrome is a rare congenital anomaly characterized by a unilateral congenital absence of the sternocostal head of the pectoralis major muscle. The absence of the pectoralis major does not only result in chest asymmetry but also in a missing anterior axillary fold, which is essential for natural anatomical appearance in both male and female patients. In Poland syndrome patients, we perform bipolar latissimus dorsi flap transfer, which can be associated with a sublatissimus implant in women. All procedures are performed through a single short midaxillary incision, and tendon translocation in this technique allows the creation of the anterior axillary fold and thus a natural chest appearance. Moreover, this technique can be performed by any plastic surgeon operating under a basic operating room setting. PMID:27622115

  19. Carbene-catalysed reductive coupling of nitrobenzyl bromides and activated ketones or imines via single-electron-transfer process

    NASA Astrophysics Data System (ADS)

    Li, Bao-Sheng; Wang, Yuhuang; Proctor, Rupert S. J.; Zhang, Yuexia; Webster, Richard D.; Yang, Song; Song, Baoan; Chi, Yonggui Robin

    2016-09-01

    Benzyl bromides and related molecules are among the most common substrates in organic synthesis. They are typically used as electrophiles in nucleophilic substitution reactions. These molecules can also be activated via single-electron-transfer (SET) process for radical reactions. Representative recent progress includes α-carbon benzylation of ketones and aldehydes via photoredox catalysis. Here we disclose the generation of (nitro)benzyl radicals via N-heterocyclic carbene (NHC) catalysis under reductive conditions. The radical intermediates generated via NHC catalysis undergo formal 1,2-addition with ketones to eventually afford tertiary alcohol products. The overall process constitutes a formal polarity-inversion of benzyl bromide, allowing a direct coupling of two initially electrophilic carbons. Our study provides a new carbene-catalysed reaction mode that should enable unconventional transformation of (nitro)benzyl bromides under mild organocatalytic conditions.

  20. Carbene-catalysed reductive coupling of nitrobenzyl bromides and activated ketones or imines via single-electron-transfer process

    PubMed Central

    Li, Bao-Sheng; Wang, Yuhuang; Proctor, Rupert S. J.; Zhang, Yuexia; Webster, Richard D.; Yang, Song; Song, Baoan; Chi, Yonggui Robin

    2016-01-01

    Benzyl bromides and related molecules are among the most common substrates in organic synthesis. They are typically used as electrophiles in nucleophilic substitution reactions. These molecules can also be activated via single-electron-transfer (SET) process for radical reactions. Representative recent progress includes α-carbon benzylation of ketones and aldehydes via photoredox catalysis. Here we disclose the generation of (nitro)benzyl radicals via N-heterocyclic carbene (NHC) catalysis under reductive conditions. The radical intermediates generated via NHC catalysis undergo formal 1,2-addition with ketones to eventually afford tertiary alcohol products. The overall process constitutes a formal polarity-inversion of benzyl bromide, allowing a direct coupling of two initially electrophilic carbons. Our study provides a new carbene-catalysed reaction mode that should enable unconventional transformation of (nitro)benzyl bromides under mild organocatalytic conditions. PMID:27671606

  1. Correlated blinking via time dependent energy transfer in single CdSe quantum dot-dye nanoassemblies

    NASA Astrophysics Data System (ADS)

    Gerlach, Frank; Täuber, Daniela; von Borczyskowski, Christian

    2013-05-01

    Optical confocal spectroscopy on self-assembled single nanoassemblies from CdSe/ZnS quantum dots (QD) and perylene diimide dye molecules demonstrates efficient Förster resonance energy transfer (FRET). Intramolecular dynamics of the flexible dye molecule change the FRET efficiency in course of the detection period of several minutes. This can be followed by correlated observations of luminescence intensities and related spectral shifts of both constituents. Contrary to several experiments on similar assemblies, the FRET efficiencies are by almost one order of magnitude larger in the non-polar liquid solvent TEHOS as compared e.g. to toluene. Experimental and theoretically expected efficiencies are in close agreement with each other.

  2. Bipolar Latissimus Dorsi Transfer through a Single Incision: First Key-Step in Poland Syndrome Chest Deformity.

    PubMed

    Watfa, William; di Summa, Pietro G; Raffoul, Wassim

    2016-08-01

    Poland syndrome is a rare congenital anomaly characterized by a unilateral congenital absence of the sternocostal head of the pectoralis major muscle. The absence of the pectoralis major does not only result in chest asymmetry but also in a missing anterior axillary fold, which is essential for natural anatomical appearance in both male and female patients. In Poland syndrome patients, we perform bipolar latissimus dorsi flap transfer, which can be associated with a sublatissimus implant in women. All procedures are performed through a single short midaxillary incision, and tendon translocation in this technique allows the creation of the anterior axillary fold and thus a natural chest appearance. Moreover, this technique can be performed by any plastic surgeon operating under a basic operating room setting. PMID:27622115

  3. Bipolar Latissimus Dorsi Transfer through a Single Incision: First Key–Step in Poland Syndrome Chest Deformity

    PubMed Central

    di Summa, Pietro G.; Raffoul, Wassim

    2016-01-01

    Summary: Poland syndrome is a rare congenital anomaly characterized by a unilateral congenital absence of the sternocostal head of the pectoralis major muscle. The absence of the pectoralis major does not only result in chest asymmetry but also in a missing anterior axillary fold, which is essential for natural anatomical appearance in both male and female patients. In Poland syndrome patients, we perform bipolar latissimus dorsi flap transfer, which can be associated with a sublatissimus implant in women. All procedures are performed through a single short midaxillary incision, and tendon translocation in this technique allows the creation of the anterior axillary fold and thus a natural chest appearance. Moreover, this technique can be performed by any plastic surgeon operating under a basic operating room setting.

  4. Accumulation of multiple oxidative equivalents at a single site by cross-surface electron transfer on TiO2.

    PubMed

    Song, Wenjing; Ito, Akitaka; Binstead, Robert A; Hanson, Kenneth; Luo, Hanlin; Brennaman, M Kyle; Concepcion, Javier J; Meyer, Thomas J

    2013-08-01

    The photodriven accumulation of two oxidative equivalents at a single site was investigated on TiO2 coloaded with a ruthenium polypyridyl chromophore [Ru(bpy)2((4,4'-(OH)2PO)2bpy)](2+) (Ru(II)P(2+), bpy = 2,2'-bipyridine, ((OH)2PO)2-bpy = 2,2'-bipyridine-4,4'-diyldiphosphonic acid) and a water oxidation catalyst [Ru(Mebimpy) ((4,4'-(OH)2PO-CH2)2bpy)(OH2)](2+) (Ru(II)OH2(2+), Mebimpy = 2,6-bis(1-methylbenzimidazol-2-yl)pyridine, (4,4'-(OH)2PO-CH2)2bpy) = 4,4'-bis-methlylenephosphonato-2,2'-bipyridine). Electron injection from the metal-to-ligand charge transfer (MLCT) excited state of -Ru(II)P(2+) (-Ru(II)P(2+)*) to give -Ru(III)P(3+) and TiO2(e(-)) was followed by rapid (<20 ns) nearest-neighbor -Ru(II)OH2(2+) to -Ru(III)P(3+) electron transfer. On surfaces containing both -Ru(II)P(2+) and -Ru(III)OH2(3+) (or -Ru(III)OH(2+)), -Ru(II)OH2(2+) was formed by random migration of the injected electron inside the TiO2 nanoparticle and recombination with the preoxidized catalyst, followed by relatively slow (μs-ms) non-nearest neighbor cross-surface electron transfer from -Ru(II)OH2(2+) to -Ru(III)P(3+). Steady state illumination of coloaded TiO2 photoanodes in a dye sensitized photoelectrosynthesis cell (DSPEC) configuration resulted in the buildup of -Ru(III)P(3+), -Ru(III)OH(2+), and -Ru(IV)═O(2+), with -Ru(IV)═O(2+) formation favored at high chromophore to catalyst ratios. PMID:23848562

  5. Heat transfer of R-134a in single-tube spray evaporation including lubricant effects and enhanced surface results

    SciTech Connect

    Moeykens, S.A.; Huebsch, W.W.; Pate, M.B.

    1995-08-01

    Single-tube spray evaporation experimental tests were conducted in order to evaluate the average wall heat transfer coefficients for seven different commercially available tubes. Liquid film supply rates were held constant in order to evaluate the effects of the enhancement on shell-side heat transfer under similar conditions. Because the spray evaporation phenomenon is so different from pool boiling, both condensation-type and evaporation-type enhanced surfaces were evaluated. A comparison of the results for all of the tubes showed that the enhanced condensation surfaces performed better than the enhanced boiling surfaces. In addition, the 26-fpi surface tested marginally better than the 40-fpi surface. Small concentrations of a polyol-ester lubricant cause a foaming effect that increases the heat transfer performance. This tendency was seen with both 32-cs and 68-cs polyol-ester oils. The 68-cs lubricant was tested at concentrations of 0.0, 0.5, 1.0, 3.0, and 5.0 with the W-40 fpi and Tu-Cii surfaces. Results with this lubricant show the performance continues to increase through the 3% concentration for most of the heat flux range tested At the upper end of the range tested, the 1.0% mass fraction yielded the best performance. The 32-cs lubricant generated trends similar to those of the 68-cs lubricant. Lubricant concentrations of 1.0%, 2.0%, and 3.0% were evaluated with plain, W-40 fpi, and Tu-Cii surfaces. The 2.0% concentration, not the 1.0 %, generated the best performance at the highest heat flux tested. This difference must be attributed to the difference in the lubricant viscosity.

  6. Studying 10Be and 11Be Halo States through the (p,d) Single-Neutron Transfer Reaction

    NASA Astrophysics Data System (ADS)

    Kuhn, Keri; Sarazin, Fred; (Pcb)2 Collaboration; Tigress Collaboration

    2015-10-01

    One-neutron transfer reactions are being used to study single-particle neutron states in nuclei. For one-neutron halo nuclei, such as 11Be, the (p,d) reaction enables the removal of the halo neutron or of one of the core neutrons. This way, it is possible to simultaneously study the halo wavefunction of the 11Be ground-state but also a possible excited halo state in 10Be. The 11Be(p, d)10Be transfer reaction at 10 MeV/nucleon is being investigated at the TRIUMF-ISAC II facility with the Printed Circuit Board Based Charged Particle ((PCB)2) array inside the TRIUMF ISAC Gamma-Ray Escape-Suppressed Spectrometer (TIGRESS). The ground state and first excited state of 10Be can be directly identified using deuteron identification and kinematics from the charged particle array, while the four excited states in10Be around 6 MeV, including the suspected halo state (2- state), are identified using coincident gamma rays from TIGRESS with the identified deuterons. Angular distributions for the 10Be populated states will be shown along with their FRESCO fits. This work is partially supported by the US Department of Energy through Grant/Contract No. DE-FG03-93ER40789 (Colorado School of Mines).

  7. Ab initio characterization of electron transfer coupling in photoinduced systems: generalized Mulliken-Hush with configuration-interaction singles.

    PubMed

    Chen, Hung-Cheng; Hsu, Chao-Ping

    2005-12-29

    To calculate electronic couplings for photoinduced electron transfer (ET) reactions, we propose and test the use of ab initio quantum chemistry calculation for excited states with the generalized Mulliken-Hush (GMH) method. Configuration-interaction singles (CIS) is proposed to model the locally excited (LE) and charge-transfer (CT) states. When the CT state couples with other high lying LE states, affecting coupling values, the image charge approximation (ICA), as a simple solvent model, can lower the energy of the CT state and decouple the undesired high-lying local excitations. We found that coupling strength is weakly dependent on many details of the solvent model, indicating the validity of the Condon approximation. Therefore, a trustworthy value can be obtained via this CIS-GMH scheme, with ICA used as a tool to improve and monitor the quality of the results. Systems we tested included a series of rigid, sigma-linked donor-bridge-acceptor compounds where "through-bond" coupling has been previously investigated, and a pair of molecules where "through-space" coupling was experimentally demonstrated. The calculated results agree well with experimentally inferred values in the coupling magnitudes (for both systems studied) and in the exponential distance dependence (for the through-bond series). Our results indicate that this new scheme can properly account for ET coupling arising from both through-bond and through-space mechanisms.

  8. Large-Eddy Simulation of Heat Transfer from a Single Cube Mounted on a Very Rough Wall

    NASA Astrophysics Data System (ADS)

    Boppana, V. B. L.; Xie, Zheng-Tong; Castro, Ian P.

    2013-06-01

    The local thermal effects in the wake of a single cube with a strong heated rear face, representing a large building in an urban area, are studied using large-eddy simulations (LES) for various degrees of heating, which are characterized by the local Richardson number, Ri. New wall models are implemented for momentum and temperature and comparison of the flow and thermal fields with the wind-tunnel data of Richards et al. (J Wind Eng Ind Aerodyn 94, 621-636, 2006) shows fair agreement. Buoyancy effects are quite evident at low Ri and a significant increase in the turbulence levels is observed for such flows. Apart from the comparisons with experiments, further analysis included the estimation of the thermal boundary-layer thickness and heat transfer coefficient for all Ri. For sufficiently strong heating, the heat transfer coefficient at the leeward face is found to be higher than the roof surface. This suggests that, beyond a certain Ri value, buoyancy forces from the former surface dominate the strong streamwise convection of the latter. Quadrant analysis along the shear layer behind the cube showed that the strength of sweeps that contribute to momentum flux is considerably enhanced by heating. The contribution of different quadrants to the heat flux is found to be very different to that of the momentum flux for lower Ri.

  9. Heat transfer and oil flow studies on a single-stage-to-orbit control-configured winged entry vehicle

    NASA Technical Reports Server (NTRS)

    Helms, V. T., III; Bradley, P. F.

    1984-01-01

    Results are presented for oil flow and phase change paint heat transfer tests conducted on a 0.006 scale model of a proposed single stage to orbit control configured vehicle. The data were taken at angles of attack up to 40 deg at a free stream Mach number of 10 for Reynolds numbers based on model length of 0.5 x 10 to the 6th power, 1.0 x 10 to the 6th power and 2.0 x 10 to the 6th power. The magnitude and distribution of heating are characterized in terms of angle of attack and Reynolds number aided by an analysis of the flow data which are used to suggest the presence of various three dimensional flow structures that produce the observed heating patterns. Of particular interest are streak heating patterns that result in high localized heat transfer rates on the wing windward surface at low to moderate angles of attack. These streaks are caused by the bow-shock/wing-shock interaction and formation of the wing-shock. Embedded vorticity was found to be associated with these interactions.

  10. Spectroscopic Factors from the Single Neutron Transfer Reaction 111Cd(d,p)112Cd

    NASA Astrophysics Data System (ADS)

    Jamieson, D. S.; Garrett, P. E.; Demand, G. A.; Finlay, P.; Green, K. L.; Leach, K. G.; Phillips, A. A.; Svensson, C. E.; Sumithrarachchi, C. S.; Triambak, S.; Wong, J.; Ball, G.; Faestermann, T.; Krücken, R.; Hertenberger, R.; Wirth, H.-F.

    2013-03-01

    The cadmium isotopes have been cited as excellent examples of vibrational nuclei for decades, with multi-phonon quadrupole, quadrupole-octupole, and mixed-symmetry states proposed. From a variety of experimental studies, a large amount of spectroscopic data has been obtained, recently focused on γ-ray studies. In the present work, the single-particle structure of 112Cd has been investigated using the 111Cd(ěcd, p)112Cd reaction. The investigation was carried out using a 22 MeV beam of polarized deuterons obtained from the Maier-Leibnitz Laboratory at Garching, Germany. The reaction ejectiles were momentum analyzed using a Q3D spectrograph, and 115 levels have been identified up to 4.2 MeV of excitation energy. Spin-parity has been assigned to each analyzed level, and angular distributions for the reaction cross sections and analyzing powers were obtained. Many additional levels have been observed compared with the previous (d,p) study performed with 8 MeV deuterons,1 including strongly populated 5- and 6- states. The former was previously assigned as a member of the quadrupole-octupole quintuplet, based on a strongly enhanced B(E2) value to the 3- state, but is now re-assigned as being predominately s1/2 ⊗ h11/2 configuration.

  11. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    PubMed

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

  12. Single software platform used for high speed data transfer implementation in a 65k pixel camera working in single photon counting mode

    NASA Astrophysics Data System (ADS)

    Maj, P.; Kasiński, K.; Gryboś, P.; Szczygieł, R.; Kozioł, A.

    2015-12-01

    Integrated circuits designed for specific applications generally use non-standard communication methods. Hybrid pixel detector readout electronics produces a huge amount of data as a result of number of frames per seconds. The data needs to be transmitted to a higher level system without limiting the ASIC's capabilities. Nowadays, the Camera Link interface is still one of the fastest communication methods, allowing transmission speeds up to 800 MB/s. In order to communicate between a higher level system and the ASIC with a dedicated protocol, an FPGA with dedicated code is required. The configuration data is received from the PC and written to the ASIC. At the same time, the same FPGA should be able to transmit the data from the ASIC to the PC at the very high speed. The camera should be an embedded system enabling autonomous operation and self-monitoring. In the presented solution, at least three different hardware platforms are used—FPGA, microprocessor with real-time operating system and the PC with end-user software. We present the use of a single software platform for high speed data transfer from 65k pixel camera to the personal computer.

  13. Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models

    PubMed Central

    Zarrabi, Nawid; Ernst, Stefan; Verhalen, Brandy; Wilkens, Stephan; Börsch, Michael

    2013-01-01

    Single-molecule Förster resonance energy (smFRET) transfer has become a powerful tool for observing conformational dynamics of biological macromolecules. Analyzing smFRET time trajectories allows to identify the state transitions occuring on reaction pathways of molecular machines. Previously, we have developed a smFRET approach to monitor movements of the two nucleotide binding domains (NBDs) of P-glycoprotein (Pgp) during ATP hydrolysis driven drug transport in solution. One limitation of this initial work was that single-molecule photon bursts were analyzed by visual inspection with manual assignment of individual FRET levels. Here a fully automated analysis of Pgp smFRET data using hidden Markov models (HMM) for transitions up to 9 conformational states is applied. We propose new estimators for HMMs to integrate the information of fluctuating intensities in confocal smFRET measurements of freely diffusing lipid bilayer bound membrane proteins in solution. HMM analysis strongly supports that under conditions of steady state turnover, conformational states with short NBD distances and short dwell times are more populated compared to conditions without nucleotide or transport substrate present. PMID:23891547

  14. Quantum dots for single-pair fluorescence resonance energy transfer in membrane- integrated EFoF1.

    PubMed

    Galvez, Eva; Düser, Monika; Börsch, Michael; Wrachtrup, Jörg; Gräber, Peter

    2008-10-01

    spFRET (single-pair fluorescence resonance energy transfer) with organic fluorophores has been used to demonstrate rotation of the subunits gamma and epsilon in membrane-integrated FoF1 during proton transport-coupled ATP synthesis. Owing to the high light intensities used in single-molecule spectroscopy, organic fluorophores show a high probability for photobleaching. Luminescent CdSe/ZnS nanocrystals with a hydrophilic shell have been covalently bound to FoF1 either to the stator subunit b or to the rotor subunit c. TIRFM (total internal reflection microscopy) shows that covalent binding of the QD (quantum dot) via cysteine to FoF1 leads to a significant decrease in the blinking probability in the microsecond-to-second time range. This effect allows the observation of subunit movements in an extended time range. If the QD is bound to the rotor subunit c, the fluorescence anisotropy shows fluctuations in the presence of ATP, in contrast with the constant anisotropy observed in the absence of ATP.

  15. On the charge transfer in the "single-sheet graphene-intercalated metal layer-SiC substrate" system

    NASA Astrophysics Data System (ADS)

    Davydov, S. Yu.

