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Sample records for single blastocyst transfer

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

    PubMed Central

    Sadasivam, Nirmala; Sadasivam, Narayanan M.

    2008-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. Clinical outcomes of frozen-thawed single blastocyst transfer in patients requiring whole embryo freezing.

    PubMed

    He, Qiao-Hua; Wang, Lu; Liang, Lin-Lin; Zhang, He-Long; Zhang, Cui-Lian; Li, Hang-Sheng; Cui, Shi-Hong

    2016-04-01

    We compared clinical outcomes amongst frozen-thawed cleavage-stage embryo, double and single blastocyst transfers in patients requiring whole embryo freezing. Data of infertile patients undergoing in-vitro fertilization and embryo transfer (IVF-ET) in our Reproductive Medicine Center from January 2010 to December 2012 were retrospectively analyzed. According to patients' wishes, patients were divided into cleavage-stage embryo transfer groups (group A, n = 456), double blastocyst transfer group (group B, n = 106), and single blastocyst transfer group (group C, n = 402). We found that the number of frozen embryos was significantly less in groups B and C than in group A (all p < 0.05), but the implantation rate was significantly higher in groups B and C as compared to group A (all p < 0.05). The clinical pregnancy rate and pregnancy rate per included cycle were significantly higher in group B than in groups A and C (all p < 0.05). The multiple pregnancy rate was significantly lower in group C than in groups A and B (all p < 0.05). The rate of early abortion was significantly lower in group C as compared to group A (p < 0.05). The data support the view that it may be the best clinical strategy for patients who require whole embryo freezing and have four or more Day 3 embryos available, to incubate Day 3 embryos into blastocysts, which are then vitrified for elective single blastocyst transfer. PMID:26889741

  4. Prognostic value of three pro-nuclei (3PN) incidence in elective single blastocyst-stage embryo transfer

    PubMed Central

    Li, Mingzhao; Zhao, Wanqiu; Li, Wei; Zhao, Xiaoli; Shi, Juanzi

    2015-01-01

    Objectives: To investigate whether 3PN incidence could lead to a worse pregnancy outcome of elective single blastocyst stage embryo transfer (eSBET). Methods: This study included 706 eSBET cycles from January 2013 to March 2015. The patients were divided into two groups as follows: group 1 included 286 patients with no 3PN zygotes and group 2 included 420 patients with 3PN zygotes. Main outcome measure: female age, endometrial thickness, infertility duration, type of infertility, main etiology of infertility, clinical pregnancy and abortion rate. Results: We observed that there was no significant difference in female age, endometrial thickness, infertility duration and abortion rate between two groups. Its worth noting that the clinical pregnancy rate in 3PN > 0% group was significantly lower than 3PN = 0 group (P = 0.021). Conclusions: 3PN incidence might make anegative effect on pregnancy outcomes in elective single blastocyst stage embryo transfer. PMID:26885129

  5. Blastocyst transfer in frozen-thawed cycles

    PubMed Central

    Han, Ae Ra; Lee, Hyoung-Song; Yang, Kwang Moon; Song, In Ok; Koong, Mi Kyoung

    2012-01-01

    Objective It is well known that fresh blastocyst transfer results in better pregnancy outcomes with a smaller number of transferred embryos compared with cleavage stage embryo transfer. However, in terms of frozen-thawed blastocyst transfer, only a few studies are available. We aimed to evaluate clinical outcomes of frozen-thawed embryo transfer (FET) with blastocysts. Methods Retrospective analysis of FET cycles with blastocysts (B-FET) between Jan 2007 and June 2009 was performed. Age-matched FET cycles with cleavage stage embryos (C-FET) during the same period were collected as controls. A total of 58 B-FET cycles were compared with 172 C-FET cycles and also compared with those of post-thaw extended culture blastocysts from frozen pronuclear stage embryos (22 cycles). Results There was no difference in the patient characteristics of each group. The embryos' survival rates after thawing were comparable (>90%) and there was no difference in the implantation rate or clinical and ongoing pregnancy rate among the three groups. Conclusion In FET, blastocyst transfers may not present better pregnancy outcomes than cleavage stage embryo transfers. A further large-scale prospective study is needed. PMID:23106042

  6. Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer : a multicentre randomized controlled trial.

    PubMed

    Ohl, J; de Mouzon, J; Nicollet, B; Benoussadh, A; Gouze, J N; Viville, S; Menezo, Y

    2015-01-01

    Despite excellent published results, the lack of well-designed, multicentre, randomized clinical trials results in an absence of general consensus on the efficacy of autologous endometrial cells coculture (AECC) in Assisted Reproductive Technology (ART). An open, multicentre, prospective, randomized controlled trial was designed to compare the pregnancy rate (PR) after the transfer of one blastocyst on day 5 after AECC to the transfer of one embryo on day 3 (control group). Patients were women aged 18 to 36, undergoing an ART cycle with no more than 1 embryo transfer failure. Sample size was calculated at 720 for a superiority trial involving an intermediate analysis at 300 patients. We present the results of the intermediate analysis that resulted in the study ending considering the observed difference. Three hundred thirty nine patients were randomized: 170 in the AECC group and 169 in the control group. The clinical PR per transfer was 53.4% with AECC and 37.3% in the control group (p=0.025). The quality of embryos was improved with AECC. These results suggest that implementation of the AECC technique to a large number of In-Vitro Fertilization (IVF) centres could lead to a substantial improvement in the proportion of successful assisted reproduction. The study was supported by the Laboratoires Genvrier, France. PMID:26718434

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

    PubMed

    Maheshwari, Abha; Hamilton, Mark; Bhattacharya, Siladitya

    2016-02-01

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

  8. 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

  9. 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

    2015-01-01

    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. Quantifying difference in gene expression profile between bovine blastocysts derived by in vitro fertilization and somatic cell nuclear transfer.

    PubMed

    Kwon, Sujin; Jeong, Sangkyun; Park, Jung Sun; Kang, Yong-Kook

    2015-01-01

    Epigenetic reprogramming intensely occurs in somatic-cell nuclear transfer (SCNT) embryos, which highlights the importance of proper expressions of reprogramming-related genes in SCNT embryos. We here assessed gene expression profiles (GEPs) difference between bovine blastocyst groups derived by in-vitro fertilization (IVF) or SCNT; in SCNT, cumulus cells and ear skin fibroblasts were used for cSCNT and fSCNT blastocysts, respectively. We obtained GEPs of 15 reprogramming-related genes in single blastocysts using multiplex PCR and found a broad range of variations in their GEPs. Weighted root-mean-square deviation (wRMSD) analysis, which calculates the deviation of SCNT blastocysts' GEPs from IVF blastocysts' mean GEP, found a significant difference between IVF and fSCNT and between cSCNT and fSCNT blastocysts (p < 0.001) but not between IVF and cSCNT. Since the fibroblasts' GEP was more distant from the IVF blastocysts' than the cumulus cells', it might partly explain the less similarity of fSCNT blastocysts' GEPs to the IVF's mean GEP. Our wRMSD method succeeds in expressing in figures how different two comparable embryo groups of different derivations are in GEP, which would be useful to select a better embryo derivation protocol among the candidates prior to field applications. PMID:26101995

  11. 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 variants 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

  12. Blastocyst stage transfer: the real benefits compared with early embryo transfer.

    PubMed

    Plachot, M; Belaisch-Allart, J; Mayenga, J M; Chouraqui, A; Serkine, A M; Tesquier, L

    2000-12-01

    With the development of commercially available sequential media it is now possible to grow human embryos to the blastocyst stage without feeder cells. The transfer of blastocysts offers several advantages, the most important being synchronization of the embryos with the uterine endometrium and selection of the best quality embryos with a high implantation potential. This study was conducted to compare the efficiency of day 2 and day 5 transfer in a prospective randomized trial involving patients for whom embryo selection was possible (i.e. those with more than three embryos on day 2 following insemination). We obtained equivalent clinical pregnancy rates per cycle for day 2 (41.7%) and day 5 (38.8%) transfer, but fewer embryos were transferred on day 5 (2.24 versus 3.03). The implantation rates were 18.9% on day 2 and 24.1% on day 5. Selected patients with a good response to gonadotrophins (at least eight good quality metaphase II oocytes) may therefore benefit from blastocyst transfer by a reduction in the multiple pregnancy rate, provided no more than two (or even one) blastocyst is transferred. PMID:11261480

  13. [Heterotopic pregnancy with intrauterine dizygotic twins following embryo transfer in the blastocyst phase].

    PubMed

    Barrón Vallejo, J; Ortega Díaz, R; Kably Ambe, A

    1999-04-01

    Ectopic pregnancy is a common complication of in vitro fertilization and embryo transfer (IVF-ET). On other hand, heterotopic pregnancy complicates 1-2% of all IVF-ET pregnancies. Tubal damage as reason for treatment and multiple embryo transfer might predispose patients to this complication. We present a successful treated case of an infertile patient that developed simultaneous twin intra- and single extra- uterine pregnancy after blastocyst-stage embryo transfer. In IVF-ET patients presence of an intrauterine gestation not exclude the possibility of a concomitant extrauterine pregnancy. Awareness of the possibility of heterotopic pregnancy after IVF-ET plays an important role in the successful treatment of this reproductive complication. Transfer of good quality embryos can be a risk factor to develop heterotopic pregnancy. PMID:10363416

  14. [Pregnancy and delivery with transfer of vitrified blastocysts following trophectoderm biopsy].