    2014-02-01

    The proposed scheme for the consideration of charge transfer in the three-layer Gr/Me/SiC system (where Gr is a single-sheet graphene, Me is an intercalated metal layer, and SiC is a substrate) contains three stages. At the first stage, a metal monolayer adsorbed on silicon carbide is considered and the charge of adatoms in this monolayer is calculated. At the second stage, the shift of the Dirac point of free-standing single-layer graphene in an electrostatic field induced by charged adatoms of the monolayer is estimated. At the third stage, a weak interaction between Me/SiC and free-standing graphene is included, which allows electrons to tunnel but does not significantly distort the density of states of free-standing graphene. Estimations are performed for n- and p-type 6 H-SiC(0001) substrates and Cu, Ag, and Au layers. The charge state of the graphene sheet and the shift of the Dirac point with respect to the Fermi level of the system are calculated. A comparison with the available experimental and theoretical results shows that the proposed scheme works quite satisfactorily.

  16. Experimental and theoretical studies of the He(2+)-He system - Differential cross sections for direct, single-, and double-charge-transfer scattering at keV energies

    NASA Technical Reports Server (NTRS)

    Gao, R. S.; Dutta, C. M.; Lane, N. F.; Smith, K. A.; Stebbings, R. F.; Kimura, M.

    1992-01-01

    Measurements and calculations of differential cross sections for direct scattering, single-charge transfer, and double-charge transfer in collisions of 1.5-, 2.0-, 6.0-, and 10.0-keV (He-3)2+ with an He-4 target are reported. The measurements cover laboratory scattering angles below 1.5 deg with an angular resolution of about 0.03 deg. A quantum-mechanical molecular-state representation is employed in the calculations; in the case of single-charge transfer a two-state close-coupling calculation is carried out taking into account electron-translation effects. The theoretical calculations agree well with the experimental results for direct scattering and double-charge transfer. The present calculation identifies the origins of oscillatory structures observed in the differential cross sections.

  17. 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

  18. 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.

  19. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  20. 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

  1. The impact of exposure to serum lipids during in vitro culture on the transcriptome of bovine blastocysts.

    PubMed

    Cagnone, Gael; Sirard, Marc-André

    2014-03-15

    In vitro culture has a detrimental impact on early embryonic development, and serum addition to IVC is recognized to compromise blastocyst quality. Particularly, serum fatty acids affect embryonic lipid composition and reduce cryopreservation survival. To understand the molecular pathways of serum-induced embryonic stress, this study examined the early development of bovine embryos produced in different protein- or lipid-supplemented culture media: BSA alone (control), BSA + serum lipid fraction (SELF), delipidated serum and total serum. These protein-lipid treatments were applied from the eight to 16 cell stages to the blastocyst stage. As planned, SELF treatment increased the fatty acid concentration in the medium compared with control medium but did not induce embryo toxicity. However, microarray comparison between blastocysts cultured in BSA without or with SELF revealed differential transcriptomic profile associated with ceramide-induced oxidative stress and inflammation. Moreover, the SELF treatment had a significant impact on genes involved in cholesterol metabolism (LDLR, HMGCS1), with the potential upstream control of the transcription factors SREBP and PPARA, two major regulators of cholesterol metabolism. In addition, the expression of pluripotence-related genes (APEX, CLDN6) was downregulated in blastocysts subjected to either SELF or total serum. Taken together, these results illustrate how the early embryonic transcriptome responds to increased lipid exposure through an inflammatory and metabolic signature.

  2. Histone modifications and mRNA expression in the inner cell mass and trophectoderm of bovine blastocysts.

    PubMed

    Herrmann, Doris; Dahl, John Arne; Lucas-Hahn, Andrea; Collas, Philippe; Niemann, Heiner

    2013-03-01

    Normal development depends on the precise sequence of changes in the configuration of chromatin; these are primarily related to specific biochemical modifications such as acetylation or methylation of histones and DNA methylation. While the role of DNA methylation during preimplantation development has been studied extensively, little is known about histone modifications related to early embryonic development. Here, we investigated gene-specific histone modifications in in vitro produced bovine blastocysts. Selected genes thought to be critical for bovine preimplantation development were examined and included POU5F1 (OCT4), NANOG, INFT, GAPDH, SLC2A3 and IGF1. We used chromatin immunoprecipitation from pools of bovine blastocysts to unravel several modifications of histone H3 in relation to mRNA expression profiles. We focused on the two cell compartments of the blastocyst, the inner cell mass (ICM) and the trophectoderm (TE). We show that gene expression patterns in the ICM and TE of the bovine blastocyst are consistent with histone modification patterns on the promoter of the corresponding genes. The data show a complex epigenetic pattern of promoter occupancy by transcriptionally permissive and repressive H3 modifications. These results pave the way to in-depth epigenetic studies of preimplantation embryos that are crucial to gain a better understanding of the epigenetic changes frequently observed after use of assisted reproductive technologies. PMID:23406883

  3. 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

  4. 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

  5. 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

  6. Contribution of oocyte source and culture conditions to phenotypic and transcriptomic variation in commercially produced bovine blastocysts.

    PubMed

    Plourde, Dany; Vigneault, Christian; Lemay, Alexandra; Breton, Lévéke; Gagné, Dominic; Laflamme, Isabelle; Blondin, Patrick; Robert, Claude

    2012-07-01

    Bovine embryo production is practiced worldwide for commercial purposes. A major concern of embryo suppliers is the impact of in vitro production systems on embryo quality. In the present study, we compared Buffalo Rat Liver cell coculture with semidefined, medium-based culture, oocytes recovered postmortem with those obtained from live animals, and in vitro with in vivo embryo development. Gene expression levels in expanded blastocysts were measured using microarray and quantitative RT-PCR. The systems were similar in terms of blastocyst yield and rate of development, whereas embryo productivity was greater for immature oocytes collected in vivo. Although immature oocytes collected in vivo had greater developmental competence, they yielded blastocysts that were indistinguishable (in terms of level of gene expression) from embryos derived from immature oocytes recovered postmortem. Culture conditions had a significant impact on gene expression, particularly among genes involved in lipid metabolism. Numerous uncharacterized novel transcript regions were also influenced by in vitro treatments. In conclusion, ovum pick-up combined with in vitro culture in semidefined medium provided a high blastocyst yield, without the deleterious effects associated with coculture.

  7. 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.

  8. Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies.

    PubMed

    An, Ran; Wang, Chunmin; Turek, John; Machaty, Zoltan; Nolte, David D

    2015-03-01

    The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates.

  9. Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies

    PubMed Central

    An, Ran; Wang, Chunmin; Turek, John; Machaty, Zoltan; Nolte, David D.

    2015-01-01

    The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates. PMID:25798318

  10. Molecular cytogenetic analysis of human blastocysts andcytotrophoblasts by multi-color FISH and Spectra Imaging analyses

    SciTech Connect

    Weier, Jingly F.; Ferlatte, Christy; Baumgartner, Adolf; Jung,Christine J.; Nguyen, Ha-Nam; Chu, Lisa W.; Pedersen, Roger A.; Fisher,Susan J.; Weier, Heinz-Ulrich G.

    2006-02-08

    Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing technologies to score all 24 chromosomes in single cells within a 3 day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found aneuploidy, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.

  11. Amino Acid Supplementation Affects Imprinted Gene Transcription Patterns in Parthenogenetic Porcine Blastocysts

    PubMed Central

    Park, Chi-Hun; Jeong, Young-Hee; Jeong, Yeun-Ik; Kwon, Jeong-Woo; Shin, Taeyoung; Hyun, Sang-Hwan; Jeung, Eui-Bae; Kim, Nam-Hyung; Seo, Sang-Kyo; Lee, Chang-Kyu; Hwang, Woo-Suk

    2014-01-01

    To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine

  12. Maternal obesity promotes a proinflammatory signature in rat uterus and blastocyst.

    PubMed

    Shankar, Kartik; Zhong, Ying; Kang, Ping; Lau, Franchesca; Blackburn, Michael L; Chen, Jin-Ran; Borengasser, Sarah J; Ronis, Martin J J; Badger, Thomas M

    2011-11-01

    Maternal obesity at conception increases the risk of offspring obesity, thus propagating an intergenerational vicious cycle. Male offspring born to obese dams are hyperresponsive to high fat-diets, gaining greater body weight, fat mass, and additional metabolic sequelae compared to lean controls. In this report, we identify the impact of maternal obesity before conception, on the embryo, and intrauterine milieu during the periimplantation period. We conducted global transcriptomic profiling in the uterus and periimplantation blastocyst, gene/protein expression analyses of inflammatory pathways in conjunction with endocrine and metabolic characterization in the dams at implantation. Uterine gene expression profiles of lean and obese dams revealed distinct signatures for genes regulating inflammation and lipid metabolism. Both pathway and gene-set enrichment analysis revealed uterine nuclear factor-κB and c-Jun N-terminal kinase signaling to be up-regulated in the uterus of obese dams, which was confirmed via immunoblotting. Obese uteri also evidenced an inflammatory secretome with higher chemokine mRNA abundance (CCL2, CCL5, CCL7, and CxCL10) and related regulators (TLR2, CD14, and Ccr1). Increased inflammation in the uterus was associated with ectopic lipid accumulation and expression of lipid metabolic genes. Gene expression in sex-identified male periimplantation blastocyst at day postcoitum 4.5 was clearly influenced by maternal obesity (359 transcripts, ±1.4-fold), including changes in developmental and epigenetic regulators. Akin to the uterus, nuclear factor-κB-regulated proinflammatory genes (CCL4 and CCL5) increased and expression of antioxidant (GPx3) and mitochondrial (TFAM and NRF1) genes decreased in the obese embryos. Our results suggest that ectopic lipid and inflammation may link maternal obesity to increased predisposition of offspring to obesity later in life.

  13. Expression of E-Cadherin, Leukemia Inhibitory Factor and Progesterone Receptor in Mouse Blastocysts after Ovarian Stimulation

    PubMed Central

    Movaghar, Bahar; Askarian, Saeedeh

    2012-01-01

    Objective: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. Materials and Methods: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student’s t-test (one sample t-test). Results: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). Conclusion: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from

  14. Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor.

    PubMed

    Wimmer, T; Lorenz, B; Stieger, K

    2016-12-15

    Neovascular pathologies in the eye like age-related macular degeneration (AMD), the diabetic retinopathie (DR), retinopathie of prematurity (ROP) or the retinal vein occlusion (RVO) are caused through a hypoxia induced upregulation of the vascular endothelial growth factor (VEGF). So far a correlation of intraocular VEGF concentrations to the impact of the pathologies is limited because of invasive sampling. Therefore, a minimally invasive, repeatable quantification of VEGF levels in the eye is needed to correlate the stage of VEGF induced pathologies as well as the efficacy of anti-VEGF treatment. Here we describe the development of three variants of enhanced BRET2 (eBRET2) based, single molecule biosensors by fusing a Renilla luciferase mutant with enhanced light output (RLuc8) to the N-terminus and a suitable eBRET2 acceptor fluorophore (GFP2) to the C-terminus of a VEGF binding domain, directly fused or separated with two different peptide linkers for the quantification of VEGF in vitro. The VEGF binding domain consists of a single chain variable fragment (scFv) based on ranibizumab in which the light- and the heavy- F(ab) chains were connected with a peptide linker to generate one open reading frame (orf). All three variants generate measureable eBRET2 ratios by transferring energy from the luciferase donor to the GFP2 acceptor, whereas only the directly fused and the proline variant permit VEGF quantification. The directly fused biosensor variant allows the quantification of VEGF with higher sensitivity, compared to the widely used ELISA systems and a wide dynamic quantification range in vitro. Our system demonstrates not only an additional in vitro application on VEGF quantification but also a promising step towards an applicable biosensor in an implantable device able to quantify VEGF reliably after implantation in vivo. PMID:27459244

  15. Quantification of the vascular endothelial growth factor with a bioluminescence resonance energy transfer (BRET) based single molecule biosensor.

    PubMed

    Wimmer, T; Lorenz, B; Stieger, K

    2016-12-15

    Neovascular pathologies in the eye like age-related macular degeneration (AMD), the diabetic retinopathie (DR), retinopathie of prematurity (ROP) or the retinal vein occlusion (RVO) are caused through a hypoxia induced upregulation of the vascular endothelial growth factor (VEGF). So far a correlation of intraocular VEGF concentrations to the impact of the pathologies is limited because of invasive sampling. Therefore, a minimally invasive, repeatable quantification of VEGF levels in the eye is needed to correlate the stage of VEGF induced pathologies as well as the efficacy of anti-VEGF treatment. Here we describe the development of three variants of enhanced BRET2 (eBRET2) based, single molecule biosensors by fusing a Renilla luciferase mutant with enhanced light output (RLuc8) to the N-terminus and a suitable eBRET2 acceptor fluorophore (GFP2) to the C-terminus of a VEGF binding domain, directly fused or separated with two different peptide linkers for the quantification of VEGF in vitro. The VEGF binding domain consists of a single chain variable fragment (scFv) based on ranibizumab in which the light- and the heavy- F(ab) chains were connected with a peptide linker to generate one open reading frame (orf). All three variants generate measureable eBRET2 ratios by transferring energy from the luciferase donor to the GFP2 acceptor, whereas only the directly fused and the proline variant permit VEGF quantification. The directly fused biosensor variant allows the quantification of VEGF with higher sensitivity, compared to the widely used ELISA systems and a wide dynamic quantification range in vitro. Our system demonstrates not only an additional in vitro application on VEGF quantification but also a promising step towards an applicable biosensor in an implantable device able to quantify VEGF reliably after implantation in vivo.

  16. Description of electron transfer in the ground and excited states of organic donor–acceptor systems by single-reference and multi-reference density functional methods

    SciTech Connect

    Filatov, Michael

    2014-09-28

    Electron transfer in the ground and excited states of a model donor–acceptor (D–A) system is investigated using the single-reference and multi-reference density functional theory (DFT) methods. To analyze the results of the calculations, a simple two-site multi-reference model was derived that predicts a stepwise electron transfer in the S{sub 0} state and a wave-like dependence of the S{sub 1} electron transfer on the external stimulus. The standard single-reference Kohn-Sham (KS) DFT approach and the time-dependent DFT (TDDFT) method failed to describe the correct dependence of the S{sub 0} and S{sub 1} electron transfer on the external electric field applied along the donor–acceptor system. The multi-reference DFT approach, the spin-restricted ensemble-referenced KS (REKS) method, was able to successfully reproduce the correct behavior of the S{sub 0} and S{sub 1} electron transfer on the applied field. The REKS method was benchmarked against experimentally measured gas phase charge transfer excitations in a series of organic donor–acceptor complexes and displayed its ability to describe this type of electronic transitions with a very high accuracy, mean absolute error of 0.05 eV with the use of the standard range separated density functionals. On the basis of the calculations undertaken in this work, it is suggested that the non-adiabatic coupling between the S{sub 0} and S{sub 1} states may interfere with the electron transfer in a weakly coupled donor–acceptor system. It is also suggested that the electronic excitation of a D{sup +}–A{sup −} system may play a dual role by assisting the further electron transfer at certain magnitudes of the applied electric field and causing the backward transfer at lower electric field strengths.

  17. The performance of single- and multi-proxy transfer functions (testate amoebae, bryophytes, vascular plants) for reconstructing mire surface wetness and pH

    NASA Astrophysics Data System (ADS)

    Mitchell, Edward A. D.; Payne, Richard J.; van der Knaap, Willem O.; Lamentowicz, Łukasz; Gąbka, Maciej; Lamentowicz, Mariusz

    2013-01-01

    Peatlands are widely exploited archives of paleoenvironmental change. We developed and compared multiple transfer functions to infer peatland depth to the water table (DWT) and pH based on testate amoeba (percentages, or presence/absence), bryophyte presence/absence, and vascular plant presence/absence data from sub-alpine peatlands in the SE Swiss Alps in order to 1) compare the performance of single-proxy vs. multi-proxy models and 2) assess the performance of presence/absence models. Bootstrapping cross-validation showing the best performing single-proxy transfer functions for both DWT and pH were those based on bryophytes. The best performing transfer functions overall for DWT were those based on combined testate amoebae percentages, bryophytes and vascular plants; and, for pH, those based on testate amoebae and bryophytes. The comparison of DWT and pH inferred from testate amoeba percentages and presence/absence data showed similar general patterns but differences in the magnitude and timing of some shifts. These results show new directions for paleoenvironmental research, 1) suggesting that it is possible to build good-performing transfer functions using presence/absence data, although with some loss of accuracy, and 2) supporting the idea that multi-proxy inference models may improve paleoecological reconstruction. The performance of multi-proxy and single-proxy transfer functions should be further compared in paleoecological data.

  18. Robust and Efficient Population Transfer in Ultracold Rubidium Using A Single Linearly Chirped Laser Pulse With a Novel Pulse Envelope

    NASA Astrophysics Data System (ADS)

    Collins, Thomas; Malinovskaya, Svetlana

    2012-06-01

    The ability to manipulate the state of a quantum system is the at very heart of the field of quantum control. As quantum control is an essential aspect of the emerging field of quantum computing, it is necessary to find techniques for manipulating quantum systems that are both robust and efficient to implement industrially. In this work the population dynamics of the valence electron of Rubidium, interacting with a single linearly chirped laser pulse, are studied. The pulse envelope is constructed from overlapping Gaussian waveforms and is described analytically by the formula: E0∑β=-n^nExp-[t-(T-n*ɛ)]^22τ0^2 with the parameter ɛ being the separation in time between each peak with the oscillating electric field is phase locked to the central peak. The response of the quantum yield obtained at the end of the pulse to changes in the parameters of the oscillating electric field and pulse envelope are studied. For certain values of these parameters, achievement of a transfer of over 99% of the population to a desired quantum state within the hyperfine structure of the 5S shell via adiabatic passage using beam intensities which are on the order of 100W/cm^2 is demonstrated. Results are robust in the adiabatic regime.

  19. Charge transfer of single laser crystallized intrinsic and phosphorus-doped Si-nanocrystals visualized by Kelvin probe force microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Jie; Xu, Jun; Lu, Peng; Shan, Dan; Li, Wei; Chen, Kunji

    2014-10-01

    Isolated intrinsic and phosphorus doped (P-doped) Si-nanocrystals (Si-NCs) on n- and p-Si substrates are fabricated by excimer laser crystallization techniques. The formation of Si-NCs is confirmed by atomic force microscopy (AFM) and conductive AFM measurements. Kelvin probe force microscopy (KPFM) is then carried out to visualize the trapped charges in a single Si-NC dot which derives from the charge transfer between Si-NCs and Si substrates due to their different Fermi levels. The laser crystallized P-doped Si-NCs have a similar Fermi level around the mid-gap to the intrinsic counterparts, which might be caused by the inactivated impurity atoms or the surface states-related Fermi level pinning. A clear rise of the Fermi level in P-doped Si-NCs is observed after a short time thermal annealing treatment, indicating the activation of dopants in Si-NCs. Moreover, the surface charge quantity can be estimated using a simple parallel plate capacitor model for a quantitative understanding of the KPFM results at the nanoscale.

  20. Ce(3+) /Tb(3+) non-/single-/co-doped K-Lu-F materials: synthesis, optical properties, and energy transfer.

    PubMed

    Cao, Chunyan; Xie, An; Noh, Hyeon Mi; Jeong, Jung Hyun

    2016-08-01

    Using a hydrothermal method, Ce(3+) /Tb(3+) non-/single-/co-doped K-Lu-F materials have been synthesized. The X-ray diffraction (XRD) results suggest that the Ce(3+) and/or Tb(3+) doping had great effects on the crystalline phases of the final samples. The field emission scanning electron microscopy (FE-SEM) images indicated that the samples were in hexagonal disk or polyhedron morphologies in addition to some nanoparticles, which also indicated that the doping also had great effects on the sizes and the morphologies of the samples. The energy-dispersive spectroscopy (EDS) patterns illustrated the constituents of different samples. The enhanced emissions of Tb(3+) were observed in the Ce(3+) /Tb(3+) co-doped K-Lu-F materials. The energy transfer (ET) efficiency ηT were calculated based on the fluorescence yield. The ET mechanism from Ce(3+) to Tb(3+) was confirmed to be the dipole-quadrupole interaction inferred from the theoretical analysis and the experimental data. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Expression of the Alpha Tocopherol Transfer Protein gene is regulated by Oxidative Stress and Common Single Nucleotide Polymorphisms

    PubMed Central

    Ulatowski, Lynn; Dreussi, Cara; Noy, Noa; Barnholtz-Sloan, Jill; Klein, Eric; Manor, Danny

    2012-01-01

    Vitamin E (α-tocopherol) is the major lipid soluble antioxidant in most animal species. By controlling the secretion of vitamin E from the liver, the α-tocopherol transfer protein (αTTP) regulates whole-body distribution and levels of this vital nutrient. However, the mechanism(s) that regulate the expression of this protein are poorly understood. Here we report that transcription of the TTPA gene in immortalized human hepatocytes (IHH) is induced by oxidative stress and by hypoxia, by agonists of the nuclear receptors PPARα and RXR, and by increased cAMP levels. The data show further that induction of TTPA transcription by oxidative stress is mediated by an already-present transcription factor, and does not require de novo protein synthesis. Silencing of the cAMP response element binding (CREB) transcription factor attenuated transcriptional responses of the TTPA gene to added peroxide, suggesting that CREB mediates responses of this gene to oxidative stress. Using a 1.9 Kb proximal segment of the human TTPA promoter together with site-directed mutagenesis approach, we found that single nucleotide polymorphisms (SNPs) that are commonly found in healthy humans dramatically affect promoter activity. These observations suggest that oxidative stress and individual genetic makeup contribute to vitamin E homeostasis in humans. These findings may explain the variable responses to vitamin E supplementation observed in human clinical trials. PMID:23079030

  2. Electron-transfer sensitization of H2 oxidation and CO2 reduction catalysts using a single chromophore.

    PubMed

    La Porte, Nathan T; Moravec, Davis B; Hopkins, Michael D

    2014-07-01

    Energy-storing artificial-photosynthetic systems for CO2 reduction must derive the reducing equivalents from a renewable source rather than from sacrificial donors. To this end, a homogeneous, integrated chromophore/two-catalyst system is described that is thermodynamically capable of photochemically driving the energy-storing reverse water-gas shift reaction (CO2 + H2 → CO + H2O), where the reducing equivalents are provided by renewable H2. The system consists of the chromophore zinc tetraphenylporphyrin (ZnTPP), H2 oxidation catalysts of the form [Cp(R)Cr(CO)3](-), and CO2 reduction catalysts of the type Re(bpy-4,4'-R2)(CO)3Cl. Using time-resolved spectroscopic methods, a comprehensive mechanistic and kinetic picture of the photoinitiated reactions of mixtures of these compounds has been developed. It has been found that absorption of a single photon by broadly absorbing ZnTPP sensitizes intercatalyst electron transfer to produce the substrate-active forms of each. The initial photochemical step is the heretofore unobserved reductive quenching of the low-energy T1 state of ZnTPP. Under the experimental conditions, the catalytically competent state decays with a second-order half-life of ∼15 μs, which is of the right magnitude for substrate trapping of sensitized catalyst intermediates.

  3. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.

    PubMed

    Imamura, Hiromi; Nhat, Kim P Huynh; Togawa, Hiroko; Saito, Kenta; Iino, Ryota; Kato-Yamada, Yasuyuki; Nagai, Takeharu; Noji, Hiroyuki

    2009-09-15

    Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 muM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of F(o)F(1)-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

  4. Electron-transfer sensitization of H2 oxidation and CO2 reduction catalysts using a single chromophore

    PubMed Central

    La Porte, Nathan T.; Moravec, Davis B.; Hopkins, Michael D.

    2014-01-01

    Energy-storing artificial-photosynthetic systems for CO2 reduction must derive the reducing equivalents from a renewable source rather than from sacrificial donors. To this end, a homogeneous, integrated chromophore/two-catalyst system is described that is thermodynamically capable of photochemically driving the energy-storing reverse water–gas shift reaction (CO2 + H2 → CO + H2O), where the reducing equivalents are provided by renewable H2. The system consists of the chromophore zinc tetraphenylporphyrin (ZnTPP), H2 oxidation catalysts of the form [CpRCr(CO)3]–, and CO2 reduction catalysts of the type Re(bpy-4,4′-R2)(CO)3Cl. Using time-resolved spectroscopic methods, a comprehensive mechanistic and kinetic picture of the photoinitiated reactions of mixtures of these compounds has been developed. It has been found that absorption of a single photon by broadly absorbing ZnTPP sensitizes intercatalyst electron transfer to produce the substrate-active forms of each. The initial photochemical step is the heretofore unobserved reductive quenching of the low-energy T1 state of ZnTPP. Under the experimental conditions, the catalytically competent state decays with a second-order half-life of ∼15 μs, which is of the right magnitude for substrate trapping of sensitized catalyst intermediates. PMID:24961370

  5. Self-Assembled Colloidal Particle Clusters from In Situ Pickering-Like Emulsion Polymerization via Single Electron Transfer Mechanism.

    PubMed

    Yuan, Jinfeng; Zhao, Weiting; Pan, Mingwang; Zhu, Lei

    2016-08-01

    A simple route is reported to synthesize colloidal particle clusters (CPCs) from self-assembly of in situ poly(vinylidene fluoride)/poly(styrene-co-tert-butyl acrylate) [PVDF/P(St-co-tBA)] Janus particles through one-pot seeded emulsion single electron transfer radical polymerization. In the in situ Pickering-like emulsion polymerization, the tBA/St/PVDF feed ratio and polymerization temperature are important for the formation of well-defined CPCs. When the tBA/St/PVDF feed ratio is 0.75 g/2.5 g/0.5 g and the reaction temperature is 35 °C, relatively uniform raspberry-like CPCs are obtained. The hydrophobicity of the P(St-co-tBA) domains and the affinity of PVDF to the aqueous environment are considered to be the driving force for the self-assembly of the in situ formed PVDF/P(St-co-tBA) Janus particles. The resultant raspberry-like CPCs with PVDF particles protruding outward may be promising for superhydrophobic smart coatings. PMID:27226331

  6. Nonspherical nanoparticles with controlled morphologies via seeded surface-initiated single electron transfer radical polymerization in soap-free emulsion.

    PubMed

    Yuan, Jinfeng; Wang, Lixia; Zhu, Lei; Pan, Mingwang; Wang, Wenjie; Liu, Ying; Liu, Gang

    2015-04-14

    This work reports a facile novel approach to prepare asymmetric poly(vinylidene fluoride)/polystyrene (PVDF/PS) composite latex particles with controllable morphologies using one-step soap-free seeded emulsion polymerization, i.e., surface-initiated single electron transfer radical polymerization (SET-RP) of styrene (St) at the surface of PVDF seed particles. It was observed that the morphology was influenced mainly by the St/PVDF feed ratio, the polymerization temperature, and the length of the catalyst Cu(0) wire (Φ 1.00 mm). When the feed ratio was St/PVDF = 5.0 g/1.0 g, snowman-like Janus particles were exclusively obtained. Raspberry-like and popcorn-like composite particles were observed at a higher reaction temperature or a shorter length of the catalyst wire. The reaction kinetics plots demonstrated some unique features. The formation of nonspherical composite nanoparticles can be ascribed to the surface nucleation of PS bulges following the SET-RP. PMID:25797695

  7. Charge transfer of single laser crystallized intrinsic and phosphorus-doped Si-nanocrystals visualized by Kelvin probe force microscopy

    SciTech Connect

    Xu, Jie; Xu, Jun Lu, Peng; Shan, Dan; Li, Wei; Chen, Kunji

    2014-10-07

    Isolated intrinsic and phosphorus doped (P-doped) Si-nanocrystals (Si-NCs) on n- and p-Si substrates are fabricated by excimer laser crystallization techniques. The formation of Si-NCs is confirmed by atomic force microscopy (AFM) and conductive AFM measurements. Kelvin probe force microscopy (KPFM) is then carried out to visualize the trapped charges in a single Si-NC dot which derives from the charge transfer between Si-NCs and Si substrates due to their different Fermi levels. The laser crystallized P-doped Si-NCs have a similar Fermi level around the mid-gap to the intrinsic counterparts, which might be caused by the inactivated impurity atoms or the surface states-related Fermi level pinning. A clear rise of the Fermi level in P-doped Si-NCs is observed after a short time thermal annealing treatment, indicating the activation of dopants in Si-NCs. Moreover, the surface charge quantity can be estimated using a simple parallel plate capacitor model for a quantitative understanding of the KPFM results at the nanoscale.

  8. Ouabain Stimulates a Na+/K+-ATPase-Mediated SFK-Activated Signalling Pathway That Regulates Tight Junction Function in the Mouse Blastocyst

    PubMed Central

    Giannatselis, Holly; Calder, Michele; Watson, Andrew J.

    2011-01-01

    The Na+/K+-ATPase plays a pivotal role during preimplantation development; it establishes a trans-epithelial ionic gradient that facilitates the formation of the fluid-filled blastocyst cavity, crucial for implantation and successful pregnancy. The Na+/K+-ATPase is also implicated in regulating tight junctions and cardiotonic steroid (CTS)-induced signal transduction via SRC. We investigated the expression of SRC family kinase (SFK) members, Src and Yes, during preimplantation development and determined whether SFK activity is required for blastocyst formation. Embryos were collected following super-ovulation of CD1 or MF1 female mice. RT-PCR was used to detect SFK mRNAs encoding Src and Yes throughout preimplantation development. SRC and YES protein were localized throughout preimplantation development. Treatment of mouse morulae with the SFK inhibitors PP2 and SU6656 for 18 hours resulted in a reversible blockade of progression to the blastocyst stage. Blastocysts treated with 10−3 M ouabain for 2 or 10 minutes and immediately immunostained for phosphorylation at SRC tyr418 displayed reduced phosphorylation while in contrast blastocysts treated with 10−4 M displayed increased tyr418 fluorescence. SFK inhibition increased and SFK activation reduced trophectoderm tight junction permeability in blastocysts. The results demonstrate that SFKs are expressed during preimplantation development and that SFK activity is required for blastocyst formation and is an important mediator of trophectoderm tight junction permeability. PMID:21901128

  9. Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification.

    PubMed

    Slaughter, Brian D; Unruh, Jay R; Allen, Michael W; Bieber Urbauer, Ramona J; Johnson, Carey K

    2005-03-15

    A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.

  10. Energy transfer between injection-locked single-mode diode lasers by two-beam coupling in BaTiO/sub 3/

    SciTech Connect

    Christian, W.R.; Beckwith, P.H.; McMichael, I.

    1989-01-01

    Two-beam coupling in photorefractive BaTiO/sub 3/ is used to combine beams coherently from two injection-locked single-mode diode lasers operating at 830 nm. We are able to transfer more than 70% of the power in one beam to the other beam with this method.

  11. A high-yield two-step transfer printing method for large-scale fabrication of organic single-crystal devices on arbitrary substrates.

    PubMed

    Deng, Wei; Zhang, Xiujuan; Pan, Huanhuan; Shang, Qixun; Wang, Jincheng; Zhang, Xiaohong; Zhang, Xiwei; Jie, Jiansheng

    2014-01-01

    Single-crystal organic nanostructures show promising applications in flexible and stretchable electronics, while their applications are impeded by the large incompatibility with the well-developed photolithography techniques. Here we report a novel two-step transfer printing (TTP) method for the construction of organic nanowires (NWs) based devices onto arbitrary substrates. Copper phthalocyanine (CuPc) NWs are first transfer-printed from the growth substrate to the desired receiver substrate by contact-printing (CP) method, and then electrode arrays are transfer-printed onto the resulting receiver substrate by etching-assisted transfer printing (ETP) method. By utilizing a thin copper (Cu) layer as sacrificial layer, microelectrodes fabricated on it via photolithography could be readily transferred to diverse conventional or non-conventional substrates that are not easily accessible before with a high transfer yield of near 100%. The ETP method also exhibits an extremely high flexibility; various electrodes such as Au, Ti, and Al etc. can be transferred, and almost all types of organic devices, such as resistors, Schottky diodes, and field-effect transistors (FETs), can be constructed on planar or complex curvilinear substrates. Significantly, these devices can function properly and exhibit closed or even superior performance than the device counterparts fabricated by conventional approach. PMID:24942458

  12. A High-yield Two-step Transfer Printing Method for Large-scale Fabrication of Organic Single-crystal Devices on Arbitrary Substrates

    PubMed Central

    Deng, Wei; Zhang, Xiujuan; Pan, Huanhuan; Shang, Qixun; Wang, Jincheng; Zhang, Xiaohong; Zhang, Xiwei; Jie, Jiansheng

    2014-01-01

    Single-crystal organic nanostructures show promising applications in flexible and stretchable electronics, while their applications are impeded by the large incompatibility with the well-developed photolithography techniques. Here we report a novel two-step transfer printing (TTP) method for the construction of organic nanowires (NWs) based devices onto arbitrary substrates. Copper phthalocyanine (CuPc) NWs are first transfer-printed from the growth substrate to the desired receiver substrate by contact-printing (CP) method, and then electrode arrays are transfer-printed onto the resulting receiver substrate by etching-assisted transfer printing (ETP) method. By utilizing a thin copper (Cu) layer as sacrificial layer, microelectrodes fabricated on it via photolithography could be readily transferred to diverse conventional or non-conventional substrates that are not easily accessible before with a high transfer yield of near 100%. The ETP method also exhibits an extremely high flexibility; various electrodes such as Au, Ti, and Al etc. can be transferred, and almost all types of organic devices, such as resistors, Schottky diodes, and field-effect transistors (FETs), can be constructed on planar or complex curvilinear substrates. Significantly, these devices can function properly and exhibit closed or even superior performance than the device counterparts fabricated by conventional approach. PMID:24942458

  13. ATOMIC AND MOLECULAR PHYSICS: Single atoms transferring between a magneto-optical trap and a far-off-resonance optical dipole trap

    NASA Astrophysics Data System (ADS)

    He, Jun; Wang, Jing; Yang, Bao-Dong; Zhang, Tian-Cai; Wang, Jun-Min

    2009-08-01

    Based on our work on single cesium atoms trapped in a large-magnetic-gradient vapour-cell magneto-optical trap (MOT), the signal-to-noise ratio (SNR) is remarkably improved. Also a far-off-resonance optical dipole trap (FORT) formed by a strongly-focused 1064 nm single frequency Nd:YVO4 laser beam is introduced. One cesium atom is prepared in the MOT, and then it can transfer successfully between the MOT and the FORT which is overlapped with the MOT. Utilizing the effective transfer, the lifetime of single atoms trapped in the FORT is measured to be 6.9 ± 0.3 s. Thus we provide a system where the atomic qubit can be coherently manipulated.

  14. Modelling and optimization of transient processes in line focusing power plants with single-phase heat transfer medium

    NASA Astrophysics Data System (ADS)

    Noureldin, K.; González-Escalada, L. M.; Hirsch, T.; Nouri, B.; Pitz-Paal, R.

    2016-05-01

    A large number of commercial and research line focusing solar power plants are in operation and under development. Such plants include parabolic trough collectors (PTC) or linear Fresnel using thermal oil or molten salt as the heat transfer medium (HTM). However, the continuously varying and dynamic solar condition represent a big challenge for the plant control in order to optimize its power production and to keep the operation safe. A better understanding of the behaviour of such power plants under transient conditions will help reduce defocusing instances, improve field control, and hence, increase the energy yield and confidence in this new technology. Computational methods are very powerful and cost-effective tools to gain such understanding. However, most simulation models described in literature assume equal mass flow distributions among the parallel loops in the field or totally decouple the flow and thermal conditions. In this paper, a new numerical model to simulate a whole solar field with single-phase HTM is described. The proposed model consists of a hydraulic part and a thermal part that are coupled to account for the effect of the thermal condition of the field on the flow distribution among the parallel loops. The model is specifically designed for large line-focusing solar fields offering a high degree of flexibility in terms of layout, condition of the mirrors, and spatially resolved DNI data. Moreover, the model results have been compared to other simulation tools, as well as experimental and plant data, and the results show very good agreement. The model can provide more precise data to the control algorithms to improve the plant control. In addition, short-term and accurate spatially discretized DNI forecasts can be used as input to predict the field behaviour in-advance. In this paper, the hydraulic and thermal parts, as well as the coupling procedure, are described and some validation results and results of simulating an example field are

  15. Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer.

    PubMed

    Sun, Yueying; Lu, Xiaohui; Su, Fengxia; Wang, Limei; Liu, Chenghui; Duan, Xinrui; Li, Zhengping

    2015-12-15

    Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis.

  16. Heat-transfer resistance at solid-liquid interfaces: a tool for the detection of single-nucleotide polymorphisms in DNA.

    PubMed

    van Grinsven, Bart; Vanden Bon, Natalie; Strauven, Hannelore; Grieten, Lars; Murib, Mohammed; Monroy, Kathia L Jiménez; Janssens, Stoffel D; Haenen, Ken; Schöning, Michael J; Vermeeren, Veronique; Ameloot, Marcel; Michiels, Luc; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2012-03-27

    In this article, we report on the heat-transfer resistance at interfaces as a novel, denaturation-based method to detect single-nucleotide polymorphisms in DNA. We observed that a molecular brush of double-stranded DNA grafted onto synthetic diamond surfaces does not notably affect the heat-transfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of single-stranded DNA cause, surprisingly, a substantially higher heat-transfer resistance and behave like a thermally insulating layer. This effect can be utilized to identify ds-DNA melting temperatures via the switching from low- to high heat-transfer resistance. The melting temperatures identified with this method for different DNA duplexes (29 base pairs without and with built-in mutations) correlate nicely with data calculated by modeling. The method is fast, label-free (without the need for fluorescent or radioactive markers), allows for repetitive measurements, and can also be extended toward array formats. Reference measurements by confocal fluorescence microscopy and impedance spectroscopy confirm that the switching of heat-transfer resistance upon denaturation is indeed related to the thermal on-chip denaturation of DNA.

  17. [Frequency ratio of two forms of amitotic division of trophoblast cell nuclei in the mink blastocysts during the period of delayed implantation].

    PubMed

    Isakova, G K; Shilova, I E

    2003-01-01

    A comparative study of amitotic division activity of trophoblast cells by constriction and by extrusion in blastocysts of American mink during the obligatory period of delayed implantation has been carried out. The frequency of occurrence of amitotic figures was found to be nearly 10% at the onset of renewal of blastocyst growth (the blastocyst size was 0.4 mm in diameter), and nearly 20% at the stage of active growth (0.9 mm), as well as at the stage of expansion prior to blastocyst attachment to the uterine wall (1.7 mm). The ratios between the frequencies of division by extrusion and by constriction were 2:1, 5:1, and 4:1 at the three stages, respectively. We suggest that the cells generated via different amitotic ways play different roles in trophoblast differentiation. PMID:12942744

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. Accumulative charge separation for solar fuels production: coupling light-induced single electron transfer to multielectron catalysis.

    PubMed

    Hammarström, Leif

    2015-03-17

    The conversion and storage of solar energy into a fuel holds promise to provide a significant part of the future renewable energy demand of our societies. Solar energy technologies today generate heat or electricity, while the large majority of our energy is used in the form of fuels. Direct conversion of solar energy to a fuel would satisfy our needs for storable energy on a large scale. Solar fuels can be generated by absorbing light and converting its energy to chemical energy by electron transfer leading to separation of electrons and holes. The electrons are used in the catalytic reduction of a cheap substrate with low energy content into a high-energy fuel. The holes are filled by oxidation of water, which is the only electron source available for large scale solar fuel production. Absorption of a single photon typically leads to separation of a single electron-hole pair. In contrast, fuel production and water oxidation are multielectron, multiproton reactions. Therefore, a system for direct solar fuel production must be able to accumulate the electrons and holes provided by the sequential absorption of several photons in order to complete the catalytic reactions. In this Account, the process is termed accumulative charge separation. This is considerably more complicated than charge separation on a single electron level and needs particular attention. Semiconductor materials and molecular dyes have for a long time been optimized for use in photovoltaic devices. Efforts are made to develop new systems for light harvesting and charge separation that are better optimized for solar fuel production than those used in the early devices presented so far. Significant progress has recently been made in the discovery and design of better homogeneous and heterogeneous catalysts for solar fuels and water oxidation. While the heterogeneous ones perform better today, molecular catalysts based on transition metal complexes offer much greater tunability of electronic and

  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. Meaning and Measurability of Single-Ion Activities, the Thermodynamic Foundations of pH, and the Gibbs Free Energy for the Transfer of Ions between Dissimilar Materials

    PubMed Central

    Rockwood, Alan L

    2015-01-01

    Considering the relationship between concentration and vapor pressure (or the relationship between concentration and fugacity) single-ion activity coefficients are definable in purely thermodynamic terms. The measurement process involves measuring a contact potential between a solution and an external electrode. Contact potentials are measurable by using thermodynamically reversible processes. Extrapolation of an equation to zero concentration and ionic strength enables determination of single-ion activity coefficients. Single-ion activities can be defined and measured without using any extra-thermodynamic assumptions, concepts, or measurements. This method could serve as a gold standard for the validation of extra-thermodynamic methods for determining single-ion activities. Furthermore, it places the concept of pH on a thermodynamically solid foundation. Contact potential measurements can also be used to determine the Gibbs free energy for the transfer of ions between dissimilar materials. PMID:25919971

  5. Meaning and Measurability of Single-Ion Activities, the Thermodynamic Foundations of pH, and the Gibbs Free Energy for the Transfer of Ions between Dissimilar Materials.