    PubMed

    Mtys, Szabolcs; Varga, Tnde; Kovcs, Pter; Knya, Mrton; Rajczy, Klra; Babenko, va; Szab, Barbara; Kaali, G Steven; Szentirmay, Zoltn

    2015-11-01

    Application of preimplantation genetic diagnosis makes it possible to transfer only embryos unaffected by a certain genetic disorder. The authors have applied the combination of trophectoderm biopsy and vitrification in order to detect a monogenic disorder. Previously diagnosed type 1 neurofibromatosis of the woman was the indication for genetic examination. In vitro fertilisation and embryo culture was performed using sequential culture mediums. Seven blastocysts could be sampled on the fifth day and were vitrified subsequently. Two blastocysts turned out to be genetically normal based on the result of genetic examination using polimerase chain reaction. A healthy boy was delivered following the transfer of warmed blastocysts and an uneventful singleton pregnancy. PMID:26498899

  15. Aberrant DNA Methylation in Porcine in vitro-, Parthenogenetic-, and Somatic Cell Nuclear Transfer-Produced Blastocysts

    PubMed Central

    Bonk, Aaron J.; Li, Rongfeng; Lai, Liangxue; Hao, Yanhong; Liu, Zhonghua; Samuel, Melissa; Fergason, Emily A.; Whitworth, Kristin M.; Murphy, Clifton N.; Antoniou, Eric; Prather, Randall S.

    2008-01-01

    Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos. The relative methylation in the gamete and blastocyst samples showed that 18.5% (921/4992) of the DNA clones were found to be significantly different (P<0.01) in at least one of the samples. Furthermore, for the different blastocyst groups, the methylation profile of the in vitro-produced blastocysts was less similar to the in vivo-produced blastocysts as compared to the parthenogenetic- and somatic cell nuclear transfer (SCNT)-produced blastocysts. The microarray results were validated by using bisulfite sequencing for 12 of the genomic regions in liver, sperm, and in vivo-produced blastocysts. These results suggest that a generalized change in global methylation is not responsible for the low developmental potential of blastocysts produced by using in vitro techniques. Instead, the appropriate methylation of a relatively small number of genomic regions in the early embryo may enable early development to occur. PMID:17595009

  16. Successful live birth after transfer of blastocyst and frozen blastocyst from rescue ICSI with application of polarized light microscopy for spindle examination on unfertilized eggs.

    PubMed

    Moon, Jeong Hee; Henderson, Sara; Garcia-Cerrudo, Elena; Mahfoudh, Alina; Reinblatt, Shauna; Son, Weon-Young

    2015-01-01

    This article aims to report successful live births after transfer of fresh blastocyst or vitrified/warmed blastocyst derived from intracytoplasmic sperm injection (ICSI) on day-1 of unfertilized mature eggs (so-called "rescue ICSI") with spindle examination using polarized light microscopy. Two couples who had rescue ICSI performed achieved a positive pregnancy result after the transfer of a fresh or vitrified blastocyst. The two pregnancies led to the live births of a healthy baby boy of 2.72 kg and baby girl of 3.4 kg, respectively. PMID:25887377

  17. Timing factors affecting blastocyst development in equine somatic cell nuclear transfer.

    PubMed

    Choi, Young-Ho; Velez, Isabel C; Macas-Garca, Beatriz; Hinrichs, Katrin

    2015-04-01

    In nuclear transfer (NT), exposure of donor cell chromatin to the ooplast cytoplasm may aid reprogramming; however, the length of exposure feasible is limited by the developmental life span of the oocyte. We examined the effect of duration of nucleus-cytoplasmic exposure before activation and of in vitro maturation (IVM) in equine NT. In experiment 1, 24 h IVM and a delay of 2, 5, or 8 h between reconstruction and activation yielded 4%, 15%, and 11% blastocysts, respectively. In experiment 2, a 5-h activation delay yielded 17% and 22% blastocysts with two donor cell lines. In experiment 3, using a 5-h activation delay, the blastocyst rate was significantly higher using oocytes after 20 h IVM than after 24 h IVM; however, only 28% of oocytes were in metaphase II (MII) at 20 h. In experiment 4, oocytes were denuded of cumulus at 20 h, and those in metaphase I (MI) were returned to culture for 3 h (20+3H treatment); blastocyst rates were 30% and 27%, respectively (8-h and 5-h delay to activation, respectively). Four live foals resulted from the transfer of 17 blastocysts (24%) produced using MII oocytes and a 5- or 8-h activation delay. Use of equine oocytes immediately after reaching MII, combined with a longer delay from reconstruction to activation, increased developmental competence after equine NT. PMID:25826725

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

    PubMed

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

    2016-04-01

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

  19. 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

  20. Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (aCGH) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles

    PubMed Central

    2013-01-01

    Background In assisted reproductive treatments, embryos remaining after fresh embryo transfer are usually selected for cryopreservation based on traditional morphology assessment. Our previous report has demonstrated that array comparative genomic hybridization (aCGH) screening for IVF patients with good prognosis significantly improves clinical and ongoing pregnancy rates in fresh embryo transfer cycles. The current study further investigates the efficiency of applying aCGH in the selection of euploid embryos for cryopreservation as related to pregnancy and implantation outcomes in subsequent frozen embryo transfer (FET) cycles. Methods First-time IVF patients with good prognosis undergoing fresh single embryo transfer and having at least one remaining blastocyst for cryopreservation were prospectively randomized into two groups: 1) Group A patients had embryos assessed by morphology first and then by aCGH screening of trophectoderm cells and 2) Group B patients had embryos evaluated by morphology alone. All patients had at least one blastocyst available for cryopreservation after fresh embryo transfer. There were 15 patients in Group A and 23 patients in Group B who failed to conceive after fresh embryo transfer and completed the FET cycles. Blastocyst survival and implantation rates were compared between the two groups. Results There were no significant differences in blastocyst survival rates between Group A and Group B (90.9% vs. 91.3%, respectively; p >0.05). However, a significantly higher implantation rate was observed in the morphology assessment plus aCGH screening group compared to the morphology assessment alone group (65.0% vs. 33.3%, respectively; p?=?0.038). There was no miscarriage observed in Group A while a 16.7% miscarriage rate was recorded in Group B (0% vs. 16.7%, respectively; p >0.05). Conclusions While aCGH screening has been recently applied to select euploid blastocysts for fresh transfer in young, low-risk IVF patients, this is the first prospective study on the impact of aCGH specifically on blastocyst survival and implantation outcomes in the subsequent FET cycles of IVF patients with good prognosis. The present study demonstrates that aCGH screening of blastocysts prior to cryopreservation significantly improves implantation rates and may reduce the risk of miscarriage in subsequent FET cycles. Further randomized clinical studies with a larger sample size are needed to validate these preliminary findings. PMID:23937723

  1. High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation.

    PubMed

    Bevacqua, Romina J; Pereyra-Bonnet, Federico; Fernandez-Martin, Rafael; Salamone, Daniel F

    2010-10-01

    In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development. PMID:20570328

  2. Aspects of in vivo oocyte production, blastocyst development, and embryo transfer in the cat.

    PubMed

    Pope, C E

    2014-01-01

    A brief overview of the progress made during the past approximately 40 years on the development of methods for in vitro production of cat embryos and intra- and interspecies embryo transfer is described. The presentation is focused primarily on research done over the past 30 years at the Cincinnati Zoo (1980-1995) and at the Audubon Nature Institute, New Orleans (1996-present) beginning with original studies on determining optimal doses of porcine FSH for ovarian stimulation and uterine embryo recovery, cryopreservation, and transfer. A key early finding was the ability of cats to respond to multiple gonadotropin (porcine FSH) treatments by repeated stimulation of follicular development. With a ? 6-month interval between FSH treatments, over the past 15 years (1998-2013), we have done 1603 laparoscopic oocyte retrievals on 337 cats and recovered >38,000 mature oocytes (mean = 24.1 per laparoscopic oocyte retrieval). The limited information available on in vivo blastocyst development in the cat during the latter portion of the preimplantation period (approximately Days 8 to 12 after coitum or approximately Days 7 to 11 after ovulation) was assembled for the purpose of comparing and contrasting it with the growth, expansion, and zona functioning of in vitro-derived blastocysts. Also, results of transferring morulae and/or blastocysts into synchronous recipients are described to emphasize evidence that appears to allude to an essential role for an intact zona pellucida in successful implantation and subsequent development in the cat. Until 2003, our in vitro-derived embryos were transferred into the uterine horns of recipients to determine the feasibility of producing offspring from such primary methods as IVF, intracytoplasmic sperm injection, SCNT, and embryo cryopreservation. With the exception of SCNT embryos, pregnancy rates were satisfactory, but embryo survival rates were not. Subsequently, after finding that SCNT embryo survival rate could be improved using laparoscopic transfer of early cleavage stage embryos into the oviduct, we applied the technique to embryos derived using IVF with sex-sorted sperm, oocyte vitrification, and embryo cryopreservation. Overall, a pregnancy rate of 67% (14/21) has resulted. Most recently, with the oviductal embryo transfer technique, two litters of Black-Footed cat kittens have been born from intra- and interspecies transfer of cryopreserved embryos. PMID:24274417

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

    PubMed Central

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

    2016-01-01

    Y-27632 is a specific inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho GTPase. The present study aimed to determine the effect of the specific ROCK inhibitor, Y-27632, on fresh human embryos and on single blastomeres obtained from discarded human embryos. A total of 784 poor-quality embryos were included, of which 526 were allocated to blastocyst culture directly and the remaining 258 were allocated to blastomere isolation. Embryos and single blastomeres were cultured either with, or without, Y-27632. Embryonic development was observed and recorded daily from day 5 onwards. Y-27632 did not affect the ratio of blastocyst formation or the quality of the human embryos. The duration of blastocyst formation was compared between the two groups in the embryo culture. On day 5, the blastocyst formation ratio in the experimental group was 11.4% (26/228), which was significantly (P=0.015) lower than the corresponding rate (19.7%; 44/223) in the control group. Survival analysis of the blastocyst formation duration showed that the median formation duration in the experimental group was significantly higher than that of the control group. The present study also obtained 1,192 blastomeres from 258 discarded day 3 embryos, and sibling blastomeres of similar sizes were equally allocated to experimental and control groups (n=596 in each). Treatment with Y-27632 increased the blastocyst formation ratio of human individual blastomeres, with 82 blastocysts of 596 blastomeres (13.8%), and 51 blastocysts of 596 blastomeres (8.6%) formed in the presence and absence of Y-27632, respectively (P=0.004). Compared with the control group, the mRNA and protein expression levels of E-cadherin in the blastocysts from blastomeres were enhanced by Y-27632 (P=0.022). In conclusion, the present study demonstrated that Y-27632 has different effects on the cleavage-stage of embryos and single blastomeres. Y-27632 increases the ratio of formation of blastocysts from single human blastomeres, but inhibits the direct formation of blastocysts from discarded human embryos. PMID:26783117

  4. 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 ?g/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. PMID:24960527

  5. 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

  6. Blastocysts derived from adult fibroblasts of a rhesus monkey ( Macaca mulatta) using interspecies somatic cell nuclear transfer.