    PubMed

    Rockwood, Alan L

    2015-06-22

    Considering the relationship between concentration and vapor pressure (or the relationship between concentration and fugacity) single-ion activity coefficients are definable in purely thermodynamic terms. The measurement process involves measuring a contact potential between a solution and an external electrode. Contact potentials are measurable by using thermodynamically reversible processes. Extrapolation of an equation to zero concentration and ionic strength enables determination of single-ion activity coefficients. Single-ion activities can be defined and measured without using any extra-thermodynamic assumptions, concepts, or measurements. This method could serve as a gold standard for the validation of extra-thermodynamic methods for determining single-ion activities. Furthermore, it places the concept of pH on a thermodynamically solid foundation. Contact potential measurements can also be used to determine the Gibbs free energy for the transfer of ions between dissimilar materials. PMID:25919971

  6. Enhancing the quality of transferred single-layer graphene with poly(4-vinylphenol) interlayer on flexible substrates

    NASA Astrophysics Data System (ADS)

    Choi, Yeonhoi; Chun, Sungwoo; Hong, Ahyoung; Ha, Chunho; Park, Wanjun

    2016-06-01

    We report the use of poly(4-vinylphenol) (PVP) as a promising contact surface of transferred graphene, capable of sustaining the original performance found in as-grown graphene. Enhancement of surface tension obtained by O2 plasma treatment of the PVP surface also increases transferred graphene quality. With an easy coating method, PVP can be applied to any flexible substrate as the interlayer to increase transferred graphene quality. Owing to the mechanical flexibility and chemical inertness of PVP, the introduction of a PVP interlayer provides a general method for graphene soft electronics to be integrated into any flexible substrate.

  7. Microchip electrophoresis-single wall carbon nanotube press-transferred electrodes for fast and reliable electrochemical sensing of melatonin and its precursors.

    PubMed

    Gomez, Federico José Vicente; Martín, Aída; Silva, María Fernanda; Escarpa, Alberto

    2015-08-01

    In the current work, single-wall carbon nanotube press-transferred electrodes (SW-PTEs) were used for detection of melatonin (MT) and its precursors tryptophan (Trp) and serotonin (5-HT) on microchip electrophoresis (ME). SW-PTEs were simply fabricated by press transferring a filtered dispersion of single-wall carbon nanotubes on a nonconductive PMMA substrate, where single-wall carbon nanotubes act as exclusive transducers. The coupling of ME-SW-PTEs allowed the fast detection of MT, Trp, and 5-HT in less than 150 s with excellent analytical features. It exhibited an impressive antifouling performance with RSD values of ≤2 and ≤4% for migration times and peak heights, respectively (n = 12). In addition, sample analysis was also investigated by analysis of 5-HT, MT, and Trp in commercial samples obtaining excellent quantitative and reproducible recoveries with values of 96.2 ± 1.8%, 101.3 ± 0.2%, and 95.6 ± 1.2% for 5-HT, MT, and Trp, respectively. The current novel application reveals the analytical power of the press-transfer technology where the fast and reliable determination of MT and its precursors were performed directly on the nanoscale carbon nanotube detectors without the help of any other electrochemical transducer.

  8. Single-molecule conductance of a chemically modified, π-extended tetrathiafulvalene and its charge-transfer complex with F4TCNQ

    PubMed Central

    García, Raúl; Herranz, M Ángeles; González, M Teresa; Bollinger, Gabino Rubio; Bürkle, Marius; Zotti, Linda A; Asai, Yoshihiro; Pauly, Fabian; Cuevas, Juan Carlos; Agraït, Nicolás

    2015-01-01

    Summary We describe the synthesis and single-molecule electrical transport properties of a molecular wire containing a π-extended tetrathiafulvalene (exTTF) group and its charge-transfer complex with F4TCNQ. We form single-molecule junctions using the in situ break junction technique using a homebuilt scanning tunneling microscope with a range of conductance between 10 G0 down to 10−7 G0. Within this range we do not observe a clear conductance signature of the neutral parent molecule, suggesting either that its conductance is too low or that it does not form a stable junction. Conversely, we do find a clear conductance signature in the experiments carried out on the charge-transfer complex. Due to the fact we expected this species to have a higher conductance than the neutral molecule, we believe this supports the idea that the conductance of the neutral molecule is very low, below our measurement sensitivity. This idea is further supported by theoretical calculations. To the best of our knowledge, these are the first reported single-molecule conductance measurements on a molecular charge-transfer species. PMID:26199662

  9. Three-color Förster resonance energy transfer within single F₀F₁-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.

    PubMed

    Ernst, Stefan; Düser, Monika G; Zarrabi, Nawid; Börsch, Michael

    2012-01-01

    Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. Förster-type resonance energy transfer, commonly referred to as FRET, is required in order to observe the dynamics of relative movements within the protein. Förster-type resonance energy transfer between two specifically attached fluorophores provides a ruler with subnanometer resolution between 3 and 8 nm, submillisecond time resolution for time trajectories of conformational changes, and single-molecule sensitivity to overcome the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary molecular machine which catalyzes the formation of adenosine triphosphate (ATP). The Escherichia coli enzyme comprises a proton driven 10 stepped rotary F(O) motor connected to a 3-stepped F(1) motor, where ATP is synthesized. This mismatch of step sizes will result in elastic deformations within the rotor parts. We present a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F(O)F(1)-ATP synthase at work. We labeled this enzyme with three fluorophores, specifically at the stator part and at the two rotors. Duty cycle-optimized with alternating laser excitation, referred to as DCO-ALEX, allowed to control enzyme activity and to unravel associated transient twisting within the rotors of a single enzyme during ATP hydrolysis and ATP synthesis. Monte Carlo simulations revealed that the rotor twisting is larger than 36 deg.

  10. Adoptive transfer of Mammaglobin-A epitope specific CD8 T cells combined with a single low dose of total body irradiation eradicates breast tumors.

    PubMed

    Lerret, Nadine M; Rogozinska, Magdalena; Jaramillo, Andrés; Marzo, Amanda L

    2012-01-01

    Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.

  11. Note: electronic circuit for two-way time transfer via a single coaxial cable with picosecond accuracy and precision.

    PubMed

    Prochazka, Ivan; Kodet, Jan; Panek, Petr

    2012-11-01

    We have designed, constructed, and tested the overall performance of the electronic circuit for the two-way time transfer between two timing devices over modest distances with sub-picosecond precision and a systematic error of a few picoseconds. The concept of the electronic circuit enables to carry out time tagging of pulses of interest in parallel to the comparison of the time scales of these timing devices. The key timing parameters of the circuit are: temperature change of the delay is below 100 fs/K, timing stability time deviation better than 8 fs for averaging time from minutes to hours, sub-picosecond time transfer precision, and a few picoseconds time transfer accuracy.

  12. Blinking fluorescence of single donor-acceptor pairs: important role of "dark'' states in resonance energy transfer via singlet levels.

    PubMed

    Osad'ko, I S; Shchukina, A L

    2012-06-01

    The influence of triplet levels on Förster resonance energy transfer via singlet levels in donor-acceptor (D-A) pairs is studied. Four types of D-A pair are considered: (i) two-level donor and two-level acceptor, (ii) three-level donor and two-level acceptor, (iii) two-level donor and three-level acceptor, and (iv) three-level donor and three-level acceptor. If singlet-triplet transitions in a three-level acceptor molecule are ineffective, the energy transfer efficiency E=I_{A}/(I_{A}+I_{D}), where I_{D} and I_{A} are the average intensities of donor and acceptor fluorescence, can be described by the simple theoretical equation E(F)=FT_{D}/(1+FT_{D}). Here F is the rate of energy transfer, and T_{D} is the donor fluorescence lifetime. In accordance with the last equation, 100% of the donor electronic energy can be transferred to an acceptor molecule at FT_{D}≫1. However, if singlet-triplet transitions in a three-level acceptor molecule are effective, the energy transfer efficiency is described by another theoretical equation, E(F)=F[over ¯](F)T_{D}/[1+F[over ¯](F)T_{D}]. Here F[over ¯](F) is a function of F depending on singlet-triplet transitions in both donor and acceptor molecules. Expressions for the functions F[over ¯](F) are derived. In this case the energy transfer efficiency will be far from 100% even at FT_{D}≫1. The character of the intensity fluctuations of donor and acceptor fluorescence indicates which of the two equations for E(F) should be used to find the value of the rate F. Therefore, random time instants of photon emission in both donor and acceptor fluorescence are calculated by the Monte Carlo method for all four types of D-A pair. Theoretical expressions for start-stop correlators (waiting time distributions) in donor and acceptor fluorescence are derived. The probabilities w_{N}^{D}(t) and w_{N}^{A}(t) of finding N photons of donor and acceptor fluorescence in the time interval t are calculated for various values of the energy

  13. Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome.

    PubMed

    Kinz-Thompson, Colin D; Sharma, Ajeet K; Frank, Joachim; Gonzalez, Ruben L; Chowdhury, Debashish

    2015-08-27

    At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describe transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies of the bacterial ribosomal pre-translocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pre-translocation complex, which are observed in a cryo-EM study, may not be observed in several smFRET studies.

  14. Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos.

    PubMed

    Cervera, R P; Martí-Gutiérrez, N; Escorihuela, E; Moreno, R; Stojkovic, M

    2009-11-01

    Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming after somatic cell nucleus transfer (SCNT) and may underlie the observed reduced viability of cloned embryos. In the current study, we tested the effects of the histone deacetylase-inhibitor trichostatin A (TSA) on preimplantation development and on histone acetylation and the gene expression of nucleus transfer (NT) porcine (Sus scrofa) embryos. Our results showed that 5 nM TSA for 26 h after reconstitution resulted in embryos (NTTSA) that reached the blastocyst stage at a higher level (48.1% vs. 20.2%) and increased number of cells (105.0 vs. 75.3) than that of the control (NTC) embryos. In addition, and unlike the NTC embryos, the treated embryos displayed a global acetylated histone H4 at lysine 8 profile similar to the in vitro-fertilized (IVF) and cultured embryos during the preimplantation development. Finally, we determined that several transcription factors exert a dramatic amount of genetic control over pluripotency, including Oct4, Nanog, Cdx2, and Rex01, the imprinting genes Igf2 and Igf2r, and the histone deacetyltransferase gene Hdac2. The NT blastocysts showed similar levels of Oct4, Cdx2, and Hdac2 but lower levels of Nanog than those of the IVF blastocyst. However, the NTTSA blastocysts showed similar levels of Rex01, Igf2, and Igf2r as those of IVF blastocysts, whereas the NTC blastocysts showed significantly lower levels for those genes. Our results suggest that TSA improves porcine SCNT preimplantation development and affects the acetylated status of the H4K8, rendering acetylation levels similar to those of the IVF counterparts.

  15. Impact of the mandatory age-based single-embryo transfer legislation in Turkey on outcome of in vitro fertilization: a multicentre study.

    PubMed

    Ergun, B; Bastu, E; Galandarov, R; Koksal, G; Yumru, H; Attar, E

    2013-08-01

    This study in Turkey evaluated the impact of age-based mandatory single-embryo transfer (SET) legislation with the subsequent increase in frozen-thawed embryo transfer (FT-EU) on pregnancy outcome of in vitro fertilization (IVF) patients. SET, FT-FT and double-embryo transfer were used in 5632 patients after legislation, while traditional IVF and FT-FT approach was used in 6029 patients before legislation. The cumulative pregnancy rate after legislation was slightly lower (38.2%) than before legislation (42.0%) but not significantly so. The single pregnancy rate for SET and traditional IVF were similar between the 2 groups (37.8% versus 28.7%), while multiple pregnancy rates were significantly higher before than after legislation (13.7% versus 0.3%). For FT-ET, the number of cycles was significantly higher after legislation (862 versus 616). SET yielded similar results to traditional IVF. In order to reduce multiple pregnancies without significantly decreasing pregnancy rates, SET might be a successful strategy.

  16. Direct Electron Transfer Kinetics of Peroxidase at Edge Plane Sites of Cup-Stacked Carbon Nanofibers and Their Comparison with Single-Walled Carbon Nanotubes.

    PubMed

    Komori, Kikuo; Tatsuma, Tetsu; Sakai, Yasuyuki

    2016-09-13

    Electron transfer kinetics at the graphene edge site is of great interest from the viewpoints of application to sensing and energy conversion and storage. Here we analyzed kinetics of direct electron transfer of horseradish peroxidase (HRP) adsorbed through surfactant sodium dodecyl sulfate at cup-stacked carbon nanofibers (CSCNFs), which provide highly ordered graphene edges, and compared it with that at single-walled carbon nanotubes (SWCNTs), which consist of a rolled-up basal plane graphene. The heterogeneous electron transfer rate constant of the Fe(2+/3+) couple of the HRP reaction center at CSCNFs (ca. 34.8 s(-1)) was an order of magnitude larger than that at SWCNTs (ca. 4.7 s(-1)). In addition, the overall rate constant of the electron transfer reaction from CSCNFs to HRP oxidized by H2O2 was higher than that from SWCNTs by a factor of 3. CSCNFs also allowed enhancement of the complex-formation reaction rate of HRP with H2O2, in comparison with that at SWCNTs. CSCNFs would therefore be applied to not only biosensors but also biofuel cells with enhanced performance. PMID:27529505

  17. Novel energy transfer mechanism in single-phased color-tunable Sr2CeO4:Eu3+ phosphors for WLEDs

    NASA Astrophysics Data System (ADS)

    Li, Haifeng; Jia, Yonglei; Sun, Wenzhi; Zhao, Ran; Fu, Jipeng; Jiang, Lihong; Zhang, Su; Pang, Ran; Li, Chengyu

    2014-09-01

    A series of color-tunable Sr2CeO4:Eu3+ phosphors have been successfully synthesized. The Rietveld refinement was firstly used to probe the phase purity and the structure of Sr2CeO4:Eu3+. The photoluminescence excitation spectra exhibit that the phosphors show a broad excitation band from 200 to 400 nm, which concurs well with the commercial near-UV LED. The photoluminescence spectra vary with the dopant content due to the energy transfer and the tunable blue-red color can be simply achieved by altering the doped Eu3+ ions concentrations. On the basis of the Inokuti-Hirayama theory, we obtained that the dipole-dipole interaction type takes charge for the energy transfer from the Ce4+-O2- charge-transfer state transition to Eu3+ ions and the schematic was plotted to illuminate the possible energy transfer process. The experimental results indicate that Sr2CeO4:Eu3+ phosphors may be potentially used as promising single-phased phosphors for near-UV white light-emitting diodes applications.

  18. Expression of epidermal growth factor receptor in the preimplantation uterus and blastocyst of the western spotted skunk.

    PubMed

    Paria, B C; Das, S K; Mead, R A; Dey, S K

    1994-08-01

    The western spotted skunk is unique in that its blastocysts undergo a 180-220-day period of arrested development before implantation. We investigated the potential role of epidermal growth factor (EGF)-related growth factors in regulating uterine and embryonic development in this species by studying the status of EGF receptor (EGF-R) in these tissues during delayed implantation and resumption of embryonic development. The cell-specific distribution of EGF binding sites and the expression of EGF-R mRNA were assayed by autoradiography and Northern blot analysis, respectively. The size of EGF-R was determined by affinity cross-linking studies, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase (PTK) activity. EGF binding sites were localized in the uterine luminal and glandular epithelium, endometrial stroma, myometrium, and blood vessels during both stages of pregnancy. As examined by Northern blot hybridization, a cRNA probe specific to mouse EGF-R hybridized to poly(A)+ RNA of skunk uteri. Transcripts similar to those of mouse uterine EGF-R were identified. [125I]-EGF was cross-linked to a 170-kDa protein both in the uterus and in blastocysts collected during the delayed implantation and periimplantation periods. However, EGF-induced PTK activity was significantly elevated above background levels during the period of renewed embryonic development, but not during arrested embryonic development. The results suggest that EGF-related growth factors may play an important role in regulating embryonic development in this species and that a change in the number and/or functional status of the EGF-R may be a prerequisite for blastocyst activation and implantation in the spotted skunk.

  19. Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection.

    PubMed

    Fernandez-Gonzalez, Lorena; Hribal, Romy; Stagegaard, Julia; Zahmel, Jennifer; Jewgenow, Katarina

    2015-04-01

    Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 μg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture.

  20. Conceptus development during blastocyst elongation in lines of pigs selected for increased uterine capacity or ovulation rate.

    PubMed

    Miles, J R; Freking, B A; Blomberg, L A; Vallet, J L; Zuelke, K A

    2008-09-01

    Lines of pigs selected for increased uterine capacity have improved conceptus survival, whereas pigs selected for increased ovulation rate have decreased conceptus survival relative to an unselected control line. The objective of this study was to evaluate conceptus development during blastocyst elongation as a potential contributing factor to differences in conceptus survival rate among these pig lines. Conceptuses were recovered from pregnant control, uterine capacity, and ovulation rate line gilts at d 10 and 12 of gestation. At d 10 of gestation, conceptus morphologic diversity was assessed by comparing within-litter average conceptus diameter and the standard deviation of conceptus diameters. At d 12 of gestation, conceptus morphologic diversity was assessed by comparing blastocyst populations obtained from individual gilts. Real-time PCR analyses for transcripts involved in steroidogenesis, cellular differentiation, and immune responsiveness were performed on spherical, ovoid, and filamentous conceptuses recovered from these selection lines. Uterine flushings were also assayed for total protein and estradiol-17beta at d 10 and 12 of gestation. Morphological data were analyzed using ANOVA with the fixed effects of line, farrowing season, and their interactions. Conceptus mortality, uterine flushing, and real-time PCR data were analyzed using ANOVA with the fixed effects of line, day or blastocyst morphology, farrowing season, and their interactions. Conceptus mortality, measured as the ratio of conceptus recovery to ovulation rate, was not different between the lines on d 10 and 12 of gestation. There were no significant line effects for conceptus morphologic diversity at d 10 and 12 of gestation. Expression of transcripts associated with steroidogenesis (steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and aromatase), cellular differentiation (cytokeratin-18 and vimentin), and immune responsiveness (interleukin-1beta) in spherical

  1. A single Tisbnd Osbnd C linkage induces interfacial charge-transfer transitions between TiO2 and a π-conjugated molecule

    NASA Astrophysics Data System (ADS)

    Fujisawa, Jun-ichi; Matsumura, Shingo; Hanaya, Minoru

    2016-07-01

    Interfacial charge-transfer (ICT) transitions between wide-band-gap semiconductors such as titanium dioxide (TiO2) and π-conjugated molecules enable the absorption of visible light with colorless organic compounds and also direct photoinduced electron transfers across the interfaces. ICT transitions have been reported to be induced by a double Tisbnd Osbnd C linkage of enediol compounds with two hydroxy groups to TiO2. In this Letter, we demonstrate that a single Tisbnd Osbnd C linkage of phenol with one hydroxy group can induce ICT transitions in the visible region. Our result widely opens up the range of organic compounds available for ICT transitions from diol compounds to mono-hydroxy compounds.

  2. Cytochrome P450-catalyzed dealkylation of atrazine by Rhodococcus sp. strain NI86/21 involves hydrogen atom transfer rather than single electron transfer.

    PubMed

    Meyer, Armin H; Dybala-Defratyka, Agnieszka; Alaimo, Peter J; Geronimo, Inacrist; Sanchez, Ariana D; Cramer, Christopher J; Elsner, Martin

    2014-08-28

    Cytochrome P450 enzymes are responsible for a multitude of natural transformation reactions. For oxidative N-dealkylation, single electron (SET) and hydrogen atom abstraction (HAT) have been debated as underlying mechanisms. Combined evidence from (i) product distribution and (ii) isotope effects indicate that HAT, rather than SET, initiates N-dealkylation of atrazine to desethyl- and desisopropylatrazine by the microorganism Rhodococcus sp. strain NI86/21. (i) Product analysis revealed a non-selective oxidation at both the αC and βC-atom of the alkyl chain, which is expected for a radical reaction, but not SET. (ii) Normal (13)C and (15)N as well as pronounced (2)H isotope effects (εcarbon: -4.0‰ ± 0.2‰; εnitrogen: -1.4‰ ± 0.3‰, KIEH: 3.6 ± 0.8) agree qualitatively with calculated values for HAT, whereas inverse (13)C and (15)N isotope effects are predicted for SET. Analogous results are observed with the Fe(iv)[double bond, length as m-dash]O model system [5,10,15,20-tetrakis(pentafluorophenyl)porphyrin-iron(iii)-chloride + NaIO4], but not with permanganate. These results emphasize the relevance of the HAT mechanism for N-dealkylation by P450.

  3. 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

  4. Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient.

    PubMed

    Yamamoto, Masaki; Matsumura, Yasufumi; Gomi, Ryota; Matsuda, Tomonari; Tanaka, Michio; Nagao, Miki; Takakura, Shunji; Uemoto, Shinji; Ichiyama, Satoshi

    2016-09-01

    Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-β-lactamase gene was detected in two different species isolated from a single patient. Metallo-β-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-β-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-β-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-β-lactamase-producing A. xylosoxidans and K. pneumoniae Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-β-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-β-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient. PMID:27381397

  5. Influence of Single Skimmer Versus Dual Funnel Transfer on the Appearance of ESI-Generated LiCl Cluster/ß-Cyclodextrin Inclusion Complexes

    NASA Astrophysics Data System (ADS)

    Kellner, Ina D.; Drewello, Thomas

    2015-08-01

    Singly and doubly charged adducts of LiCl with β-cyclodextrin (βCD) of the type (βCD)(LiCl)nLi+ and (βCD)2(LiCl)pLi2 2+ were studied using electrospray ionization mass spectrometry (ESI-MS). Insight into their structural composition was gained by analysis of their collision-induced dissociation (CID) mass spectra. The conditions the ions experience in the transfer region interfacing the ESI source and the mass analyzer were found to have a marked influence on the nature of the detected ions. In one instrument incorporating a single skimmer, individually attached LiCl ion pairs were observed, whereas the dual funnel ion guides of the second instrument allow the detection of previously unknown labile inclusion complexes of (LiCl)n clusters in βCD.

  6. Silver transfer in proustite Ag{sub 3}AsS{sub 3} at high temperatures: Conductivity and single-crystal X-ray studies

    SciTech Connect

    Gagor, Anna Pawlowski, Antoni; Pietraszko, Adam

    2009-03-15

    Single crystals of proustite Ag{sub 3}AsS{sub 3} have been characterised by impedance spectroscopy and single-crystal X-ray diffraction in the temperature ranges of 295-543 and 295-695 K, respectively. An analysis of the one-particle potential of silver atoms shows that in the whole measuring temperature range defects in the silver substructure play a major role in the conduction mechanism. Furthermore, the silver transfer is equally probable within silver chains and spirals, as well as between chains and spirals. The trigonal R3c room temperature phase does not change until the decomposition of the crystal. The electric anomaly of the first-order character which appears near 502 K is related to an increase in the electronic component of the total conductivity resulting from Ag{sub 2}S deposition at the sample surface. - Joint probability density function map of silver atoms at T=695 K.

  7. Heat and mass transfer at a free surface with diabatic boundaries in a single-species system under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Fuhrmann, Eckart; Dreyer, Michael E.

    2014-06-01

    In this paper, we analyzed the heat and mass transfer at a free surface under microgravity conditions. The SOURCE-II (Sounding Rocket COMPERE Experiment) experiment was performed on a suborbital flight in February 2012 from Esrange in North Sweden. It provided representative data with respect to solid, liquid, and vapor temperatures as well as the visible surface position. The objectives were to quantify the deformation of the free liquid surface and to correlate the apparent contact angle to a characteristic temperature difference between subcooled liquid and superheated wall. Furthermore, the influence of evaporation and condensation at the liquid/vapor interface and at the superheated wall must be taken into account to analyze heat and mass fluxes due to a characteristic temperature difference. In the following, we show evidently that the magnitude of the apparent contact angle depends on the exerted specific pressurizations of the vapor phase during the experiment and hence on the change in the saturation temperature at the free surface. The characteristic temperature difference is defined with respect to the wall temperature in the vicinity of the contact line and the saturation temperature. Therefore, apparent contact angle and temperature difference can be correlated and indicate a specific characteristic. Concerning the heat and mass transfer at the free liquid surface and the contact line, two different methods are presented to evaluate the net mass due to phase change within a certain time interval. In the first approach, the mass flow rate is calculated by means of the ideal gas law and its derivatives with respect to temperature and pressure. The second approach calculates the heat flux as well as the mass flux at the wall and in the region of the free liquid surface. In these cases, a specific heat transfer coefficient and a thermal boundary layer thickness are used.

  8. 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.

  9. 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

  10. Large aperture deformable mirror with a transferred single-crystal silicon membrane actuated using large-stroke PZT Unimorph Actuators

    NASA Technical Reports Server (NTRS)

    Hishinumat, Yoshikazu; Yang, Eui - Hyeok (EH)

    2005-01-01

    We have demonstrated a large aperture (50 mm x 50 mm) continuous membrane deformable mirror (DM) with a large-stroke piezoelectric unimorph actuator array. The DM consists of a continuous, large aperture, silicon membrane 'transferred' in its entirety onto a 20 x 20 piezoelectric unimorph actuator array. A PZT unimorph actuator, 2.5 mm in diameter with optimized PZT/Si thickness and design showed a deflection of 5.7 [m at 20V. An assembled DM showed an operating frequency bandwidth of 30 kHz and influence function of approximately 30%.

  11. Blocking Single-Stranded Transferred DNA Conversion to Double-Stranded Intermediates by Overexpression of Yeast DNA REPLICATION FACTOR A1

    PubMed Central

    Levy, Avner; Dafny, Raz; Tzfira, Tzvi

    2015-01-01

    Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1–RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process. PMID

  12. 2.0-μm emission and energy transfer of Ho3+/Yb3+ co-doped LiYF4 single crystal excited by 980 nm

    NASA Astrophysics Data System (ADS)

    Yang, Shuo; Xia, Hai-Ping; Jiang, Yong-Zhang; Zhang, Jia-Zhong; Jiang, Dong-Sheng; Wang, Cheng; Feng, Zhi-Gang; Zhang, Jian; Gu, Xue-Mei; Zhang, Jian-Li; Jiang, Hao-Chuan; Chen, Bao-Jiu

    2015-06-01

    Ho3+/Yb3+ co-doped LiYF4 single crystals with various Yb3+ concentrations and ˜ 0.98 mol% Ho3+ concentration are grown by the Bridgman method under the conditions of taking LiF and YF3 as raw materials and a temperature gradient (40 °C/cm-50 °C/cm) for the solid-liquid interface. The luminescent performances of the crystals are investigated through emission spectra, infrared transmittance spectrum, emission cross section, and decay curves under excitation by 980 nm. Compared with the Ho3+ single-doped LiYF4 crystal, the Ho3+/Yb3+ co-doped LiYF4 single crystal has an obviously enhanced emission band from 1850 nm to 2150 nm observed when excited by a 980-nm diode laser. The energy transfer from Yb3+ to Ho3+ and the optimum fluorescence emission around 2.0 μm of Ho3+ ions are investigated. The maximum emission cross section of the above sample at 2.0 μm is calculated to be 1.08×10-20 cm2 for the LiYF4 single crystal of 1-mol% Ho3+ and 6-mol% Yb3+ according to the measured absorption spectrum. The high energy transfer efficiency of 88.9% from Yb3+ to Ho3+ ion in the sample co-doped by Ho3+ (1 mol%) and Yb3+ (8 mol%) demonstrates that the Yb3+ ions can efficiently sensitize the Ho3+ ions. Project supported by the National Natural Science Foundation of China (Grant Nos. 51472125 and 51272109) and the K.C. Wong Magna Fund in Ningbo University, China (Grant No. NBUWC001).

  13. Nanofabrication of spin-transfer torque devices by a polymethylmethacrylate mask one step process: Giant magnetoresistance versus single layer devices

    NASA Astrophysics Data System (ADS)

    Parge, Anne; Niermann, Tore; Seibt, Michael; Münzenberg, Markus

    2007-05-01

    We present a method to prepare magnetic spin torque devices of low specific resistance in a one step lithography process. The quality of the pillar devices is demonstrated for a standard magnetic double layer device. For single layer devices, we found hysteretic switching and a more complex dynamical excitation pattern in higher fields. A simple model to explain the resistance spikes is presented.

  14. pH-Sensitive photoinduced energy transfer from bacteriorhodopsin to single-walled carbon nanotubes in SWNT-bR hybrids.

    PubMed

    El Hadj, Karim; Bertoncini, Patricia; Chauvet, Olivier

    2013-10-22

    Energy transfer mechanisms in noncovalently bound bacteriorhodopsin/single-walled carbon nanotube (SWNT) hybrids are investigated using optical absorption and photoluminescence excitation measurements. The morphology of the hybrids was investigated by atomic force microscopy. In this study, proteins are immobilized onto the sidewall of the carbon nanotubes using a sodium cholate suspension-dialysis method that maintains the intrinsic optical and fluorescence properties of both molecules. The hybrids are stable in aqueous solutions for pH ranging from 4.2 to 9 and exhibit photoluminescence properties that are pH-dependent. The study reveals that energy transfer from bacteriorhodopsin to carbon nanotubes takes place. So, at pH higher than 5 and up to 9, the SWNTs absorb the photons emitted by the aromatic residues of the protein, inducing a strong increase in intensity of the E11 emissions of SWNTs through their E33 and E44 excitations. From pH = 4.2 to pH = 5, the protein fluorescence is strongly quenched whatever the emission wavelengths, while additional fluorescence features appear at excitation wavelengths ranging from 660 to 680 nm and at 330 nm. The presence of these features is attributed to a resonance energy transfer mechanism that has an efficiency of 0.94 ± 0.02. More, by increasing the pH of the dispersion, the fluorescence characteristics become those observed at higher pH values and vice versa.

  15. Transfer characteristics and low-frequency noise in single- and multi-layer MoS{sub 2} field-effect transistors

    SciTech Connect

    Sharma, Deepak; Motayed, Abhishek; Shah, Pankaj B.; Amani, Matin; Georgieva, Mariela; Glen Birdwell, A.; Dubey, Madan; Li, Qiliang; Davydov, Albert V.

    2015-10-19

    Leveraging nanoscale field-effect transistors (FETs) in integrated circuits depends heavily on its transfer characteristics and low-frequency noise (LFN) properties. Here, we report the transfer characteristics and LFN in FETs fabricated with molybdenum disulfide (MoS{sub 2}) with different layer (L) counts. 4L to 6L devices showed highest I{sub ON}-I{sub OFF} ratio (≈10{sup 8}) whereas LFN was maximum for 1L device with normalized power spectral density (PSD) ≈1.5 × 10{sup −5 }Hz{sup −1}. For devices with L ≈ 6, PSD was minimum (≈2 × 10{sup −8 }Hz{sup −1}). Further, LFN for single and few layer devices satisfied carrier number fluctuation (CNF) model in both weak and strong accumulation regimes while thicker devices followed Hooge's mobility fluctuation model in the weak accumulation regime and CNF model in strong accumulation regime, respectively. Transfer-characteristics and LFN experimental data are explained with the help of model incorporating Thomas-Fermi charge screening and inter-layer resistance coupling.

  16. Meniscus-force-mediated layer transfer technique using single-crystalline silicon films with midair cavity: Application to fabrication of CMOS transistors on plastic substrates

    NASA Astrophysics Data System (ADS)

    Sakaike, Kohei; Akazawa, Muneki; Nakagawa, Akitoshi; Higashi, Seiichiro

    2015-04-01

    A novel low-temperature technique for transferring a silicon-on-insulator (SOI) layer with a midair cavity (supported by narrow SiO2 columns) by meniscus force has been proposed, and a single-crystalline Si (c-Si) film with a midair cavity formed in dog-bone shape was successfully transferred to a poly(ethylene terephthalate) (PET) substrate at its heatproof temperature or lower. By applying this proposed transfer technique, high-performance c-Si-based complementary metal-oxide-semiconductor (CMOS) transistors were successfully fabricated on the PET substrate. The key processes are the thermal oxidation and subsequent hydrogen annealing of the SOI layer on the midair cavity. These processes ensure a good MOS interface, and the SiO2 layer works as a “blocking” layer that blocks contamination from PET. The fabricated n- and p-channel c-Si thin-film transistors (TFTs) on the PET substrate showed field-effect mobilities of 568 and 103 cm2 V-1 s-1, respectively.

  17. Single-event and total-dose effects in geo-stationary transfer orbit during solar-activity maximum period measured by the Tsubasa satellite

    NASA Astrophysics Data System (ADS)

    Koshiishi, H.; Kimoto, Y.; Matsumoto, H.; Goka, T.

    The Tsubasa satellite developed by the Japan Aerospace Exploration Agency was launched in Feb 2002 into Geo-stationary Transfer Orbit GTO Perigee 500km Apogee 36000km and had been operated well until Sep 2003 The objective of this satellite was to verify the function of commercial parts and new technologies of bus-system components in space Thus the on-board experiments were conducted in the more severe radiation environment of GTO rather than in Geo-stationary Earth Orbit GEO or Low Earth Orbit LEO The Space Environment Data Acquisition equipment SEDA on board the Tsubasa satellite had the Single-event Upset Monitor SUM and the DOSimeter DOS to evaluate influences on electronic devices caused by radiation environment that was also measured by the particle detectors of the SEDA the Standard DOse Monitor SDOM for measurements of light particles and the Heavy Ion Telescope HIT for measurements of heavy ions The SUM monitored single-event upsets and single-event latch-ups occurred in the test sample of two 64-Mbit DRAMs The DOS measured accumulated radiation dose at fifty-six locations in the body of the Tsubasa satellite Using the data obtained by these instruments single-event and total-dose effects in GTO during solar-activity maximum period especially their rapid changes due to solar flares and CMEs in the region from L 1 1 through L 11 is discussed in this paper

  18. Nuclear-mitochondrial incompatibility in interorder rhesus monkey-cow embryos derived from somatic cell nuclear transfer.

    PubMed

    Kwon, Daekee; Koo, Ok-Jae; Kim, Min-Jung; Jang, Goo; Lee, Byeong Chun

    2016-10-01

    Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development. Cytochrome b is a protein of complex III of the electron transport chain (ETC). According to meta-analysis of amino acid sequences, the homology of cytochrome b is 75 % between rhesus monkeys and cattle. To maintain the function of ETC after iSCNT, 4n iSCNT embryos were produced by fusion of non-enucleated cow oocytes and rhesus monkey somatic cells. The blastocyst development rate of 4n iSCNT embryos was higher than that of 2n embryos (P < 0.01). Formation of reactive oxygen species (ROS) is an indirect indicator of ETC activity of cells. The ROS levels of 4n iSCNT embryos was higher than that of 2n embryos (P < 0.01). Collectively, rhesus monkey iSCNT embryos reconstructed with cow oocytes have nuclear-mitochondrial incompatibility due to fundamental species differences between rhesus monkeys and cattle. Nuclear-mitochondrial incompatibility seems to correlate with low ETC activity and extremely low blastocyst development of rhesus monkey-cow iSCNT embryos.

  19. A single exercise bout enhances the manufacture of viral-specific T-cells from healthy donors: implications for allogeneic adoptive transfer immunotherapy

    PubMed Central

    Spielmann, Guillaume; Bollard, Catherine M.; Kunz, Hawley; Hanley, Patrick J.; Simpson, Richard J.

    2016-01-01

    Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The adoptive transfer of donor-derived viral-specific cytotoxic T-cells (VSTs) is an effective treatment for controlling CMV and EBV infections after HSCT; however, new practical methods are required to augment the ex vivo manufacture of multi-VSTs from healthy donors. This study investigated the effects of a single exercise bout on the ex vivo manufacture of multi-VSTs. PBMCs isolated from healthy CMV/EBV seropositive participants before (PRE) and immediately after (POST) 30-minutes of cycling exercise were stimulated with CMV (pp65 and IE1) and EBV (LMP2A and BMLF1) peptides and expanded over 8 days. The number (fold difference from PRE) of T-cells specific for CMV pp65 (2.6), EBV LMP2A (2.5), and EBV BMLF1 (4.4) was greater among the VSTs expanded POST. VSTs expanded PRE and POST had similar phenotype characteristics and were equally capable of MHC-restricted killing of autologous target cells. We conclude that a single exercise bout enhances the manufacture of multi-VSTs from healthy donors without altering their phenotype or function and may serve as a simple and economical adjuvant to boost the production of multi-VSTs for allogeneic adoptive transfer immunotherapy. PMID:27181409

  20. In vitro development of bison embryos using interspecies somatic cell nuclear transfer.

    PubMed

    Seaby, R P; Alexander, B; King, W A; Mastromonaco, G F

    2013-12-01

    Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non-domestic animal species. However, problems arise during the development of these embryos, which may be related to species-specific differences in nuclear-cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria-related genes NRF1, MT-CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34-33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45-12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80-87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT-CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.

  1. Single-stage Reconstruction of Elbow Flexion Associated with Massive Soft-Tissue Defect Using the Latissimus Dorsi Muscle Bipolar Rotational Transfer

    PubMed Central

    Cuéllar, Vanessa G.; Ghiassi, Alidad; Sharpe, Frances

    2016-01-01

    Introduction: In the upper extremity, the latissimus dorsi muscle can be used as an ipsilateral rotational muscle flap for soft-tissue coverage or functional reconstruction of arm and elbow. Patients who have both major soft-tissue loss and functional deficits can be successfully treated with a single-stage functional latissimus dorsi rotational muscle transfer that provides simultaneous soft-tissue coverage and functional reconstruction. Methods: Our data base was queried for all patients undergoing a rotational latissimus dorsi muscle transfer for simultaneous soft-tissue coverage and functional reconstruction of elbow flexion. Four patients were identified. A chart review documented the mechanism of injury, associated injuries, soft-tissue defect size, number of surgical procedures, length of follow-up, last elbow range of motion, and flexion strength. Results: Four patients with loss of elbow flexion due to traumatic loss of the anterior compartment muscles and the overlying soft tissue underwent simultaneous soft-tissue coverage and elbow flexorplasty using the ipsilateral latissimus dorsi as a bipolar muscle rotational tissue transfer. All flaps survived and had a recovery of Medical Research Council Grade 4/5 elbow flexion strength. No additional procedures were required for elbow flexion. The surgical technique is described and supplemented with surgical technique video and patient outcome. Conclusions: This patient series augments the data provided in other series supporting the safety and efficacy of this procedure which provides both soft-tissue coverage and functional restoration of elbow flexion as a single-stage procedure in the setting of massive traumatic soft-tissue loss of the arm. PMID:27757363

  2. Single-molecule spectroscopy and femtosecond transient absorption studies on the excitation energy transfer process in ApcE(1-240) dimers.