    PubMed

    Kwon, Dae Kee; Kang, Jung Taek; Park, Sol Ji; Gomez, Ma Ninia Limas; Kim, Su Jin; Atikuzzaman, Mohammad; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2011-08-01

    In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms. PMID:21554770

  7. Implantation in the nine-banded armadillo: how does a single blastocyst form four embryos?

    PubMed

    Enders, A C

    2002-01-01

    In the course of a study on reproduction in the nine-banded armadillo, conceptuses between the beginning of implantation and primitive streak formation were examined to determine the manner of trophoblast differentiation during invasion of the endometrium and the sequence involved in formation of four identical quadruplets. The armadillo blastocyst implants in the fundic recess of the uterus. A single amnion and cup-shaped epiblastic plate are formed, and an exocelom develops between the amnion and trophoblast of the implantation site. Loss of the abembryonic trophoblast exposes both visceral and parietal endoderm to the uterine lumen, inverting the yolk sac. Continued expansion of the exocelom facilitates the intrusion of the forming conceptus into the uterine lumen and is accompanied by enlargement of the epiblastic plate. Separate areas of condensations of epiblast cells are the first indication of formation of the four identical quadruplets. The single layer of microvillous trophoblast with basal infoldings (designated absorptive trophoblast) is most likely to contribute extensively to movement of fluid into the exocelom. The resulting expansion of the exocelom not only enlarges the implantation site but also displaces the collapsing common amnion, limiting the amnion to the areas of the forming embryos. PMID:11869094

  8. Transfer of vitrified blastocysts from one or two superovulated Large White Hyperprolific donors to Meishan recipients: reproductive parameters at Day 30 of pregnancy.

    PubMed

    Cuello, Cristina; Berthelot, Franoise; Martinat-Bott, Franoise; Guillouet, Philippe; Furstoss, Vincent; Boisseau, Christian; Manceau, Patrick; Locatelli, Alain; Martnez, Emilio A

    2004-04-01

    The present study was designed to determine the effect of pooling embryos from two donors on the reproductive success of transfer of vitrified/warmed porcine blastocysts. Intact blastocysts were collected from superovulated Large White Hyperprolific gilts (n = 24) on Days 5-5.5 after artificial insemination. Embryos were recovered by flushing the uterine horns, and unhatched blastocysts were selected. Vitrification and warming were performed as described by Berthelot et al. [Cryobiology 41(2000) 116]. To evaluate in vitro development, 37 vitrified/warmed blastocysts were cultured, non-vitrified embryos (n = 48) were used as controls. Embryo transfers were conducted in asynchronous (-24 h) Meishan gilts (n = 20). Twenty vitrified/warmed blastocysts were surgically transferred into one uterine horn. Ten recipients received embryos from one donor (Group 1) and the other 10 transfers were performed with mixed embryos from two donors (Group 2). Pregnancy was assessed ultrasonographically at Day 25 after estrus and recipients were slaughtered at Day 30 after transfer. In vitro survival rate of the vitrified/warmed blastocysts was lower (P < 0.01) than that from control embryos (73.0% versus 93.7%). The pregnancy rate for Group 1 (70%) was not different (P > 0.05) than that from Group 2 (90%). No significant differences were detected between Groups 1 and 2 for in vivo embryo development (number fetuses/transferred embryos in pregnant recipients) or in vivo embryo survival (number viable fetuses/transferred embryos in pregnant recipients). However, the in vivo efficiency (number viable fetuses/total transferred embryos) was higher (P < 0.05) when transfers were performed with embryos from two donors (19.5% versus 30.5%). These results indicate that pooling embryos from two donors increases the in vivo efficiency after transfer of vitrified/warmed porcine blastocysts. PMID:14757470

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

    PubMed Central

    2011-01-01

    Background The in vitro culture of presumed zygotes derived from single cow ovum pick-up (OPU) is important for the production of quality blastocysts maintaining pedigree. The aim of the present study was to evaluate the agar chip-embedded helper embryo coculture system for single cow OPU-derived zygotes by assessing embryo quality. Methods Cumulus oocyte complexes (COCs) were collected from Hanwoo cows with high genetic merit twice a week using the ultra-sound guided OPU technique and from slaughterhouse ovaries. The Hanwoo cow COCs and slaughterhouse ovaries were matured in vitro, fertilized in vitro with thawed Hanwoo sperm and cultured for 24 h. The presumed zygotes were subsequently placed in three different culture systems: (1) control OPU (controlOPU) with single cow OPU-derived presumed zygotes (2~8); (2) agar chip-embedded slaughterhouse helper embryo coculture (agarOPU) with ten presumed zygotes including all presumed zygotes from a cow (2~8) and the rest from agar chip-embedded slaughterhouse presumed zygotes (8~2); and (3) slaughterhouse in vitro embryo production (sIVP) with ten slaughterhouse ovary-derived presumed zygotes, each in 50 ?L droplets. Day 8 blastocysts were assayed for apoptosis and gene expression using real time PCR. Results The coculture system promoted higher blastocyst development in OPU zygotes compared to control OPU zygotes cultured alone (35.2 vs. 13.9%; P < 0.01). Genes predicted to be involved in implantation failure and/or embryo resorption were down-regulated (P < 0.05) in control OPU zygotes (CD9, 0.4-fold; AKRAB1, 0.3-fold) and in cocultured zygotes (CD9, 0.3-fold; AKRAB1, 0.3-fold) compared to sIVP blastocysts (1.0-fold). Moreover, genes involved in implantation and/or normal calf delivery were up-regulated (P < 0.05 to P < 0.01) in control OPU zygotes (PGSH2, 5.0-fold; TXN, 4.3-fold; PLAU, 1.7-fold) and cocultured zygotes (PGSH2, 14.5-fold; TXN, 3.2-fold; PLAU, 6.8-fold) compared to sIVP (1.0-fold) blastocysts. However, the expression of PLAC8, TGF-?1, ODC1, ATP5A1 and CASP3 did not differ between the three culture groups. Conclusions Results show that the agar chip-embedded helper embryo coculture system enhances developmental competence and embryo quality in cultures of limited numbers of high pedigree single cow OPU presumed zygotes. PMID:21864328

  10. 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

  11. Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray

    PubMed Central

    Huntriss, John; Hemmings, Karen; Baskaran, Praveen; Hazelwood, Lee; Elder, Kay; Virtanen, Carl; Miller, David; Picton, Helen M.

    2015-01-01

    Objective To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts. Design A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays. Setting University research laboratory. Patient(s) Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by invitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom. Intervention(s) None. Main Outcome Measure(s) Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos. Result(s) Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs. Conclusion(s) The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts. PMID:25914096

  12. Comparing spatial expression dynamics of bovine blastocyst under three different procedures: in-vivo, in-vitro derived, and somatic cell nuclear transfer embryos.

    PubMed

    Nagatomo, Hiroaki; Akizawa, Hiroki; Sada, Ayari; Kishi, Yasunori; Yamanaka, Ken-ichi; Takuma, Tetsuya; Sasaki, Keisuke; Yamauchi, Nobuhiko; Yanagawa, Yojiro; Nagano, Masashi; Kono, Tomohiro; Takahashi, Masashi; Kawahara, Manabu

    2015-11-01

    There has been no work on spatiotemporal transcriptomic differences of blastocysts using in vivo- and in vitro-derived, and somatic cell nuclear transfer (SCNT) embryos. Here, we first compared the lineage-differentially transcriptomic profiles of in vivo- and in vitro-derived embryos by microarray analysis using divided into inner cell mass (ICM)-and trophectoderm (TE)-side samples, as well as those derived from SCNT in order to explore lineage-differentially expressed genes that are associated with preimplantation development in cattle. The transcriptomic profiles of the ICM-specific and TE-specific genes were similar between in vitro-derived embryos and in vivo-derived embryos, whereas SCNT embryos exhibited unusual lineage-differentially gene expression regulation at the blastocyst stage. The genes expressed in a spatiotemporal manner between developmentally normal in-vivo derived blastocysts and developmentally abnormal SCNT blastocysts might play critical roles for preimplantation development. Comparing spatial expression dynamics of bovine blastocyst under three different procedures revealed that CIITA was expressed in ICM-side samples of all the embryo types. CIITA is known as the master regulator of major histocompatibility complexes (MHC) class II genes that express in antigen-presenting cells but its biological function in preimplantation embryo is still unknown in mammals. Knockdown of CIITA expression in in vitro-derived embryos did not affect cleavage, but disrupted development of embryos into the blastocyst stage. These findings provide the novel transcriptomic information on blastocyst formation, raising the possibility that immune function-related gene directly plays important roles in bovine preimplantation development. PMID:26753242

  13. 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

  14. 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 medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa + BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa + ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa + BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first time we were able to show that IFOT is a feasible technique to generate bovine blastocysts by transferring in vitro-matured oocytes derived from slaughterhouse ovaries. These IFOT-derived blastocysts closely resemble in vivo-derived blastocysts in terms of lipid content and freeze survival. Thus, the present study laid the groundwork for newly created scientific experiments enabling novel analytical possibilities. Nevertheless, IFOT-derived embryos still reached lower pregnancy rates by trend compared with in vivo-derived embryos, also implicating an important role for the maturational environment in further developmental characteristics. PMID:25926438

  15. Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

    PubMed Central

    de Semir, D.; Maurisse, R.; Du, F.; Xu, J.; Yang, X.; Illek, B.; Gruenert, D. C.