    PubMed

    Long, Saran; Zhou, Meng; Tang, Kun; Zeng, Xiao-Li; Niu, Yingli; Guo, Qianjin; Zhao, Kai-Hong; Xia, Andong

    2015-05-28

    ApcE(1-240) dimers with one intrinsic phycocyanobilin (PCB) chromophore in each monomer that is truncated from the core-membrane linker (ApcE) of phycobilisomes (PBS) in Nostoc sp. PCC 7120 show a sharp and significantly red-shifted absorption. Two explanations either conformation-dependent Förster resonance energy transfer (FRET) or the strong exciton coupling limit have been proposed for red-shifted absorption. This is a classic example of the special pair in the photosynthetic light harvesting proteins, but the mechanism of this interaction is still a matter of intense debate. We report the studies using single-molecule and transient absorption spectra on the interaction in the special pair of ApcE dimers. Our results demonstrate the presence of conformation-dependent FRET between the two PCB chromophores in ApcE dimers. The broad distributions of fluorescence intensities, lifetimes and polarization difference from single-molecule measurements reveal the heterogeneity of local protein-pigment environments in ApcE dimers, where the same molecular structures but different protein environments are the main reason for the two PCB chromophores with different spectral properties. The excitation energy transfer rate between the donor and the acceptor about (110 ps)(-1) is determined from transient absorption measurements. The red-shifted absorption in ApcE dimers could result from more extending conformation, which shows another type of absorption redshift that does not depend on strong exciton coupling. The results here stress the importance of conformation-controlled spectral properties of the chemically identical chromophores, which could be a general feature to control energy/electron transfer, widely existing in the light harvesting complexes. PMID:25925197

  3. Agrobacterium-Mediated Gene Transfer Results Mainly in Transgenic Plants Transmitting T-DNA as a Single Mendelian Factor

    PubMed Central

    Budar, F.; Thia-Toong, L.; Van Montagu, M.; Hernalsteens, J.-P.

    1986-01-01

    Forty-four independent transformed tobacco plants were obtained from a cocultivation experiment with Agrobacterium tumefaciens strains carrying modified Ti-plasmids. The transformed plants were either self-fertilized or crossed with nontransformed plants or with other transformed plants. The segregation of a phenotypic marker (kanamycin resistance) in the progenies of these plants was determined. In 40 cases out of 44, the segregation of the kanamycin resistance marker is consistent with Mendelian genetics. Among these 40 clones, 35 contain a single kanamycin resistance locus. The five others segregate two independent resistance loci. In two of the single insert clones, the segregation ratio after selfing indicates that the T-DNA insertion may have caused a recessive lethal mutation. PMID:17246346

  4. Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen.

    PubMed

    Liu, Jie; Westhusin, Mark; Long, Charles; Johnson, Gregory; Burghardt, Robert; Kraemer, Duane

    2010-12-01

    Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos.

  5. Beyond the single-file fluid limit using transfer matrix method: Exact results for confined parallel hard squares

    SciTech Connect

    Gurin, Péter; Varga, Szabolcs

    2015-06-14

    We extend the transfer matrix method of one-dimensional hard core fluids placed between confining walls for that case where the particles can pass each other and at most two layers can form. We derive an eigenvalue equation for a quasi-one-dimensional system of hard squares confined between two parallel walls, where the pore width is between σ and 3σ (σ is the side length of the square). The exact equation of state and the nearest neighbor distribution functions show three different structures: a fluid phase with one layer, a fluid phase with two layers, and a solid-like structure where the fluid layers are strongly correlated. The structural transition between differently ordered fluids develops continuously with increasing density, i.e., no thermodynamic phase transition occurs. The high density structure of the system consists of clusters with two layers which are broken with particles staying in the middle of the pore.

  6. Beyond the single-file fluid limit using transfer matrix method: Exact results for confined parallel hard squares

    NASA Astrophysics Data System (ADS)

    Gurin, Péter; Varga, Szabolcs

    2015-06-01

    We extend the transfer matrix method of one-dimensional hard core fluids placed between confining walls for that case where the particles can pass each other and at most two layers can form. We derive an eigenvalue equation for a quasi-one-dimensional system of hard squares confined between two parallel walls, where the pore width is between σ and 3σ (σ is the side length of the square). The exact equation of state and the nearest neighbor distribution functions show three different structures: a fluid phase with one layer, a fluid phase with two layers, and a solid-like structure where the fluid layers are strongly correlated. The structural transition between differently ordered fluids develops continuously with increasing density, i.e., no thermodynamic phase transition occurs. The high density structure of the system consists of clusters with two layers which are broken with particles staying in the middle of the pore.

  7. Beyond the single-file fluid limit using transfer matrix method: Exact results for confined parallel hard squares.

    PubMed

    Gurin, Péter; Varga, Szabolcs

    2015-06-14

    We extend the transfer matrix method of one-dimensional hard core fluids placed between confining walls for that case where the particles can pass each other and at most two layers can form. We derive an eigenvalue equation for a quasi-one-dimensional system of hard squares confined between two parallel walls, where the pore width is between σ and 3σ (σ is the side length of the square). The exact equation of state and the nearest neighbor distribution functions show three different structures: a fluid phase with one layer, a fluid phase with two layers, and a solid-like structure where the fluid layers are strongly correlated. The structural transition between differently ordered fluids develops continuously with increasing density, i.e., no thermodynamic phase transition occurs. The high density structure of the system consists of clusters with two layers which are broken with particles staying in the middle of the pore.

  8. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing

    PubMed Central

    Liu, Wenqiang; Liu, Xiaoyu; Wang, Chenfei; Gao, Yawei; Gao, Rui; Kou, Xiaochen; Zhao, Yanhong; Li, Jingyi; Wu, You; Xiu, Wenchao; Wang, Su; Yin, Jiqing; Liu, Wei; Cai, Tao; Wang, Hong; Zhang, Yong; Gao, Shaorong

    2016-01-01

    Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos. PMID:27462457

  9. Identification of key factors conquering developmental arrest of somatic cell cloned embryos by combining embryo biopsy and single-cell sequencing.

    PubMed

    Liu, Wenqiang; Liu, Xiaoyu; Wang, Chenfei; Gao, Yawei; Gao, Rui; Kou, Xiaochen; Zhao, Yanhong; Li, Jingyi; Wu, You; Xiu, Wenchao; Wang, Su; Yin, Jiqing; Liu, Wei; Cai, Tao; Wang, Hong; Zhang, Yong; Gao, Shaorong

    2016-01-01

    Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer. However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. Here, we first developed a somatic cell nuclear transfer embryo biopsy system at two- or four-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Then, through single-cell transcriptome sequencing of somatic cell nuclear transfer embryos with different developmental fates, we identified that inactivation of Kdm4b, a histone H3 lysine 9 trimethylation demethylase, functions as a barrier for two-cell arrest of cloned embryos. Moreover, we discovered that inactivation of another histone demethylase Kdm5b accounts for the arrest of cloned embryos at the four-cell stage through single-cell analysis. Co-injection of Kdm4b and Kdm5b can restore transcriptional profiles of somatic cell nuclear transfer embryos and greatly improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study therefore provides an effective approach to identify key factors responsible for the developmental arrest of somatic cell cloned embryos. PMID:27462457

  10. Fe2+-Ti4+ vs. Fe2+-Fe3+ charge-transfer and short-range order in single chains of face-sharing octahedra: ellenbergerite and dumortierite

    NASA Astrophysics Data System (ADS)

    Chopin, C.; Langer, K.; Khomenko, V.

    2009-04-01

    In zoned pyrope megacrysts from the Dora-Maira UHP terrane, new, dark-violet colour varieties of the hexagonal, high-pressure silicate ellenbergerite extend the range of known Fe contents for this mineral from 0-0.1 to 0-0.4 atom pfu, for Ti contents commonly in the range 0.2-0.4 pfu. The new varieties show an extremely intense pleochroism, colourless for E perpendicular to c to deep Prussian blue for E//c, as compared to colourless to lilac or reddish purple for classical Fe-poor ellenbergerite. These features were the incentive for an electronic absorption spectroscopic study and a reappraisal of the interpretation of the charge transfers (CT), colour and ordering schemes in this group and the structurally related borosilicate dumortierite. Both structures are characterized by the presence of infinite single chains of face-sharing, partly vacant octahedra along the 6-fold screw axis and pseudo-hexad axis, respectively, in which the Fe and Ti atoms are partitioned. In the spectra of Fe-poor ellenbergerite, the presence of a single Fe2+-Ti4+ CT band near 19000 cm˘1 was taken as evidence for complete short-range ordering of Mg(Fe), Ti and vacancies in the octahedral single chain [1]. The E//c spectra of Fe-rich ellenbergerite show the same absorption band near 19000 cm˘1 but consistently flanked by another CT band near 14000 cm˘1 , the intensity of which increases with total Fe content. The latter is similar to the 12400 cm˘1 CT band observed as the single feature in E//c spectra of the isotructural (Ti-free and Fe-bearing) phosphoellenbergerite, and clearly assigned to Fe2+-Fe3+ CT in the octahedral single chain [1]. The same colour pattern occurs in the dumortierite group, with red Fe-poor, Ti-rich crystals showing a single CT band near 20000 cm˘1, blue Ti-poor crystals showing a single CT band near 16500 cm˘1, and violet Fe- and Ti-rich crystals showing a combination of the two bands [2]. In the light of the new data, we reinterpret the dumortierite colour

  11. Comparison of HYDRA predictions to temperature data from two single-assembly spent fuel heat transfer tests

    SciTech Connect

    McCann, R.A.

    1986-12-01

    The HYDRA computer code was used to simulate the thermal performance of an actual and a model spent fuel assembly. The HYDRA-predicted temperatures were then compared with measured data from two single-assembly test sections. The objective of this effort was to further verify the predictive capabilities of the HYDRA code for use in assessments of the hydrothermal performance of spent fuel dry storage systems. After HYDRA has been adequately evaluated and validated, the code will be documented to permit design and licensing safety analyses.

  12. Fluorescence energy transfer distance measurements using site-directed single cysteine mutants. The membrane insertion of colicin A.

    PubMed

    Lakey, J H; Baty, D; Pattus, F

    1991-04-01

    The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of insertion of a soluble protein into a lipid bilayer. The soluble structure is known from X-ray crystallography and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this work fluorescence spectroscopy was used to study the membrane-bound structure. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulfol-naphthyl)ethylenediamine (IAEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Three mutants K39C (helix 2), T127C (between helices 6 and 7) and S16Crpt (helix 1, which bears a decapeptide repeat before the mutation) gave useful derivatives. In the soluble protein they showed emission wavelengths decreasing in the order K39C greater than T127C greater than S16Crpt and although all showed blue shifts on addition of dimyristoylphosphatidylglycerol (DMPG) this order was maintained in the membrane-bound state. These shifts were not indicative of deep membrane insertion. Polarization of IAEDANS revealed differences in mobility between mutants. The three tryptophan residues were used as a compound donor to IAEDANS in resonance energy transfer distance determinations. The values obtained for the soluble form were 1.2 A to 3.2 A longer than in the crystal structure. On addition of lipids the indicated distances increased: S16Crpt-I(AEDANS) 6.45 A (22%), K39C-I 5.45 A (18%) and T127C-I 2.4 A (14%). N-bromosuccinimide (NBS) completely abolishes the tryptophan emission from the thermolytic fragment. When lipids were added to a mixture containing ten NBS-treated channel-forming fragments to one IAEDANS labelled fragment the indicated distances increased rather more: S16Crpt-I 9.7 A (38%), K39C-I 8.1 A (36%) and T127C-I 2.5 A (16%). This showed that intermolecular transfer reduces the distance estimated in samples containing only labelled protein. The ensemble of results shows that the amphipathic

  13. 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

  14. 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)

  15. 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

  16. Dynamics of single and multiple bubbles and associated heat transfer in nucleate boiling under low gravity conditions.

    PubMed

    Qiu, D; Son, G; Dhir, V K; Chao, D; Logsdon, K

    2002-10-01

    Experimental studies and numerical simulation of growth and lift-off processes of single bubbles formed on designed nucleation sites have been conducted under low-gravity conditions. Merging of multiple bubbles and lift-off processes during boiling of water in the parabola flights of KC-135 aircraft were also experimentally studied. The heating area of the flat heater surface was discretized and equipped with a number of small heating elements that were separately powered in the temperature-control mode. As such, the wall superheat remained nearly constant during the growth and departure of the bubbles, whereas the local heat flux varied during the boiling process. From numerical calculation it is found that peak of heat flux occurs locally at the contact line of bubble and heater surface. Dry conditions exist inside the bubble base area, which is characterized through a zero heat flux region in the numerical calculation and a lower heat flux period in the experimental results. During the merger of multiple bubbles, dry-out continues. In both the numerical calculations and experimental results, the bubble lift-off is associated with an apparent increase in heat flux. Wall heat flux variation with time and spatial distribution during the growth of a single bubble from numerical simulations are compared with experimental data.

  17. Hydrogen peroxide elimination from C4a-hydroperoxyflavin in a flavoprotein oxidase occurs through a single proton transfer from flavin N5 to a peroxide leaving group.

    PubMed

    Sucharitakul, Jeerus; Wongnate, Thanyaporn; Chaiyen, Pimchai

    2011-05-13

    C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.

  18. Secondary deuterium kinetic isotope effects in irreversible additions of hydride and carbon nucleophiles to aldehydes: A spectrum of transition states from complete bond formation to single electron transfer

    SciTech Connect

    Gajewski, J.J.; Bocian, W.; Harris, N.J.; Olson, L.P.; Gajewski, J.P.

    1999-01-20

    The competitive kinetics of hydride and organometallic additions to benzaldehyde-H and -D were determined at {minus}78 C using LiAlH{sub 4}, LiBEt{sub 3}H, NaBH{sub 4}, LiBH{sub 4}, LiAl(O-tert-butoxy){sub 3}H, NaB(OMe){sub 3}H, NaB-(Ac){sub 3}H (at 20 C) methyl, phenyl, and allyl Grignard, and methyl-, phenyl-, n-butyl-, tert-butyl-, and allyllithium. The additions of hydride were found to have an inverse secondary deuterium kinetic isotope effects in all cases, but the magnitude of the effect varied inversely with the apparent reactivity of the hydride. In the additions of methyl Grignard reagent and of methyllithium and phenyllithium, inverse secondary deuterium isotope effects were observed; little if any isotope effect was observed with phenyl Grignard or n-butyl- and tert-butyllithium. With allyl Grignard and allyllithium, a normal secondary deuterium kinetic isotope effect was observed. The results indicate that rate-determining single-electron transfer occurs with allyl reagents, but direct nucleophilic reaction occurs with all of the other reagents, with the extent of bond formation dependent on the reactivity of the reagent. In the addition of methyllithium to cyclohexanecarboxyaldehyde, a less inverse secondary deuterium kinetic isotope effect was observed than that observed in the addition of methyllithium to benzaldehyde, and allyllithium addition to cyclohexanecarboxaldehyde had a kinetic isotope effect near unity. The data with organometallic additions, which are not incompatible with observations of carbonyl carbon isotope effects, suggest that electrochemically determined redox potentials which indicate endoergonic electron transfer with energies less than ca. 13 kcal/mol allow electron-transfer mechanisms to compete well with direct polar additions to aldehydes, provided that the reagent is highly stabilized, like allyl species. Methyllithium and phenyllithium and methyl and phenyl Grignard reagents are estimated to undergo electron transfer with

  19. Excitation energy transfer in natural photosynthetic complexes and chlorophyll trefoils: hole-burning and single complex/trefoil spectroscopic studies

    SciTech Connect

    Ryszard Jankowiak, Kansas State University, Department of Chemistry, CBC Bldg., Manhattan KS, 66505; Phone: 532-6785

    2012-09-12

    In this project we studied both natural photosynthetic antenna complexes and various artificial systems (e.g. chlorophyll (Chl) trefoils) using high resolution hole-burning (HB) spectroscopy and excitonic calculations. Results obtained provided more insight into the electronic (excitonic) structure, inhomogeneity, electron-phonon coupling strength, vibrational frequencies, and excitation energy (or electron) transfer (EET) processes in several antennas and reaction centers. For example, our recent work provided important constraints and parameters for more advanced excitonic calculations of CP43, CP47, and PSII core complexes. Improved theoretical description of HB spectra for various model systems offers new insight into the excitonic structure and composition of low-energy absorption traps in very several antenna protein complexes and reaction centers. We anticipate that better understanding of HB spectra obtained for various photosynthetic complexes and their simultaneous fits with other optical spectra (i.e. absorption, emission, and circular dichroism spectra) provides more insight into the underlying electronic structures of these important biological systems. Our recent progress provides a necessary framework for probing the electronic structure of these systems via Hole Burning Spectroscopy. For example, we have shown that the theoretical description of non-resonant holes is more restrictive (in terms of possible site energies) than those of absorption and emission spectra. We have demonstrated that simultaneous description of linear optical spectra along with HB spectra provides more realistic site energies. We have also developed new algorithms to describe both nonresonant and resonant hole-burn spectra using more advanced Redfield theory. Simultaneous description of various optical spectra for complex biological system, e.g. artificial antenna systems, FMO protein complexes, water soluble protein complexes, and various mutants of reaction centers

  20. A self-contained culture platform using carbon dioxide produced from a chemical reaction supports mouse blastocyst development in vitro.

    PubMed

    Swain, Jason E

    2011-09-01

    Elevated CO(2) is required for in vitro embryo culture to maintain proper media pH and to supply embryo metabolic pathways. As an alternative to current approaches using gas cylinders, we examined use of a chemical reaction to supply CO(2). A closed culture system was constructed and chemicals added to generate CO(2), which was then supplied to developing embryos. This system was shown to provide a stable pH (7.2-7.4) over 4 days of use. One-cell mouse embryos were cultured in the device and no difference in blastocyst formation or cell number was apparent between embryos grown in a closed system with CO(2) supplied by a chemical reaction or positive controls grown in a an open system in a CO(2) incubator. This approach provides a highly purified, inexpensive, and easily obtainable gas source and offers potential for development of new, self-contained culture platforms. PMID:21532256

  1. Fine structures of embryonic discs of in vivo post-hatching porcine blastocysts at the pre-primitive streak stage.

    PubMed

    Xia, P; Liu, Z; Qin, P

    2011-04-01

    To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc.

  2. High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization

    PubMed Central

    Yoshikawa, Toshiyuki; Piao, Yulan; Zhong, Jinhui; Matoba, Ryo; Carter, Mark G.; Wang, Yuxia; Goldberg, Ilya; Ko, Minoru S.H.

    2006-01-01

    Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos (∼60 μm diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the ICM and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos. PMID:16325481

  3. Synthesis, transfer printing, electrical and optical properties, and applications of materials composed of self-assembled, aligned single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Pint, Cary L.

    Super growth of single-walled carbon nanotubes (SWNTs) has emerged as a unique method for synthesizing self-assembled, pristine, aligned SWNT materials composed of ultra-long (millimeter-long) nanotubes. This thesis focuses on novel routes of synthesizing such self-assembled SWNTs and the challenges that arise in integrating this material into next-generation applications. First of all, this work provides unique insight into growth termination of aligned SWNTs, emphasizing the mechanism that inhibits the growth of infinitely long nanotubes. Exhaustive real-time growth studies, combined with ex-situ and in-situ TEM characterization emphasizes that Ostwald ripening and subsurface diffusion of catalyst particles play a key role in growth termination. As a result, rational steps to solving this problem can enhance growth, and may ultimately lead to the meter or kilometer-long SWNTs that are necessary for a number of applications. In addition, other novel synthesis routes are discussed, such as the ability to form macroscopic fibrils of SWNTs, called "flying carpets" from 40 nm thick substrates, and the ability to achieve supergrowth of SWNTs that are controllably doped with nitrogen. In the latter case, molecular heterojunctions of doped and undoped sections in a single strand of ultralong SWNTs are demonstrated Secondly, as supergrowth is conducted on alumina coated SiO2 substrates, any applications will require that one can transfer the SWNTs to host surfaces with minimal processing. This work demonstrates a unique contact transfer route by which both patterned arrays of SWNTs, or homogenous SWNT carpets, can be transferred to any host surface. In the first case, the SWNTs are grown vertically aligned, and transferred in patterns of horizontally aligned SWNT. This transfer process relies on simple water-vapor etching of amorphous carbons at the catalyst following growth, and strong van der Waals adhesion of the high surface-area SWNT to host surfaces (gecko effect

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

    PubMed

    Schulz, Laura C; Roberts, R Michael

    2011-06-01

    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 the blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased toward a particular fate as early as the two-cell stage. In this study, 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. The presence or absence of leptin polarization was not linked to differences in the ability of embryos to normally develop to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm, but on blastocoel expansion, it was lost from the 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.