    2013-01-01

    The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl? ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology. PMID:22234514

  16. 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

  17. 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 sibling oocytes to evaluate the efficiency of selecting competent blastocysts for transfer by combining time-lapse monitoring and array CGH testing for PGS patients. Our data clearly demonstrate that the combination of these two advanced technologies to select competent blastocysts for transfer results in improved implantation and ongoing pregnancy rates for PGS patients. PMID:24954518

  18. 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-? signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-? 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

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

    PubMed

    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

    2015-09-15

    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

  20. Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation.

    PubMed

    Talbot, Neil C; Powell, Anne M; Camp, Mary; Ealy, Alan D

    2007-02-01

    Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming. PMID:17570020

  1. Cytokines and Blastocyst Hatching.

    PubMed

    Seshagiri, Polani B; Vani, Venkatappa; Madhulika, Pathak

    2016-03-01

    Blastocyst implantation into the uterine endometrium establishes early pregnancy. This event is regulated by blastocyst- and/or endometrium-derived molecular factors which include hormones, growth factors, cell adhesion molecules, cytokines and proteases. Their coordinated expression and function are critical for a viable pregnancy. A rate-limiting event that immediately precedes implantation is the hatching of blastocyst. Ironically, blastocyst hatching is tacitly linked to peri-implantation events, although it is a distinct developmental phenomenon. The exact molecular network regulating hatching is still unclear. A number of implantation-associated molecular factors are expressed in the pre-implanting blastocyst. Among others, cytokines, expressed by peri-implantation blastocysts, are thought to be important for hatching, making blastocysts implantation competent. Pro-inflammatory (IL-6, LIF, GM-CSF) and anti-inflammatory (IL-11, CSF-1) cytokines improve hatching rates; they modulate proteases (MMPs, tPAs, cathepsins and ISP1). However, functional involvement of cytokines and their specific mediation of hatching-associated proteases are unclear. There is a need to understand mechanistic roles of cytokines and proteases in blastocyst hatching. This review will assess the available knowledge on blastocyst-derived pro-inflammatory and anti-inflammatory cytokines and their role in potentially regulating blastocyst hatching. They have implications in our understanding of early embryonic loss and infertility in mammals, including humans. PMID:26706391

  2. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 better define the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst...

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

    PubMed Central

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

    2014-01-01

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

  4. Successful cryopreservation of expanded equine blastocysts.

    PubMed

    Choi, Y H; Velez, I C; Riera, F L; Roldn, J E; Hartman, D L; Bliss, S B; Blanchard, T L; Hayden, S S; Hinrichs, K

    2011-07-01

    Effective cryopreservation of expanded equine blastocysts (> 300 ?m in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 ?m) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300-730 ?m in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered ("Central") biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 ?m in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts. PMID:21458049

  5. 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

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

    2015-08-01

    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

  6. Comprehensive chromosome screening of trophectoderm with vitrification facilitates elective single-embryo transfer for infertile women with advanced maternal age.

    PubMed

    Schoolcraft, William B; Katz-Jaffe, Mandy G

    2013-09-01

    The universal goal of assisted reproduction technologies is a singleton delivery of a healthy full-term baby. For younger women (<35 years of age) single-embryo transfer is a viable option resulting in clinical success similar to multiple-embryo transfers. In contrast, older women have significantly lower pregnancy rates following single-embryo transfer. To provide effective single-embryo transfer options for older women, improved methods of embryo selection are required to overcome the marked differences in outcome of single- versus double-embryo transfer. With the development of comprehensive chromosome screening, blastocyst vitrification, and trophectoderm biopsy techniques, older women have the opportunity of elective single-embryo transfer with live birth rates as high as those reported for younger good-prognosis infertility patients. PMID:23993664

  7. The mammalian blastocyst.

    PubMed

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

    2016-03-01

    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. WIREs Dev Biol 2016, 5:210-232. doi: 10.1002/wdev.220 For further resources related to this article, please visit the WIREs website. PMID:26799266

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

    PubMed Central

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

    2015-01-01

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

  9. 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

    2015-10-21

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

  10. Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

    PubMed Central

    McElroy, Sohyun L.; Byrne, James A.; Chavez, Shawn L.; Behr, Barry; Hsueh, Aaron J.; Westphal, Lynn M.; Reijo Pera, Renee A.

    2010-01-01

    Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer. PMID:20539753

  11. Freezing mouse blastocysts. The influence of the preparations prior to freezing on the survival rate of the blastocysts.

    PubMed

    Hoogenkamp, H

    1982-01-01

    A good survival rate in culturing mouse blastocysts can be obtained in Ovum Culture Medium, enriched with 20 per cent inactivated Foetal Bovine Serum or Sheep Serum under air. The transfer of fresh blastocysts gives the best results if the recipients are on day 3 of the pseudo-pregnancy, but with 20 hours' cultured blastocysts it is better to use recipients on day 4. Exposure to 1.5 M DMSO has no harmful effect, provided that the DMSO is added at 5 degrees C in 6 steps and is removed, again in 6 steps, at 35 degrees C. The crystallization of the medium containing the embryos at -5 degrees C to -6 degrees C doet not appear te have a harmful influence on culture results of the blastocysts. PMID:15861587

  12. Vitrification: a simple and successful method for cryostorage of human blastocysts.

    PubMed

    Liebermann, Juergen

    2015-01-01

    Cryopreservation is one of the keystones in clinical infertility treatment. Especially vitrification has become a well-established and widely used routine procedure that allows important expansion of therapeutic strategies when in vitro fertilization (IVF) is used to treat infertility. Vitrification of human blastocysts allows us to maximize the potential for conception from any one in vitro fertilization cycle and prevents wastage of embryos. This goes even further toward to best utilize a patient's supernumerary oocytes after retrieval, maximizing the use of embryos from a single stimulation cycle. The technology may even be used to eliminate fresh embryo transfers for reasons of convenience, uterine receptivity, fertility preservation, preimplantation genetic diagnosis, or emergency management. In this chapter, the application of vitrification technology for cryopreserving human blastocyst will be revealed through step-by-step protocols. The results that are presented using the described protocols underscore the robustness of the vitrification technology for embryo cryopreservation. PMID:25428012

  13. 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

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

    NASA Astrophysics Data System (ADS)

    Wieche?, 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 ?-helix typical peak was twice more intensive than the ?-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 available material for research purposes is limited.

  15. Live birth after single embryo transfer of autologous cryopreserved oocytes from a patient with myelodysplastic syndrome who underwent allogenic peripheral blood stem cell transplantation.

    PubMed

    Tsai, Yi-Yi; Chen, Shee-Uan; Shieh, Chia-Jen; Yao, Yi-Lin; Yang, Yu-Shih; Chen, Chin-Der

    2014-12-01

    We report a live birth after single embryo transfer derived from autologous cryopreserved oocytes of a patient with myelodysplastic syndrome who had undergone allogenic peripheral blood stem cell transplantation (PBSCT). In 2006, a 24-year-old female diagnosed with myelodysplastic syndrome was referred for fertility preservation before she underwent PBSCT. After controlled ovarian stimulation, 38 oocytes were retrieved for cryopreservation using a slow-freezing protocol. She was cured by PBSCT and entered menopause. After seven years, she requested thawing of the oocytes. She was prepared for a thawing cycle using hormone replacement therapy. Twenty-two cryopreserved oocytes were thawed, and 20 (91%) oocytes survived. Thirteen mature oocytes were inseminated by intracytoplasmic sperm injection. Ten (77%) oocytes were normally fertilized and 6 (60%) oocytes developed into blastocysts. Embryo transfer to her own uterus with one blastocyst was performed. Five blastocysts were vitrified. A sonographic exam at 7 weeks of gestation revealed one gestational sac with positive cardiac motion. A normal female baby weighing 2704g was delivered at 40 weeks of gestation. A successful pregnancy from autologous cryopreserved oocytes is encouraging for cancer patients undergoing fertility preservation. For infertile cancer patients after PBSCT, we suggest the transfer of one embryo to reduce the risk of multiple pregnancies. PMID:25294099

  16. Chromosomal analysis of blastocysts from balanced chromosomal rearrangement carriers.

    PubMed

    Gui, Baoheng; Yao, Zhongyuan; Li, Yanping; Liu, Donge; Liu, Nenghui; Xia, Yan; Huang, Yanru; Mei, Libin; Ma, Ruiyu; Lu, Sijia; Liang, Desheng; Wu, Lingqian