  5. Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes.

    PubMed

    Warzych, E; Peippo, J; Szydlowski, M; Lechniak, D

    2007-02-01

    The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.

  6. State transition analysis of spontaneous branch migration of the Holliday junction by photon-based single-molecule fluorescence resonance energy transfer.

    PubMed

    Okamoto, Kenji; Sako, Yasushi

    2016-02-01

    Branch migration of Holliday junction (HJ) DNA in solution is a spontaneous conformational change between multiple discrete states. We applied single-molecule fluorescence resonance energy transfer (smFRET) measurement to three-state branch migration. The photon-based variational Bayes-hidden Markov model (VB-HMM) method was applied to fluorescence signals to reproduce the state transition trajectories and evaluate the transition parameters, such as transition rate. The upper limit of time resolution suggested in simulation was nearly achieved for the state dynamics with relatively small FRET changes, and the distinctions in the populations of different states were successfully retrieved. We also discuss the suitability of the HJ as a standard sample for smFRET dynamics measurements and data analysis.

  7. State transition analysis of spontaneous branch migration of the Holliday junction by photon-based single-molecule fluorescence resonance energy transfer.

    PubMed

    Okamoto, Kenji; Sako, Yasushi

    2016-02-01

    Branch migration of Holliday junction (HJ) DNA in solution is a spontaneous conformational change between multiple discrete states. We applied single-molecule fluorescence resonance energy transfer (smFRET) measurement to three-state branch migration. The photon-based variational Bayes-hidden Markov model (VB-HMM) method was applied to fluorescence signals to reproduce the state transition trajectories and evaluate the transition parameters, such as transition rate. The upper limit of time resolution suggested in simulation was nearly achieved for the state dynamics with relatively small FRET changes, and the distinctions in the populations of different states were successfully retrieved. We also discuss the suitability of the HJ as a standard sample for smFRET dynamics measurements and data analysis. PMID:26687325

  8. Single-component and white light-emitting phosphor BaAl2Si2O8: Dy3+, Eu3+ synthesis, luminescence, energy transfer, and tunable color

    NASA Astrophysics Data System (ADS)

    Ma, Pingchuan; Song, Yanhua; Sheng, Ye; Yuan, Bo; Guan, Hongxia; Xu, Chengyi; Zou, Haifeng

    2016-10-01

    A series of Dy3+ - Eu3+ co-doped BaAl2Si2O8 phosphors were prepared via the conventional solid-state reaction method. Their crystal structure, luminescent characteristic and lifetime were investigated. The optimum doping concentrations of Dy3+and Eu3+ are both 0.05 for Dy3+ or Eu3+ singly doped BaAl2Si2O8. Furthermore, BaAl2Si2O8: 0.05Dy3+ and BaAl2Si2O8: 0.05Eu3+ emits yellow and red light. The emission color of BaAl2Si2O8: Dy3+, Eu3+ could be tuned from yellow to white due to the energy transfer. This energy transfer from Dy3+ to Eu3+ was confirmed and investigated by photoluminescence spectra and the decay time of energy donor Dy3+ ions. With constantly increasing Eu3+ concentration, the energy transfer efficiency from Dy3+ to Eu3+ in BaAl2Si2O8 host increased gradually and reached as high as 81%, the quantum yield was about 47.43%. BaAl2Si2O8: Dy3+, Eu3+ phosphors can be effectively excited by UV (about 348 nm) light and emit visible light from yellow to white by altering the concentration ratio of Dy3+ and Eu3+, indicating that the phosphors have potential applications as a white light-emitting phosphor for display and lighting.

  9. Bio-compatible organic humidity sensor transferred to arbitrary surfaces fabricated using single-cell-thick onion membrane as both the substrate and sensing layer.

    PubMed

    Sajid, Memoon; Aziz, Shahid; Kim, Go Bum; Kim, Soo Wan; Jo, Jeongdai; Choi, Kyung Hyun

    2016-01-01

    A bio-compatible disposable organic humidity sensor has been fabricated that can be transferred to any arbitrary target surface. Single cell thick onion membrane has been used as the substrate while it also doubles as the active layer of the sensor. Two different types of sensors were fabricated. In type-1, the membrane was fixed into a plastic frame with IDT patterns on one side while the other side was also exposed to environment. In type-2, onion membrane was attached to a glass substrate with one side exposed to environment having an IDT screen-printed on top of it. The electrical output response of the sensors showed their ability to detect relative humidity between 0% RH and 80% RH with stable response and good sensitivity. The impedance of the sensors changed from 16 MΩ to 2 MΩ for type-1 and 6 MΩ to 20 KΩ for type-2. The response times of type-1 and type-2 were ~1 and 1.5 seconds respectively. The recovery times were ~10.75 seconds and ~11.25 seconds for type-1 and type-2 respectively. The device was successfully transferred to various randomly shaped surfaces without damaging the device. PMID:27436586

  10. Bio-compatible organic humidity sensor transferred to arbitrary surfaces fabricated using single-cell-thick onion membrane as both the substrate and sensing layer

    PubMed Central

    Sajid, Memoon; Aziz, Shahid; Kim, Go Bum; Kim, Soo Wan; Jo, Jeongdai; Choi, Kyung Hyun

    2016-01-01

    A bio-compatible disposable organic humidity sensor has been fabricated that can be transferred to any arbitrary target surface. Single cell thick onion membrane has been used as the substrate while it also doubles as the active layer of the sensor. Two different types of sensors were fabricated. In type-1, the membrane was fixed into a plastic frame with IDT patterns on one side while the other side was also exposed to environment. In type-2, onion membrane was attached to a glass substrate with one side exposed to environment having an IDT screen-printed on top of it. The electrical output response of the sensors showed their ability to detect relative humidity between 0% RH and 80% RH with stable response and good sensitivity. The impedance of the sensors changed from 16 MΩ to 2 MΩ for type-1 and 6 MΩ to 20 KΩ for type-2. The response times of type-1 and type-2 were ~1 and 1.5 seconds respectively. The recovery times were ~10.75 seconds and ~11.25 seconds for type-1 and type-2 respectively. The device was successfully transferred to various randomly shaped surfaces without damaging the device. PMID:27436586

  11. Cooperative interaction of Agrobacterium VirE2 protein with single-stranded DNA: implications for the T-DNA transfer process.

    PubMed

    Citovsky, V; Wong, M L; Zambryski, P

    1989-02-01

    Induction of Agrobacterium tumefaciens vir gene expression by wounded plant cells results in production of a free transferable single-stranded (ss) copy of T-DNA, the T-strand. One of the Vir proteins, the VirE2 polypeptide, is a ssDNA-binding protein. In the present work, interaction of nopaline-specific VirE2 protein (Mr 69,000) with ssDNA was studied by using nitrocellulose filter binding, gel retardation, and electron microscopy techniques. The VirE2 protein was found to bind to ssDNA molecules with strong cooperativity, forming VirE2-ssDNA complexes with a binding site of 28-30 nucleotides. The VirE2-ssDNA complexes are stable at high salt concentrations and resistant to exonucleolytic activity. When examined under the electron microscope, the VirE2 protein converted collapsed free ssDNA molecules into unfolded and extended structures. The structure and properties of VirE2-ssDNA complexes predict possible functions in Agrobacterium virulence to (i) protect the T-strands from cellular nucleases and (ii) facilitate transfer of the T-strands through bacterial membranes possibly by specific interaction with putative membrane pores formed in plant-induced Agrobacterium cells. PMID:2919168

  12. Color-tunable photoluminescence and energy transfer properties of single-phase Ba10(PO4)6O:Eu2+, Mn2+ phosphors

    NASA Astrophysics Data System (ADS)

    Guo, Qingfeng; Liao, Libing; Mei, Lefu; Liu, Haikun

    2015-12-01

    Single-phase Ba10-x-y(PO4)6O:xEu2+,yMn2+ samples with apatite structure have been synthesized via a solid-state reaction method. The phase structure, luminescence properties, lifetime, PL thermal stability, as well as fluorescence decay curves of the samples were investigated. Effective energy transfer occurs from Eu2+ to Mn2+ in Ba10(PO4)6O and a possible mechanism of the energy-transfer from Eu2+ to Mn2+ is proposed. The critical distances Rc was calculated by concentration quenching and turned out to be about 0.817 nm (xc=0.21). The CIE and thermally stable luminescence behaviors of Ba9.94(PO4)6O:0.06Eu2+ phosphor were also studied in detail. All the results indicate that Ba10-x-y(PO4)6O:xEu2+, yMn2+ phosphors have potential applications as near UV-convertible phosphors for white light-emitting diodes.

  13. Bio-compatible organic humidity sensor transferred to arbitrary surfaces fabricated using single-cell-thick onion membrane as both the substrate and sensing layer

    NASA Astrophysics Data System (ADS)

    Sajid, Memoon; Aziz, Shahid; Kim, Go Bum; Kim, Soo Wan; Jo, Jeongdai; Choi, Kyung Hyun

    2016-07-01

    A bio-compatible disposable organic humidity sensor has been fabricated that can be transferred to any arbitrary target surface. Single cell thick onion membrane has been used as the substrate while it also doubles as the active layer of the sensor. Two different types of sensors were fabricated. In type-1, the membrane was fixed into a plastic frame with IDT patterns on one side while the other side was also exposed to environment. In type-2, onion membrane was attached to a glass substrate with one side exposed to environment having an IDT screen-printed on top of it. The electrical output response of the sensors showed their ability to detect relative humidity between 0% RH and 80% RH with stable response and good sensitivity. The impedance of the sensors changed from 16 MΩ to 2 MΩ for type-1 and 6 MΩ to 20 KΩ for type-2. The response times of type-1 and type-2 were ~1 and 1.5 seconds respectively. The recovery times were ~10.75 seconds and ~11.25 seconds for type-1 and type-2 respectively. The device was successfully transferred to various randomly shaped surfaces without damaging the device.

  14. Transfer between an Algerian and a French hospital of four multi-drug resistant bacterial strains together via a single patient

    PubMed Central

    Moissenet, Didier; Richard, Patrick; Granados, Maria; Mérens, Audrey; Fournier, Damien; Fines-Guyon, Marguerite; Arlet, Guillaume; Vu-Thien, Hoang

    2015-01-01

    A 5 years-old girl, seriously burnt with fire, was first hospitalized during four days in an hospital at Alger, and then transferred to our hospital at Paris. Admitted in our intensive care burns unit, she was third degree burnt on 78% of total body surface area, already treated with imipenem and vancomycin at her arrival. Clinical aggravation was rapidly observed and death occurred within 24 hours. Cultures of blood and multiple wound swabs yielded 3 multi-drug resistant bacterial strains: Acinetobacter baumannii with carbapenemase OXA-23, Pseudomonas aeruginosa serotype O11 with metallo-ß-lactamase VIM-4 and Klebsiella pneumoniae with CTX-M-15 extended-spectrum ß-lactamase. Culture of a rectal swab showed colonization by Enterococcus faecium with vanA glycopeptides resistance. Patients colonized with one or two multi-drug-resistant strains were not rare in our burns unit, especially those transferred from Algeria, but this case of a single patient harboring four multi-drug-resistant strains is exceptional. PMID:26550534

  15. Transfer between an Algerian and a French hospital of four multi-drug resistant bacterial strains together via a single patient.

    PubMed

    Moissenet, Didier; Richard, Patrick; Granados, Maria; Mérens, Audrey; Fournier, Damien; Fines-Guyon, Marguerite; Arlet, Guillaume; Vu-Thien, Hoang

    2015-01-01

    A 5 years-old girl, seriously burnt with fire, was first hospitalized during four days in an hospital at Alger, and then transferred to our hospital at Paris. Admitted in our intensive care burns unit, she was third degree burnt on 78% of total body surface area, already treated with imipenem and vancomycin at her arrival. Clinical aggravation was rapidly observed and death occurred within 24 hours. Cultures of blood and multiple wound swabs yielded 3 multi-drug resistant bacterial strains: Acinetobacter baumannii with carbapenemase OXA-23, Pseudomonas aeruginosa serotype O11 with metallo-ß-lactamase VIM-4 and Klebsiella pneumoniae with CTX-M-15 extended-spectrum ß-lactamase. Culture of a rectal swab showed colonization by Enterococcus faecium with vanA glycopeptides resistance. Patients colonized with one or two multi-drug-resistant strains were not rare in our burns unit, especially those transferred from Algeria, but this case of a single patient harboring four multi-drug-resistant strains is exceptional. PMID:26550534

  16. Microscopic origin of the charge transfer in single crystals based on thiophene derivatives: A combined NEXAFS and density functional theory approach.

    PubMed

    Chernenkaya, A; Morherr, A; Backes, S; Popp, W; Witt, S; Kozina, X; Nepijko, S A; Bolte, M; Medjanik, K; Öhrwall, G; Krellner, C; Baumgarten, M; Elmers, H J; Schönhense, G; Jeschke, H O; Valentí, R

    2016-07-21

    We have investigated the charge transfer mechanism in single crystals of DTBDT-TCNQ and DTBDT-F4TCNQ (where DTBDT is dithieno[2,3-d;2',3'-d'] benzo[1,2-b;4,5-b']dithiophene) using a combination of near-edge X-ray absorption spectroscopy (NEXAFS) and density functional theory calculations (DFT) including final state effects beyond the sudden state approximation. In particular, we find that a description that considers the partial screening of the electron-hole Coulomb correlation on a static level as well as the rearrangement of electronic density shows excellent agreement with experiment and allows to uncover the details of the charge transfer mechanism in DTBDT-TCNQ and DTBDT-F4 TCNQ, as well as a reinterpretation of previous NEXAFS data on pure TCNQ. Finally, we further show that almost the same quality of agreement between theoretical results and experiment is obtained by the much faster Z+1/2 approximation, where the core hole effects are simulated by replacing N or F with atomic number Z with the neighboring atom with atomic number Z+1/2. PMID:27448899

  17. Microscopic origin of the charge transfer in single crystals based on thiophene derivatives: A combined NEXAFS and density functional theory approach

    NASA Astrophysics Data System (ADS)

    Chernenkaya, A.; Morherr, A.; Backes, S.; Popp, W.; Witt, S.; Kozina, X.; Nepijko, S. A.; Bolte, M.; Medjanik, K.; Öhrwall, G.; Krellner, C.; Baumgarten, M.; Elmers, H. J.; Schönhense, G.; Jeschke, H. O.; Valentí, R.

    2016-07-01

    We have investigated the charge transfer mechanism in single crystals of DTBDT-TCNQ and DTBDT-F4TCNQ (where DTBDT is dithieno[2,3-d;2',3'-d'] benzo[1,2-b;4,5-b']dithiophene) using a combination of near-edge X-ray absorption spectroscopy (NEXAFS) and density functional theory calculations (DFT) including final state effects beyond the sudden state approximation. In particular, we find that a description that considers the partial screening of the electron-hole Coulomb correlation on a static level as well as the rearrangement of electronic density shows excellent agreement with experiment and allows to uncover the details of the charge transfer mechanism in DTBDT-TCNQ and DTBDT-F4 TCNQ, as well as a reinterpretation of previous NEXAFS data on pure TCNQ. Finally, we further show that almost the same quality of agreement between theoretical results and experiment is obtained by the much faster Z+1/2 approximation, where the core hole effects are simulated by replacing N or F with atomic number Z with the neighboring atom with atomic number Z+1/2.

  18. Microscopic origin of the charge transfer in single crystals based on thiophene derivatives: A combined NEXAFS and density functional theory approach.

    PubMed

    Chernenkaya, A; Morherr, A; Backes, S; Popp, W; Witt, S; Kozina, X; Nepijko, S A; Bolte, M; Medjanik, K; Öhrwall, G; Krellner, C; Baumgarten, M; Elmers, H J; Schönhense, G; Jeschke, H O; Valentí, R

    2016-07-21

    We have investigated the charge transfer mechanism in single crystals of DTBDT-TCNQ and DTBDT-F4TCNQ (where DTBDT is dithieno[2,3-d;2',3'-d'] benzo[1,2-b;4,5-b']dithiophene) using a combination of near-edge X-ray absorption spectroscopy (NEXAFS) and density functional theory calculations (DFT) including final state effects beyond the sudden state approximation. In particular, we find that a description that considers the partial screening of the electron-hole Coulomb correlation on a static level as well as the rearrangement of electronic density shows excellent agreement with experiment and allows to uncover the details of the charge transfer mechanism in DTBDT-TCNQ and DTBDT-F4 TCNQ, as well as a reinterpretation of previous NEXAFS data on pure TCNQ. Finally, we further show that almost the same quality of agreement between theoretical results and experiment is obtained by the much faster Z+1/2 approximation, where the core hole effects are simulated by replacing N or F with atomic number Z with the neighboring atom with atomic number Z+1/2.

  19. Thermal single-well injection-withdrawal tracer tests for determining fracture-matrix heat transfer area

    SciTech Connect

    Pruess, K.; Doughty, C.

    2010-01-15

    Single-well injection-withdrawal (SWIW) tracer tests involve injection of traced fluid and subsequent tracer recovery from the same well, usually with some quiescent time between the injection and withdrawal periods. SWIW are insensitive to variations in advective processes that arise from formation heterogeneities, because upon withdrawal, fluid parcels tend to retrace the paths taken during injection. However, SWIW are sensitive to diffusive processes, such as diffusive exchange of conservative or reactive solutes between fractures and rock matrix. This paper focuses on SWIW tests in which temperature itself is used as a tracer. Numerical simulations demonstrate the sensitivity of temperature returns to fracture-matrix interaction. We consider thermal SWIW response to the two primary reservoir improvements targeted with stimulation, (1) making additional fractures accessible to injected fluids, and (2) increasing the aperture and permeability of pre-existing fractures. It is found that temperature returns in SWIW tests are insensitive to (2), while providing a strong signal of more rapid temperature recovery during the withdrawal phase for (1).

  20. Impact of Molecular Organization on Exciton Diffusion in Photosensitive Single-Crystal Halogenated Perylenediimides Charge Transfer Interfaces.

    PubMed

    Pinto, Rui M; Gouveia, Wilson; Maçôas, Ermelinda M S; Santos, Isabel C; Raja, Sebastian; Baleizão, Carlos; Alves, Helena

    2015-12-23

    The efficiency of organic photodetectors and optoelectronic devices is strongly limited by exciton diffusion, in particular for acceptor materials. Although mechanisms for exciton diffusion are well established, their correlation to molecular organization in real systems has received far less attention. In this report, organic single-crystals interfaces were probed with wavelength-dependent photocurrent spectroscopy and their crystal structure resolved using X-ray diffraction. All systems present a dynamic photoresponse, faster than 500 ms, up to 650 nm. A relationship between molecular organization and favorable exciton diffusion in substituted butyl-perylenediimides (PDIB) is established. This is demonstrated by a set of PDIBs with different intra- and interstack distances and short contacts and their impact on photoresponse. Given the short packing distances between PDIs cores along the same stacking direction (3.4-3.7 Å), and across parallel stacks (2.5 Å), singlet exciton in these PDIBs can follow both Förster and Dexter exciton diffusion, with the Dexter-type mechanism assuming special relevance for interstack exciton diffusion. Yet, the response is maximized in substituted PDIBs, where a 2D percolation network is formed through strong interstack contacts, allowing for PDIBs primary excitons to reach with great efficiency the splitting interface with crystalline rubrene. The importance of short contacts and molecular distances, which is often overlooked as a parameter to consider and optimize when choosing materials for excitonic devices, is emphasized. PMID:26599347

  1. Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose.