    2016-04-01

    Balanced chromosomal rearrangements (CRs) are among the most common genetic abnormalities in humans. In the present study, we have investigated the degree of consistency between the chromosomal composition of the blastocyst inner cell mass (ICM) and trophectoderm (TE) in carriers with balanced CR, which has not been previously addressed. As a secondary aim, we have also evaluated the validity of cleavage-stage preimplantation genetic diagnosis (PGD) based on fluorescence in situ hybridization (FISH) of blastocysts from CR carriers. Blastocyst ICM and TE were screened for chromosomal aneuploidy and imbalance of CR-associated chromosomes based on whole-genome copy number variation analysis by low-coverage next-generation sequencing (NGS) following single-cell whole-genome amplification by multiple annealing and looping-based amplification cycling. The NGS results were analyzed without knowledge of cleavage-stage FISH results. NGS results for blastocyst ICM and TE from CR carriers were 86.49% (32/37) consistent. Of the 1702 (37×46) chromosomes examined, 99.47% (1693/1702) showed consistency. However, only 40.0% (18/45) of all embryos had consistent results for chromosomes involved in CR, as determined by blastocyst NGS and cleavage-stage FISH. Of the 85 CR-affected chromosomes analyzed by FISH, 37.65% (32/85) were incongruous with NGS results, with 87.5% (28/32) showing imbalanced composition by FISH but balanced composition by NGS. These results indicate that chromosomal composition of blastocyst ICM and TE in balanced CR carriers is highly consistent, and that PGD based on cleavage-stage FISH is inaccurate; therefore, using blastocyst TE biopsies for NGS-based PGD is recommended for identifying chromosomal imbalance in embryos from balanced CR carriers. PMID:26825930

  17. Incidence of chromosomal abnormalities in bovine blastocysts derived from unsorted and sex-sorted spermatozoa.

    PubMed

    Garcia-Herreros, M; Carter, T F; Villagmez, D A F; Macaulay, A D; Rath, D; King, W A; Lonergan, P

    2010-01-01

    The aim of the present study was to examine the incidence of chromosomal abnormalities in bovine blastocysts produced by IVF with unsorted, X-sorted or Y-sorted spermatozoa. In Experiment 1, individual blastocysts were processed to examine the incidence of mixoploidy using fluorescent in situ hybridisation. Overall, 80% (44/55) of blastocysts were mixoploid (10/15, 14/15 and 20/25 for X-sorted, Y-sorted and unsorted spermatozoa, respectively; P > 0.05). However, the prevalence of abnormal XY chromosome complements was relatively low in all groups; on average, only a small fraction of the total nuclei per embryo appeared polyploid (1.64%, 5.62% and 6.0% for X-sorted, Y-sorted and unsorted spermatozoa, respectively). Interestingly, 20% (5/25) of blastocysts derived from unsorted spermatozoa were found to be chimeric (XX/XY). In Experiment 2, chimeric embryos were detected among the blastocysts derived from two of five sires tested. In addition, one chimeric blastocyst was detected among nine in vivo-derived blastocysts obtained following AI. In conclusion, based on the results of the present study, the incidence of chromosomal abnormalities did not different between blastocysts derived from sex-sorted or unsorted spermatozoa. In addition, the occurrence of mixed sex chimeras was not limited to a single sire and was not unique to blastocysts derived from IVF. PMID:20883653

  18. Deliveries from trophectoderm biopsied, fresh and vitrified blastocysts derived from polar body biopsied, vitrified oocytes.

    PubMed

    Grifo, Jamie; Adler, Alexis; Lee, Hsiao Ling; Morin, Scott J; Smith, Meghan; Lu, Lucy; Hodes-Wertz, Brooke; McCaffrey, Caroline; Berkeley, Alan; Munn, Santiago

    2015-08-01

    This longitudinal study reports preliminary findings of six patients who underwent first polar body biopsy followed by oocyte vitrification. All oocytes were warmed, inseminated by intracytoplasmic sperm injection and cultured to blastocyst. All suitable blastocysts underwent trophectoderm biopsy for aneuploidy screening, and supernumerary blastocysts were vitrified. Euploid blastocysts were transferred either fresh or in a subsequent programmed cycle. Of the 91 metaphase II oocytes, 30 had euploid first polar bodies. Development to blastocyst was more likely in oocytes with a euploid first polar body (66.7% versus 24.6%; P < 0.001). Nineteen euploid blastocysts were produced: 10 from oocytes with a euploid first polar body and nine from oocytes with an aneuploid first polar body. Five out of six patients (83%) had a live birth or ongoing pregnancy at the time of analysis. Eleven euploid blastocysts have been transferred and seven implanted (64%). Although the chromosomal status of the first polar body was poorly predictive of embryonic ploidy, an association was found between chromosomal status of the first polar body and development to blastocyst. Further study is required to characterize these relationships, but proof of concept is provided that twice biopsied, twice cryopreserved oocytes and embryos can lead to viable pregnancies. PMID:26096028

  19. Multiple pregnancy after single or multiple embryo transfer performed according to Korean guidelines

    PubMed Central

    Han, E Jung; Kim, Seul Ki; Lee, Jung Ryeol; Suh, Chang Suk; Kim, Seok Hyun

    2015-01-01

    Objective To assess compliance with Korean guidelines for embryo transfer, the possible reasons for non-compliance, and multiple pregnancies according to each specific condition in compliant cycles. Methods A single-institution, retrospective study was conducted of 256 fresh in vitro fertilization cycles during 2012-2014. To assess compliance with Korean guidelines, the maximum recommended number of embryos transferred (according to criteria of age, transfer day, and presence of favorable conditions) was compared with the actual number of embryos transferred. Clinical pregnancy rate (PR) was assessed as the percentage of pregnant women resulting from each set of transfer conditions, including the number of embryos transferred. The multiple pregnancy rate (MPR) was calculated as the percentage of pregnant women with a multifetal pregnancy. Results The compliance rate with the Korean guidelines was 96.5% (247/256). Non-compliance occurred in nine cycles owing to poor embryo quality, repeated implantation failure, or hostile endometrium. In compliant cycles, the PR was 31.2% (77/247), and the MPR was 27.3% (21/77; 20 twins and one triplet). Higher MPR was noted in two types of transfer conditions: transfer of three cleavage embryos in women aged 35-39 years with favorable conditions (66.7%; primarily from those aged 35-37 years) and transfer of two blastocysts in women aged ≥40 years with favorable conditions (50%). Conclusion Under the Korean guidelines, compliance rate was high in our center. Multiple pregnancies occurred primarily in group with favorable conditions. In high-risk groups for multiple pregnancies, reducing number of embryos transferred should be considered than suggested in the guideline. PMID:26816876

  20. Maternal age effect on early human embryonic development and blastocyst formation.

    PubMed

    Janny, L; Menezo, Y J

    1996-09-01

    In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([blastocysts/embryos on day 2]* 100) and the blastocyst expansion rate ([expanded blastocysts/blastocysts]* 100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (< 30 years, 82%; > or = 40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. PMID:8873066

  1. Dihydrolipoic Acid Induces Cytotoxicity in Mouse Blastocysts through Apoptosis Processes

    PubMed Central

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

    2012-01-01

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

  2. Identification of a suitable endogenous control gene in porcine blastocysts for use in quantitative PCR analysis of microRNAs.

    PubMed

    Li, Jun; Wei, Hengxi; Li, Yan; Li, Qiuyan; Li, Ning

    2012-02-01

    To obtain reliable results in quantitative PCR (qPCR) reactions, an endogenous control (EC) gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA (miRNA) genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of miRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types (P-value>0.05). These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm (a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes) and NormFinder (an excel plug-in that uses an ANOVA-based model to estimate intra- and inter-group variation to indicate the single most stable EC gene) programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts. PMID:22415683

  3. Cell signalling during blastocyst morphogenesis.

    PubMed

    Eckert, Judith J; Velazquez, Miguel A; Fleming, Tom P

    2015-01-01

    Blastocyst morphogenesis is prepared for even before fertilisation. Information stored within parental gametes can influence both maternal and embryonic gene expression programmes after egg activation at fertilisation. A complex network of intrinsic, cell-cell mediated and extrinsic, embryo-environment signalling mechanisms operates throughout cleavage, compaction and cavitation. These signalling events not only ensure developmental progression, cell differentiation and lineage allocation to inner cell mass (embryo proper) and trophectoderm (future extraembryonic lineages) but also provide a degree of developmental plasticity ensuring survival in prevailing conditions by adaptive responses. Indeed, many cellular functions including differentiation, metabolism, gene expression and gene expression regulation are subject to plasticity with short- or long-term consequences even into adult life. The interplay between intrinsic and extrinsic signals impacting on blastocyst morphogenesis is becoming clearer. This has been best studied in the mouse which will be the focus of this chapter but translational significance to human and domestic animal embryology will be a focus in future years. PMID:25956293

  4. Identification of Inappropriately Reprogrammed Genes by Large-Scale Transcriptome Analysis of Individual Cloned Mouse Blastocysts

    PubMed Central

    Morita, Shinnosuke; Yamada, Kaori; Jincho, Yuko; Tane, Shouji; Sotomaru, Yusuke; Kono, Tomohiro

    2010-01-01

    Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos. PMID:20614022

  5. Effect of Vitrification on the MicroRNA Transcriptome in Mouse Blastocysts

    PubMed Central

    Zhao, Xueming; Hao, Haisheng; Du, Weihua; Zhu, Huabin

    2015-01-01

    Vitrification is commonly used in the cryopreservation of mammalian blastocysts to overcome the temporal and spatial limitations of embryo transfer. Previous studies have shown that the implantation ability of vitrified blastocysts is impaired and that microRNAs (miRNAs) regulate the critical genes for embryo implantation. However, little information is available about the effect of vitrification on the miRNA transcriptome in blastocysts. In the present study, the miRNA transcriptomes in fresh and vitrified mouse blastocysts were analyzed by miRNA Taqman assay based method, and the results were validated using quantitative real-time PCR (qRT-PCR). Then, the differentially expressed miRNAs were assessed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 760 known mouse miRNAs were detected in the vitrified and fresh mouse blastocysts. Of these, the expression levels of five miRNAs differed significantly: in the vitrified blastocysts, four miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p) were upregulated, and one (mmu-miR-212-3p) was downregulated. The expression levels of all miRNAs measured by the miRNA Taqman assay based method and qRT-PCR were consistent. The four upregulated miRNAs were predicted to regulate 877 candidate target genes, and the downregulated miRNA was predicted to regulate 231 genes. The biological analysis further showed that the differentially expressed miRNAs mainly regulated the implantation of embryos. In conclusion, the results of our study showed that vitrification significantly altered the miRNA transcriptome in mouse blastocysts, which may decrease the implantation potential of vitrified blastocysts. PMID:25853900

  6. Clinical effectiveness of elective single versus double embryo transfer: meta-analysis of individual patient data from randomised trials

    PubMed Central

    2010-01-01

    Objective To compare the effectiveness of elective single embryo transfer versus double embryo transfer on the outcomes of live birth, multiple live birth, miscarriage, preterm birth, term singleton birth, and low birth weight after fresh embryo transfer, and on the outcomes of cumulative live birth and multiple live birth after fresh and frozen embryo transfers. Design One stage meta-analysis of individual patient data. Data sources A systematic review of English and non-English articles from Medline, Embase, and the Cochrane Central Register of Controlled Trials (up to 2008). Additional studies were identified by contact with clinical experts and searches of bibliographies of all relevant primary articles. Search terms included embryo transfer, randomised controlled trial, controlled clinical trial, single embryo transfer, and double embryo transfer. Review methods Comparisons of the clinical effectiveness of cleavage stage (day 2 or 3) elective single versus double embryo transfer after fresh or frozen in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) treatments were included. Trials were included if the intervention differed only in terms of the intended number of embryos to be transferred. Trials that involved only blastocyst (day five) transfers were excluded. Results Individual patient data were received for every patient recruited to all eight eligible trials (n=1367). A total of 683 and 684 women randomised to the single and double embryo transfer arms, respectively, were included in the analysis. Baseline characteristics in the two groups were comparable. The overall live birth rate in a fresh IVF cycle was lower after single (181/683, 27%) than double embryo transfer (285/683, 42%) (adjusted odds ratio 0.50, 95% confidence interval 0.39 to 0.63), as was the multiple birth rate (3/181 (2%) v 84/285 (29%)) (0.04, 0.01 to 0.12). An additional frozen single embryo transfer, however, resulted in a cumulative live birth rate not significantly lower than the rate after one fresh double embryo transfer (132/350 (38%) v 149/353 (42%) (0.85, 0.62 to 1.15), with a minimal cumulative risk of multiple birth (1/132 (1%) v 47/149 (32%)). The odds of a term singleton birth (that is, over 37 weeks) after elective single embryo transfer was almost five times higher than the odds after double embryo transfer (4.93, 2.98 to 8.18). Conclusions Elective single embryo transfer results in a higher chance of delivering a term singleton live birth compared with double embryo transfer. Although this strategy yields a lower pregnancy rate than a double embryo transfer in a fresh IVF cycle, this difference is almost completely overcome by an additional frozen single embryo transfer cycle. The multiple pregnancy rate after elective single embryo transfer is comparable with that observed in spontaneous pregnancies. PMID:21177530

  7. Pig blastocyst-uterine interactions.

    PubMed

    Bazer, Fuller W; Johnson, Gregory A

    2014-01-01

    The litter-bearing pig is an invaluable model for research in reproductive biology. Spherical pig blastocysts on Day 10 of pregnancy undergo rapid morphological changes to tubular and then filamentous forms by Day 12 and a filamentous conceptus of almost 1m in length by Day 16 of pregnancy. Thus, trophectoderm of each conceptus achieves intimate contact with luminal uterine epithelium (LE) for exchange of nutrients, gases, hormones, growth factors and other key molecules for survival and development. Estrogens secreted between Days 11 and 13 of pregnancy signals pregnancy recognition to ensure that nutrients and prostaglandin F2-alpha (PGF) are secreted into the uterine lumen (exocrine secretion) rather than into the uterine vein (endocrine secretion) which would lead to regression of the corpora lutea (CL) and failure to maintain pregnancy. Pigs have a true epitheliochorial placenta. The fluid filled amnion bouys the embryo so that it develops symmetrically. The allantois fills with allantoic fluid to expand contact of the chorioallantois with uterine LE, and the allanotois supports the vascular system of the placenta. The chorion/trophectoderm in direct contact with uterine LE exchanges gases and nutrients and forms unique structures call areolae that absorb nutrient-rich secretions from uterine glands and transports them directly into fetal blood. The period from Days 20 to 70 of pregnancy is for placental growth in preparation for rapid fetal growth between Days 70 and 114 (term) of gestation. Maturation of the fetal hypothalamic-pituitary-adrenal axis leads to increases in secretion of cortisol from the fetal adrenal glands. Cortisol sets in motion secretion of estrogens, oxytocin, relaxin and prolactin, as well as increases in their receptors, which are required for delivery of piglets and for initiation of lactation and expression of maternal behavior. This review provides details of gestation in the pig with respect to uterine biology, implantation, placentation, fetal development and parturition. PMID:24388881

  8. Inadequacy of single-impulse transfers for path constrained rendezvous

    NASA Technical Reports Server (NTRS)

    Stern, S. A.; Soileau, K. M.

    1987-01-01

    The use of single-impulse techniques to maneuver from point to point about a large space structure (LSS) with an arbitrary geometrical configuration and spin is examined. Particular consideration is given to transfers with both endpoints on the forbidden zone surface. Clohessy-Wiltshire equations of relative motion are employed to solve path constrained rendezvous problems. External and internal transfers between arbitrary points are analyzed in terms of tangential departure and arrival conditions. It is observed that single-impulse techniques are inadequate for transferring about the exterior of any LSS; however, single-impulse transfers are applicable for transfers in the interior of LSSs. It is concluded that single-impulse transducers are not applicable for path constrained rendezvous guidance.

  9. Diverse Roles of Prostaglandins in Blastocyst Implantation

    PubMed Central

    2014-01-01

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

  10. Trends and Correlates of Monozygotic Twinning After Single Embryo Transfer

    PubMed Central

    Kanter, Jessica R.; Boulet, Sheree L.; Kawwass, Jennifer F.; Jamieson, Denise J.; Kissin, Dmitry M.

    2015-01-01

    OBJECTIVE To evaluate trends of monozygotic twinning after single embryo transfer and its association with patient and treatment factors. METHODS Our retrospective cohort study included 28,596 pregnancies after fresh, nondonor single embryo transfer during 20032012 reported to the National ART Surveillance System. We examined trends of monozygotic twin pregnancies (number of fetal heart tones on first-trimester ultrasonography more than one or number of neonates born more than one) and assessed patient and treatment factors for monozygotic twin compared with singleton pregnancies. Modified Poisson regression models were used to estimate adjusted risk ratios (RRs) and 95% confidence intervals (CIs) for association between monozygotic twinning and selected factors stratified by day 23 and day 56 transfer. RESULTS During 20032012, the incidence of monozygotic twinning after single embryo transfer was lower for day 23 transfers than for day 56 transfers (1.71%, 95% CI 1.451.98, n=162 compared with 2.50%, 95% CI 2.282.73, n=472); the incidence did not change significantly over the study period. Among day 23 transfers, assisted hatching increased the risk for monozygotic twinning compared with singletons (adjusted RR 2.16, 95% CI 1.533.06); use of intracytoplasmic sperm injection decreased the risk (adjusted RR 0.60, 95% CI 0.420.85). Having one or more prior pregnancies increased the risk for monozygotic twinning among day 56 transfers (adjusted RR 1.26, 95% CI 1.031.53). CONCLUSION Monozygotic twinning after single embryo transfers was more common among day 56 embryo transfers than day 23 transfers. Use of assisted hatching was associated with increased risk for monozygotic twinning for day 23 transfers. PMID:25560112

  11. Progesterone replacement with vaginal gel versus i.m. injection: cycle and pregnancy outcomes in IVF patients receiving vitrified blastocysts

    PubMed Central

    Shapiro, Daniel B.; Pappadakis, Jennifer A.; Ellsworth, Nancy M.; Hait, Howard I.; Nagy, Zsolt Peter