    PubMed

    Hinck, L; Van Der Smissen, P; Heusterpreute, M; Donnay, I; De Hertogh, R; Pampfer, S

    2001-02-01

    Previous investigations have shown that maternal diabetes impairs rodent embryo development during the earliest phase of gestation. Exposure to high concentrations of glucose before implantation results in a decrease in the number of cells per embryo and in a concomitant increase in two nuclear markers of apoptosis, chromatin degradation and nuclear fragmentation. In the present study, we show that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), are involved in the embryotoxicity of high glucose. Using reverse transcription-polymerase chain reaction and immunocytochemistry, we first demonstrated that these two effectors were expressed in rat blastocysts. The two effectors were detected in all the cells of the blastocysts and the immuno-signals were excluded from the nuclei. Rat blastocysts were incubated for 24 h in either 6 mM or 28 mM glucose in the presence or absence of specific inhibitors (DEVD-CHO [10 microM] against caspase-3 and aurin [1 microM] against CAD). After incubation, blastocysts were examined for the proportion of nuclei showing signs of chromatin degradation or nuclear fragmentation. Addition of DEVD-CHO or aurin was found to inhibit the increase in chromatin degradation induced by high glucose. None of these two inhibitors prevented the increase in nuclear fragmentation triggered by excess glucose. Our data indicate that chromatin degradation and nuclear fragmentation are two nuclear damages that are induced separately by high glucose in rat blastocysts. Chromatin degradation is apparently mediated by the activation of caspase-3 and CAD.

  2. 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

  3. The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632.

    PubMed

    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.

  4. Assessing the reactivity of sodium alkyl-magnesiates towards quinoxaline: single electron transfer (SET) vs. nucleophilic alkylation processes.

    PubMed

    Livingstone, Zoe; Hernán-Gómez, Alberto; Baillie, Sharon E; Armstrong, David R; Carrella, Luca M; Clegg, William; Harrington, Ross W; Kennedy, Alan R; Rentschler, Eva; Hevia, Eva

    2016-04-14

    By exploring the reactivity of sodium butyl-magnesiate (1) supported by the bulky chelating silyl(bisamido) ligand {Ph2Si(NAr*)2}(2-) (Ar* = 2,6-iPr2-C6H3) towards Quinoxaline (Qx), the ability of this bimetallic system to effectively promote SET processes has been disclosed. Thus 1 executes the single-electron reduction of Qx affording complex (2) whose structure in the solid state contains two quinaxolyl radical anions Qx˙ stabilised within a dimeric magnesiate framework. Combining multinuclear NMR and EPR measurements with DFT calculations, new insights into the constitution of 2 in solution and its magnetic behaviour have been gained. Further evidence on the SET reactivity of 1 was found when it was reacted with nitroxyl radical TEMPO which furnished contacted ion pair sodium magnesiate [(Ph2Si(NAr*)2)Mg(TEMPO(-))Na(THF)3] (4) where both metals are connected by an alkoxide bridge, resulting from reduction of TEMPO. The role that the different ligands present in 1 can play in these new SET reactions has also been assessed. Using an amination approach, the Bu group in 1 can be replaced by the more basic amide TMP allowing the isolation of (3) which was characterised by multinuclear NMR and X-ray crystallography. (1)H NMR monitoring of the reaction of 3 with Qx showed its conversion to 2, leaving the hydrogen atoms of the heterocycle untouched. Contrastingly, using sodium homoalkyl magnesiate [NaMg(CH2SiMe3)3] (5) led to the chemoselective C2 alkylation of this heterocycle, suggesting that the presence of the steric stabiliser {Ph2Si(NAr*)2}(2-) on the mixed-metal reagent is required in order to facilitate the Qx reduction.

  5. Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

    PubMed

    Gómez, Martha C; Pope, C Earle

    2015-01-01

    In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

  6. Single cell monitoring of growth arrest and morphological changes induced by transfer of wild-type p53 alleles to glioblastoma cells.

    PubMed Central

    Van Meir, E G; Roemer, K; Diserens, A C; Kikuchi, T; Rempel, S A; Haas, M; Huang, H J; Friedmann, T; de Tribolet, N; Cavenee, W K

    1995-01-01

    Mutation of the p53 tumor suppressor gene is one of the earliest identified genetic lesions during malignant progression of human astrocytomas. To assess the functional significance of these mutations, wild-type (WT) p53 genes were introduced into glioblastoma cell lines having mutant, WT, or null endogenous p53 alleles. Populations of cells with mutant or null endogenous p53 alleles and exogenous WT p53 were spontaneously selected in culture for cells expressing only mutant p53 or no p53, which then displayed a growth and tumorigenic phenotype identical to the parental cells. To determine the phenotypic consequences of WT p53 expression before the occurrence of mutations, we developed a single cell assay to monitor WT p53-dependent transcription activity. Transfer and expression of exogenous WT p53 genes to cells with endogenous mutant or deleted, but not WT, p53 alleles caused growth arrest and morphological changes, including increased cell size and acquisition of multiple nuclei. This supports the hypothesis that genetic lesions of the p53 gene play an important role in the genesis of astrocytomas. Furthermore, the high sensitivity of the episomal single cell reporter strategy developed here has potential clinical applications in the rapid screening of patients for germ-line mutations of the p53 gene or any other gene with known targets for transcriptional transactivation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7862624

  7. Single-Molecule Fluorescence Resonance Energy Transfer Studies of the Human Telomerase RNA Pseudoknot: Temperature-/Urea-Dependent Folding Kinetics and Thermodynamics

    PubMed Central

    2015-01-01

    The ribonucleoprotein telomerase is an RNA-dependent DNA polymerase that catalyzes the repetitive addition of a short, species-specific, DNA sequence to the ends of linear eukaryotic chromosomes. The single RNA component of telomerase contains both the template sequence for DNA synthesis and a functionally critical pseudoknot motif, which can also exist as a less stable hairpin. Here we use a minimal version of the human telomerase RNA pseudoknot to study this hairpin–pseudoknot structural equilibrium using temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) experiments. The urea dependence of these experiments aids in determination of the folding kinetics and thermodynamics. The wild-type pseudoknot behavior is compared and contrasted to a mutant pseudoknot sequence implicated in a genetic disorder–dyskeratosis congenita. These findings clearly identify that this 2nt noncomplementary mutation destabilizes the folding of the wild-type pseudoknot by substantially reducing the folding rate constant (≈ 400-fold) while only nominally increasing the unfolding rate constant (≈ 5-fold). Furthermore, the urea dependence of the equilibrium and rate constants is used to develop a free energy landscape for this unimolecular equilibrium and propose details about the structure of the transition state. Finally, the urea-dependent folding experiments provide valuable physical insights into the mechanism for destabilization of RNA pseudoknots by such chemical denaturants. PMID:24617561

  8. Trophic transfer of nano-TiO2 in a paddy microcosm: A comparison of single-dose versus sequential multi-dose exposures.

    PubMed

    Kim, Jung In; Park, Hyung-Geun; Chang, Kwang-Hyeon; Nam, D H; Yeo, Min-Kyeong

    2016-05-01

    In the present study, replicated paddy microcosm systems were used to investigate the environmental fate and trophic transfer of titanium nanoparticles (NPs) over a period of 14 days. Most TiO2 NPs immediately settled down in the sediment, and high accumulations of nano TiO2 in the sandy loam sediment and biofilm were observed. The test organisms (quillworts, water dropworts, duckweeds, biofilms, river snails, and Chinese muddy loaches) and environmental media (freshwater, sandy loam sediment) were exposed to sequential low doses (2 mg/L at 1 h, 4 days, and 9 days) or a single high-dose (6 mg/L) of TiO2 NPs. The bioconcentration factors (BCFs) of nano-TiO2 in biofilms, quillworts, duckweeds, and Chinese muddy loaches were higher in the sequential multi-dose group than in the single-dose group. Chinese muddy loaches showed higher bioaccumulation factors (BAFs) over their prey than river snails. The difference in the carbon isotope ratios between Chinese muddy loaches and river snails was less than 2‰, and an approximately 4‰ difference in the stable nitrogen isotope ratio was observed in the two aquatic predators from their major prey (e.g., biofilms or particulate organic matter). The trophic levels between biofilms and river snails and between biofilms and Chinese muddy loaches were 2.8 and 2.4 levels, respectively. These results indicate that these two predators consumed biofilm and other alternative preys at a higher level than biofilm. Although the trophic transfer rates of TiO2 are generally low, relatively higher biomagnification factors (BMFs) were found in Chinese muddy loaches (0.04-0.05) than in river snails (0.01-0.02). These results suggest that TiO2 NPs show greater movement in the sediment than in the water and that TiO2 NPs can be retained through aquatic food chains more after a sequential low-dose exposure than after a single high-dose exposure. PMID:26854701

  9. Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer.

    PubMed

    Ernst, Stefan; Düser, Monika G; Zarrabi, Nawid; Dunn, Stanley D; Börsch, Michael

    2012-10-01

    Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

  10. Unraveling a Single-Step Simultaneous Two-Electron Transfer Process from Semiconductor to Molecular Catalyst in a CoPy/CdS Hybrid System for Photocatalytic H2 Evolution under Strong Alkaline Conditions.

    PubMed

    Xu, Yuxing; Ye, Yun; Liu, Taifeng; Wang, Xiuli; Zhang, Bingqing; Wang, Mei; Han, Hongxian; Li, Can

    2016-08-31

    Electron transfer processes from semiconductor to molecular catalysts was studied in a model hybrid photocatalytic hydrogen evolution system composed of [Co((III))(dmgH)2PyCl] (CoPy) and CdS under different pH conditions. Thermodynamic and kinetic studies revealed that photocatalytic H2 evolution under high pH conditions (pH 13.5) can only account for the thermodynamically more favorable single-step simultaneous two-electron transfer from photoirradiated CdS to Co(III)Py to produce unavoidable intermediate Co(I)Py, rather than a two-step successive one-electron transfer process. This finding not only provides new insight into the charge transfer processes between semiconductors and molecular catalysts but also opens up a new avenue for the assembly and optimization of semiconductor-molecular catalyst hybrid systems processed through multielectron transfer processes. PMID:27529565

  11. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer

    SciTech Connect

    Kishigami, Satoshi . E-mail: kishigami@cdb.riken.jp; Mizutani, Eiji; Ohta, Hiroshi; Hikichi, Takafusa; Thuan, Nguyen Van; Wakayama, Sayaka; Bui, Hong-Thuy; Wakayama, Teruhiko

    2006-02-03

    The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.

  12. Single-cell analyses revealed transfer ranges of IncP-1, IncP-7, and IncP-9 plasmids in a soil bacterial community.

    PubMed

    Shintani, Masaki; Matsui, Kazuhiro; Inoue, Jun-Ichi; Hosoyama, Akira; Ohji, Shoko; Yamazoe, Atsushi; Nojiri, Hideaki; Kimbara, Kazuhide; Ohkuma, Moriya

    2014-01-01

    The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a "transient" host of pCAR1.

  13. On the sensitivity of long-term magnetotelluric monitoring in Southern Italy and source-dependent robust single station transfer function variability

    NASA Astrophysics Data System (ADS)

    Romano, Gerardo; Balasco, Marianna; Lapenna, Vincenzo; Siniscalchi, Agata; Telesca, Luciano; Tripaldi, Simona

    2014-06-01

    Since 2007, a permanent magnetotelluric (MT) monitoring station has been working in the seismic area of the Agri Valley (Basilicata region, southern Italy) in order to investigate the stability of the MT transfer function. The station was installed in a rural area near the supposed seismogenic fault of the strong earthquake (Mw = 6.9) that struck the Agri Valley in 1857. Analysing about 4 yr of MT data characterized by a low seismic activity, the long-term systematic variations of robust single station MT transfer function estimates were observed in two different sounding period ranges. First, a significant seasonal component of variability for short periods was noted; these short periods were up to 16 s and were linked to variations in wetting/drying of soil moisture in the shallower layers. Second, a connection between the monitored estimates and global geomagnetic activity, Ap index, was found, particularly in the [20-100 s] period range. Analysing remote reference results and tipper estimates in shorter monitoring window, it was shown that such effect cannot be explained by a local or incoherent noise, and a large-scale coherent source should be claimed. We show that this effect is subtle because it produces smooth estimates, satisfying the dispersion relationship between apparent resistivity and phase, with small error bars. As the global geomagnetic activity level increases, robust estimators, like the median value, can be considered as a representative of the estimates due to the natural source, and they tend to stabilize when the Ap index approaches 10. It is also worth noting that our monitored time window includes the recent global minimum of solar activity which occurred in 2009, thus enhancing the estimate dependence on the Ap index.

  14. Intrauterine embryo transfer with canine embryos cryopreserved by the slow freezing and the Cryotop method

    PubMed Central

    HORI, Tatsuya; USHIJIMA, Hitoshi; KIMURA, Taku; KOBAYASHI, Masanori; KAWAKAMI, Eiichi; TSUTSUI, Toshihiko

    2016-01-01

    Canine embryos (8-cell to blastocyst stages) frozen-thawed using the slow-freezing method with glycerol (four recipients) or dimethyl sulfoxide (three recipients) as a cryoprotectant and vitrified-warmed using the Cryotop method (five recipients) were surgically transferred into the unilateral uterine horn of recipient bitches. As a result, the morphology of embryos frozen-thawed using the slow-freezing method was judged to be normal, but no conception occurred in any of the recipient bitches. Two of the five bitches that received transferred embryos (morula to early blastocyst stages) vitrified-warmed using the Cryotop method became pregnant and produced normal pups (1/9 embryos, 11.1% and 1/6 embryos, 17.0%). It was concluded that the Cryotop method was more appropriate for canine embryo cryopreservation than the slow-freezing method, which is used for the cryopreservation of embryos of other mammalian species. PMID:27041356

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

    PubMed Central

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

    2007-01-01

    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

  16. 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

  17. Open pulled straw vitrification of goat embryos at various stages of development.

    PubMed

    Al Yacoub, A N; Gauly, M; Holtz, W

    2010-05-01

    This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of

  18. Utilization of optimized BCR three-step sequential and dilute HCl single extraction procedures for soil-plant metal transfer predictions in contaminated lands.

    PubMed

    Kubová, Jana; Matús, Peter; Bujdos, Marek; Hagarová, Ingrid; Medved', Ján

    2008-05-30

    The prediction of soil metal phytoavailability using the chemical extractions is a conventional approach routinely used in soil testing. The adequacy of such soil tests for this purpose is commonly assessed through a comparison of extraction results with metal contents in relevant plants. In this work, the fractions of selected risk metals (Al, As, Cd, Cu, Fe, Mn, Ni, Pb, Zn) that can be taken up by various plants were obtained by optimized BCR (Community Bureau of Reference) three-step sequential extraction procedure (SEP) and by single 0.5 mol L(-1) HCl extraction. These procedures were validated using five soil and sediment reference materials (SRM 2710, SRM 2711, CRM 483, CRM 701, SRM RTH 912) and applied to significantly different acidified soils for the fractionation of studied metals. The new indicative values of Al, Cd, Cu, Fe, Mn, P, Pb and Zn fractional concentrations for these reference materials were obtained by the dilute HCl single extraction. The influence of various soil genesis, content of essential elements (Ca, Mg, K, P) and different anthropogenic sources of acidification on extraction yields of individual risk metal fractions was investigated. The concentrations of studied elements were determined by atomic spectrometry methods (flame, graphite furnace and hydride generation atomic absorption spectrometry and inductively coupled plasma optical emission spectrometry). It can be concluded that the data of extraction yields from first BCR SEP acid extractable step and soil-plant transfer coefficients can be applied to the prediction of qualitative mobility of selected risk metals in different soil systems. PMID:18585191

  19. An algorithm to estimate building heights from Google street-view imagery using single view metrology across a representational state transfer system

    NASA Astrophysics Data System (ADS)

    Díaz, Elkin; Arguello, Henry

    2016-05-01

    Urban ecosystem studies require monitoring, controlling and planning to analyze building density, urban density, urban planning, atmospheric modeling and land use. In urban planning, there are many methods for building height estimation using optical remote sensing images. These methods however, highly depend on sun illumination and cloud-free weather. In contrast, high resolution synthetic aperture radar provides images independent from daytime and weather conditions, although, these images rely on special hardware and expensive acquisition. Most of the biggest cities around the world have been photographed by Google street view under different conditions. Thus, thousands of images from the principal streets of a city can be accessed online. The availability of this and similar rich city imagery such as StreetSide from Microsoft, represents huge opportunities in computer vision because these images can be used as input in many applications such as 3D modeling, segmentation, recognition and stereo correspondence. This paper proposes a novel algorithm to estimate building heights using public Google Street-View imagery. The objective of this work is to obtain thousands of geo-referenced images from Google Street-View using a representational state transfer system, and estimate their average height using single view metrology. Furthermore, the resulting measurements and image metadata are used to derive a layer of heights in a Google map available online. The experimental results show that the proposed algorithm can estimate an accurate average building height map of thousands of images using Google Street-View Imagery of any city.

  20. Cyanide-bridged [Fe8M6] clusters displaying single-molecule magnetism (M=Ni) and electron-transfer-coupled spin transitions (M=Co).

    PubMed

    Mitsumoto, Kiyotaka; Oshiro, Emiko; Nishikawa, Hiroyuki; Shiga, Takuya; Yamamura, Yasuhisa; Saito, Kazuya; Oshio, Hiroki

    2011-08-22

    Cyanide-bridged metal complexes of [Fe(8)M(6)(μ-CN)(14)(CN)(10)(tp)(8)(HL)(10)(CH(3)CN)(2)][PF(6)](4)⋅n CH(3)CN⋅m H(2)O (HL=3-(2-pyridyl)-5-[4-(diphenylamino)phenyl]-1H-pyrazole), tp(-) =hydrotris(pyrazolylborate), 1: M=Ni with n=11 and m=7, and 2: M=Co with n=14 and m=5) were prepared. Complexes 1 and 2 are isomorphous, and crystallized in the monoclinic space group P2(1)/n. They have tetradecanuclear cores composed of eight low-spin (LS) Fe(III) and six high-spin (HS) M(II) ions (M=Ni and Co), all of which are bridged by cyanide ions, to form a crown-like core structure. Magnetic susceptibility measurements revealed that intramolecular ferro- and antiferromagnetic interactions are operative in 1 and in a fresh sample of 2, respectively. Ac magnetic susceptibility measurements of 1 showed frequency-dependent in- and out-of-phase signals, characteristic of single-molecule magnetism (SMM), while desolvated samples of 2 showed thermal- and photoinduced intramolecular electron-transfer-coupled spin transition (ETCST) between the [(LS-Fe(II))(3) (LS-Fe(III))(5)(HS-Co(II))(3)(LS-Co(III))(3)] and the [(LS-Fe(III))(8)(HS-Co(II))(6)] states. PMID:21830241

  1. Dendritic, transferable, strictly monolayer MoS2 flakes synthesized on SrTiO3 single crystals for efficient electrocatalytic applications.

    PubMed

    Zhang, Yu; Ji, Qingqing; Han, Gao-Feng; Ju, Jing; Shi, Jianping; Ma, Donglin; Sun, Jingyu; Zhang, Yanshuo; Li, Minjie; Lang, Xing-You; Zhang, Yanfeng; Liu, Zhongfan

    2014-08-26

    Controllable synthesis of macroscopically uniform, high-quality monolayer MoS2 is crucial for harnessing its great potential in optoelectronics, electrocatalysis, and energy storage. To date, triangular MoS2 single crystals or their polycrystalline aggregates have been synthesized on insulating substrates of SiO2/Si, mica, sapphire, etc., via portable chemical vapor deposition methods. Herein, we report a controllable synthesis of dendritic, strictly monolayer MoS2 flakes possessing tunable degrees of fractal shape on a specific insulator, SrTiO3. Interestingly, the dendritic monolayer MoS2, characterized by abundant edges, can be transferred intact onto Au foil electrodes and serve as ideal electrocatalysts for hydrogen evolution reaction, reflected by a rather low Tafel slope of ∼73 mV/decade among CVD-grown two-dimensional MoS2 flakes. In addition, we reveal that centimeter-scale uniform, strictly monolayer MoS2 films consisting of relatively compact domains can also be obtained, offering insights into promising applications such as flexible energy conversion/harvesting and optoelectronics.

  2. Improving the drug delivery characteristics of graphene oxide based polymer nanocomposites through the "one-pot" synthetic approach of single-electron-transfer living radical polymerization

    NASA Astrophysics Data Syst