    2014-01-01

    STUDY QUESTION Does the type of luteal support affect pregnancy outcomes in recipients of vitrified blastocysts? SUMMARY ANSWER Luteal support with vaginal progesterone gel or i.m. progesterone (IMP) results in comparable implantation and pregnancy rates in IVF patients receiving vitrified blastocysts. WHAT IS KNOWN ALREADY In fresh IVF cycles, both IMP and vaginal progesterone have become the standard of care for luteal phase support. Due to conflicting data in replacement cycles, IMP is often considered to be the standard of care. STUDY DESIGN, SIZE, DURATION Retrospective analysis of 920 frozen embryo transfer (FET) cycles between 1 January 2010 and 1 September 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients from a large, private practice undergoing autologous and donor FET using IMP or vaginal progesterone gel for luteal support were included in the analysis. IMP was used for luteal support in 682 FET cycles and vaginal progesterone gel was used in 238 FET cycles. Standard clinical outcomes of positive serum hCG levels, implantation, clinical pregnancy, spontaneous abortion and live birth were reported. MAIN RESULTS AND THE ROLE OF CHANCE The IMP and vaginal progesterone gel groups had similar patient demographics for all characteristics assessed. Implantation rates (46.4 versus 45.6%, P = 0.81), clinical pregnancy rates (61.7 versus 60.5%, P = 0.80) and live birth rates (49.1 versus 48.9%, P > 0.99) were not significantly different between IMP and vaginal progesterone gel, respectively. LIMITATIONS, REASONS FOR CAUTION This study is limited by its retrospective design and by its lack of randomization to the type of luteal support. In addition, because no a priori expected rates of success could be provided for this retrospective investigation, it was not possible to estimate statistical power associated with the various outcomes presented. WIDER IMPLICATIONS OF THE FINDINGS With the recent trends toward single embryo transfer (SET) and use of vitrified blastocysts in FET cycles, our data with ?40% of cycles being SET and use of exclusively vitrified blastocysts are more relevant to current practices than previous studies. STUDY FUNDING/COMPETING INTEREST(S) Support for data collection and analysis was provided by Actavis, Inc. D.S. has received honoraria for lectures and participation in Scientific Advisory Boards for Actavis, Inc. J.P. is an employee of Actavis, Inc. N.E. has received payment from Actavis, Inc., for her time for data collection. H.H. has received payment from Actavis, Inc., for statistical analyses. Z.P.N. has nothing to disclose. PMID:24847018

  12. 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 2.510 ?M 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 2.510 ?M 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

  13. Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus.

    PubMed Central

    Tinland, B; Hohn, B; Puchta, H

    1994-01-01

    Transferred DNA (T-DNA) is transferred as a single-stranded derivative from Agrobacterium to the plant cell nucleus. This conclusion is drawn from experiments exploiting the different properties of single- and double-stranded DNA to perform extrachromosomal homologous recombination in plant cells. After transfer from Agrobacterium to plant cells, T-DNA molecules recombined much more efficiently if the homologous sequences were of opposite polarity than if they were of the same polarity. This observation reflects the properties of single-stranded DNA; single-stranded DNA molecules of opposite polarity can anneal directly, whereas single-stranded DNA molecules of the same polarity first have to become double stranded to anneal. Judging from the relative amounts of single- to double-stranded T-DNA derivatives undergoing recombination, we infer that the T-DNA derivatives enter the plant nucleus in their single-stranded form. Images PMID:11607492

  14. Comparison of Tetraploid Blastocyst Microinjection of Outbred Crl:CD1(ICR), Hybrid B6D2F1/Tac, and Inbred C57BL/6NTac Embryos for Generation of Mice Derived from Embryonic Stem Cells

    PubMed Central

    Kirchain, Sharron M; Hayward, Alison M; Mkandawire, John M; Qi, Peimin; Burds, Aurora A

    2008-01-01

    Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%). PMID:18524172

  15. The Role of Mitochondria from Mature Oocyte to Viable Blastocyst

    PubMed Central

    2013-01-01

    The oocyte requires a vast supply of energy after fertilization to support critical events such as spindle formation, chromatid separation, and cell division. Until blastocyst implantation, the developing zygote is dependent on the existing pool of mitochondria. That pool size within each cell decreases with each cell division. Mitochondria obtained from oocytes of women of advanced reproductive age harbor DNA deletions and nucleotide variations that impair function. The combination of lower number and increased frequency of mutations and deletions may result in inadequate mitochondrial activity necessary for continued embryo development and cause pregnancy failure. Previous reports suggested that mitochondrial activity within oocytes may be supplemented by donor cytoplasmic transfer at the time of intracytoplasmic sperm injection (ICSI). Those reports showed success; however, safety concerns arose due to the potential of two distinct populations of mitochondrial genomes in the offspring. Mitochondrial augmentation of oocytes is now reconsidered in light of our current understanding of mitochondrial function and the publication of a number of animal studies. With a better understanding of the role of this organelle in oocytes immediately after fertilization, blastocyst and offspring, mitochondrial augmentation may be reconsidered as a method to improve oocyte quality. PMID:23766762

  16. Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice.

    PubMed

    Wang, Xiaoqian; Catt, Sally; Pangestu, Mulyoto; Temple-Smith, Peter

    2009-09-01

    Ovarian tissue cryopreservation and transplantation can be used to preserve fertility for cancer patients. In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by solid surface vitrification or traditional slow-cooling using various in vitro and in vivo techniques, including allotransplantation, in vitro oocyte maturation, embryo culture in vitro, blastocyst cryopreservation, embryo transfer, and development. The importance of cumulus cells for oocyte maturation, fertilization, and embryo development was investigated. Graft recovery, follicle survival, and oocyte retrieval was similar in control, vitrified, and slow-cooled groups. High rates of oocyte maturation, cleavage, and blastocyst formation were achieved, with no significant differences between the control, vitrified or slow-cooled ovarian tissue grafts. The presence of cumulus cells was important for oocyte maturation, fertilization, and subsequent development. Cumulus-oocyte complexes with no surrounding cumulus cells (N-COCs) or with an incomplete layer (P-COCs) had significantly lower rates of oocyte maturation and blastocyst formation than cumulus-oocyte complexes with at least one complete layer of cumulus cells (F-COCs; maturation rate: 63, 78 vs 94%; blastocyst rate: 29, 49 vs 80%). Live births were achieved using vitrified blastocysts derived from oocytes taken from vitrified and slow-cooled ovarian tissue heterotypic allografts. Successful production of healthy offspring from these vitrified blastocysts suggests that this technique should be considered as a useful stage to pause in the assisted reproduction pathway. This provides an alternative protocol for restoring fertility and offering cancer patients a better indication of their chances of pregnancy and live birth. PMID:19556437

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

    PubMed Central

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

    2015-01-01

    Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts. PMID:26536655

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

    PubMed Central

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

    2011-01-01

    The electron transfer (ET) dynamics from core/multi-shell (CdSe/CdS3MLZnCdS2MLZnS2ML) 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. PMID:21915369

  19. Adiponectin stimulates glucose uptake in rabbit blastocysts.

    PubMed

    Fischer, Suenje; Santos, Anne Navarrete; Thieme, Ren; Ramin, Nicole; Fischer, Bernd

    2010-11-01

    Since the discovery of adipokines, the adipose tissue is no longer considered to be an inactive fat storage. It secretes a variety of bioactive molecules, which regulate body metabolism and energy homeostasis. One of these molecules is the adipokine adiponectin. In different tissues, adiponectin triggers metabolic effects through the adenosine monophosphate-activated protein kinase (PRKA), which is a master regulator in glucose and lipid metabolism. Recent studies point to a role for adiponectin in reproduction. Adiponectin and its receptors are present in female reproductive tract during pregnancy, and the preimplantation embryo is fully equipped with adiponectin. Here, we show that both receptor isoforms, ADIPOR1 and ADIPOR2, are expressed in 6-day-old rabbit blastocysts. To investigate the signaling pathway of adiponectin in preimplantation embryos, rabbit blastocysts were cultured in vitro and stimulated with adiponectin. Supplementation of adiponectin (1 ?g/ml) enhanced PRKA alpha 1/2 (PRKAA1/2) phosphorylation and decreased expression of phosphoenolpyruvate carboxykinase 2 (PCK2), a key regulator of gluconeogenesis. Inhibition of PRKAA1/2 by Compound C (10 ?M) restored PCK2 transcription. Adiponectin enhanced embryonic glucose uptake and led to a translocation of solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4), previously known as GLUT4. We conclude that adiponectin influences the glucose metabolism of rabbit blastocysts via the phosphorylation of PRKAA1/2, which in turn results in a decrease of gluconeogenesis and an increase in glycolysis. The regulatory influence of adiponectin on glucose metabolism of blastocysts may be of specific interest in pathophysiological situations, such as obesity during pregnancy. PMID:20686181

  20. 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

  1. 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.

  2. Rho-Associated Kinase Activity Is Required for Proper Morphogenesis of the Inner Cell Mass in the Mouse Blastocyst1

    PubMed Central

    Laeno, Arlene May A.; Tamashiro, Dana Ann A.; Alarcon, Vernadeth B.

    2013-01-01

    ABSTRACT The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births. PMID:23946538

  3. 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

  4. 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

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

    PubMed Central

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

    2016-01-01

    STUDY QUESTION Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners? SUMMARY ANSWER The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment. WHAT IS KNOWN ALREADY Recently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes. STUDY DESIGN, SIZE, DURATION This longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET). PARTICIPANTS/MATERIALS, SETTING, METHODS Seven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical outcomes following FEET cycles were stratified by biopsy operator and compared. Cellularity of the biopsy sample was also correlated with clinical outcomes. MAIN RESULTS AND THE ROLE OF CHANCE The seven practitioners performed 2586 biopsies, five in centre IVF-1 and one in each of the other two IVF centres (IVF-2 and IVF-3). Overall, 2437 out of 2586 (94.2%) blastocyst biopsies resulted in a conclusive diagnosis, 119 (4.6%) showed a nonconcurrent result and 30 (1.2%) failed to amplify, suggesting the absence of TE cells in the test tube or presence of degenerated/lysed cells only. Among the samples producing a conclusive diagnosis, a mean concurrence value of 0.253 (95% CI = 0.250–0.257) was observed. Logistic regression analysis adjusted for confounding factors showed no differences in the diagnosis rate and in the concurrence of the genetic analysis between different biopsy practitioners. An overall mean number of 7.32 cells (95% CI = 6.82–7.81; range 2–15) were predicted from all biopsies. Higher cellularity was significantly associated with a better quality of the CCS diagnosis (P < 0.01) and with the conclusive diagnosis rate, with nonconcurrent samples showing significantly lower numbers of cells (2.1; 95% CI=1.5–2.7) compared with samples resulting in a conclusive diagnosis (mean cells number 7.5; 95% CI = 7.1–7.9, P < 0.01). However, no differences were recorded between different biopsy practitioners regarding cellularity of the biopsy. Finally, logistic analysis showed no impact of the biopsy practitioners on the observed ongoing rates of implantation, biochemical pregnancy loss and miscarriage after the FEET cycles. LIMITATIONS, REASONS FOR CAUTION These data come from a restricted set of laboratories where all of the embryologists received identical training and use identical equipment. A single TE biopsy method and CCS technology was used and these data particularly apply to PGS programmes using blastocyst biopsy without zona opening at the cleavage stage and using qPCR-based CCS. To make firm conclusions on the potential impact of biopsy on biochemical pregnancy loss and miscarriages according to practitioner and biopsy cellularity, a larger sample size is needed. WIDER IMPLICATIONS OF THE FINDINGS We reported a very high consistency and reproducibility of the blastocyst biopsy approach coupled with qPCR-based CSS for both genetic and clinical outcomes across different practitioners working in different IVF centres when appropriate training is provided and when the same laboratory setting is used. These data are important considering the trend towards the use of blastocyst biopsy worldwide for PGD/PGS applications. STUDY FUNDING/COMPETING INTEREST(S) None. PMID:26637492

  6. Technique of Coronary Transfer for TGA with Single Coronary Artery

    PubMed Central

    Kim, Tae Ho; Jung, Jae Jun; Kim, Yong Han; Yang, Ji-Hyuk; Jun, Tae-Gook

    2014-01-01

    An eight-day-old neonate was diagnosed with dextro-transposition of the great arteries, atrial septal defect, patent ductus arteriosus, and a single sinus origin of the coronary arteries. The single coronary artery originated from the left sinus (sinus 2), had a proximal left circumflex arterial branch, and passed anteriorly to the right side of the aorta, further branching into the right coronary and left anterior descending arteries. We successfully performed an arterial switch operation and coronary transfer by tube graft reconstruction with autologous aortic tissue to treat the dextro-transposition of the great arteries and atrial septal defect with a single-sinus origin of the coronary arteries. PMID:25551074

  7. Expression of mRNA, before and after freezing, in bovine blastocysts cultured under different conditions.

    PubMed

    Kuzmany, Anna; Havlicek, Vitezslav; Wrenzycki, Christine; Wilkening, Sandra; Brem, Gottfried; Besenfelder, Urban

    2011-02-01

    Production methods and culture systems have been shown to affect blastocyst mRNA expression and cryopreservability, which may serve as sensitive indicators of embryo quality and developmental competence. In the present study, the impact of four established culture conditions for producing bovine blastocysts (in vitro production, IVP; gamete intra-fallopian transfer, GIFT; transfer of cleaved stages into the oviduct, CLVT; multiple ovulation embryo transfer, MOET) was assessed, in terms of both cryosurvival and levels of mRNA expression of several selected genes (occludin, desmocollin 2, solute carrier family 2 member 3, BAX, BCL-XL, heat shock protein 1A, aquaporin 3, DNA methyltransferase 1a) detected with RT-qPCR. At 24 hours post-thawing, blastocysts derived from in vitro production showed a significantly higher re-expansion rate compared to the other groups. At later times, this difference was no longer significant. Before freezing, embryos of the MOET group showed significantly more desmocollin 2 mRNA compared to embryos produced using other culture methods. After freezing, significant upregulation was found in transcripts of heat shock protein 1A in embryos of all groups; of solute carrier family 2 member 3, only in IVP derived embryos; of BAX, BCL-XL, occludin, desmocollin 2, only in the MOET and IVP groups. Aquaporin 3 and DNA methyltransferase 1a were neither up- nor downregulated in blastocysts of any group. In conclusion, these findings suggest that, after freezing, embryos seem to have switched on mRNA synthesis, an active metabolism, operational cell connections, and are prepared for hatching and beyond. PMID:21144573

  8. 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

  9. Time-lapse videomicrographic analyses of contractions in mouse blastocysts.

    PubMed

    Niimura, Sueo

    2003-12-01

    Contraction has been observed in cultured blastocysts of many mammals, but little is known about the features of the contraction and its physiological role in blastocysts. The author analyzed contractions of a large number of cultured mouse blastocysts by time-lapse videomicrography. The results revealed that blastocysts repeated contractions of different degrees during the expanded stage from 10 h after blastocoel formation, and that the number of contractions was greater during the hatching period than in the periods pre- and post-hatching. The results also showed that the time needed for both contraction and re-expansion to the size before contraction tended to lengthen in blastocysts severely contracted. It was inferred that contractions of blastocysts occur physiologically in relation to myosin light chain kinase, but not due to an increase in permeability between trophectoderm cells in association with their division, or the influence of culture. Furthermore, it was inferred that re-expansion of contracted blastocysts occurs due to active transport and accumulation of Na(+) from the trophectoderm cells into blastocoelic fluid as a result of the action of Na(+)/K(+)-ATPase activated in the membrane of trophectoderm cells. Our results suggested that contractions are also present in blastocysts developed in vivo, and that weak contractions (less than 20% volume reduction) play an important role in hatching, whereas strong contractions (20% or more volume reduction) have the effect of inhibiting hatching. From our results on contractions of various blastocysts, it seems possible to evaluate the developmental ability of embryos, i.e. embryo quality, based on contractions of blastocysts. PMID:14967891

  10. 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.

  11. 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. ?-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.

  12. 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."

  13. 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

  14. 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. PMID:26584541

  15. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    PubMed

    Kawamura, Kazuhiro; Chen, Yuan; Shu, Yimin; Cheng, Yuan; Qiao, Jie; Behr, Barry; Pera, Renee A Reijo; Hsueh, Aaron J W

    2012-01-01

    Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells. PMID:23152897

  16. Culturing surplus poor-quality embryos to blastocyst stage have positive predictive value of clinical pregnancy rate

    PubMed Central

    Zhu, Hai Bo; Zhang, Zhi Hong; Fadlalla, Elfateh; Wang, Rui Xue; Geng, Dong Feng; Liu, Rui Zhi

    2014-01-01

    Background: Clinical reproductive centers produce large amounts of surplus poor-quality embryos annually, how to maximize the use of these embryos, and which of them have the potential to develop into blastocyst stage and influencing factors were lack of systematic research. Objective: To investigate the fate of surplus poor-quality embryos which were cultured to obtain blastocyst, determine the factors which may influence the blastulation, and discuss their application in predicting of the pregnancy outcomes. Materials and Methods: Day 3 (D3) after embryo transfer and freezing, surplus poor-quality embryos from IVF/ICSI cycles were cultured to blastocyst by the sequential method, then the blastulation outcomes were observed. Focusing on the blastulation rate of those embryos with different number cells and different embryonic grade; and last the relationship between the pregnancy outcomes of remained poor-quality embryos with successful blastulation or failed blastulation groups were studied. Results: Of 127 patients with 569 poor-quality in vitro cultured embryos, there were formation of 248 blastocysts from 91 patients (43.59%), which lead to development of 138 high-quality blastocysts (24.25%). With the increase in cells number of D 3 blastomeres, the blastulation rate gradually increased, that, 7-cell blastomeres blastulation rate was the highest (70.59%), and 8-cell blastomeres is a little below (70.37%); while the embryonic levels and blastulation rate did not show this positive relationship. The clinical pregnancy rate and implantation rate of those who had successful blastulation (67.03% and 42.39%) were higher than of those who failed to develop to blastocyst (p=0.039). Conclusion: Day 3 poor-quality embryos with successful blastulation or with failed blastulation had predictive value on pregnancy outcomes. For embryo transfer 7-8 cells grade III-IV embryo is better than 4-5 cells grade I-II embryo, in case of lack good-quality embryos. PMID:25469133

  17. A novel approach to sexing bovine blastocysts using male-specific gene expression.

    PubMed

    Hamilton, C K; Combe, A; Caudle, J; Ashkar, F A; Macaulay, A D; Blondin, P; King, W A

    2012-05-01

    When examining gene expression profiles for the purposes of assessing embryo quality, it is imperative that sex be considered, because many embryonic transcripts have sex-related expression patterns. The objective of this study was to systematically examine eight Y chromosome linked genes (DDX3Y, EIF1AY, HSFY, SRY, TSPY, USP9Y, ZFY, and ZRSR2Y) to characterize their expression in bovine blastocysts and to examine the usefulness of this expression for the purpose of RNA-based embryo sexing. In order to examine the expression of these genes, pools of blastocysts (groups of 10 and 20) as well as single embryos (N = 50) were analyzed with reverse transcriptase polymerase chain reaction (RT-PCR) and reverse transcriptase quantitative PCR (RT-qPCR). Of the 50 single embryos, 32 were concurrently sexed with DNA-based methods. Transcripts of DDX3Y, EIF1AY, TSPY, USP9Y, ZFY and ZRSR2Y were detected in the pooled and single blastocysts, but no transcripts were detected for HSFY or SRY. After performing DNA-based sexing experiments, we concluded that this expression was restricted to the male embryos. The consistency of the expression varied according to the gene as well as the specific primer set. Three genes were expressed in the full set of male embryos, DDX3Y, USP9Y, and ZRSR2Y and therefore represent good candidates for RNA-based sexing methods. PMID:22341705

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    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.

  19. A caprine chimera produced by injection of embryonic germ cells into a blastocyst.

    PubMed

    Jia, W; Yang, W; Lei, A; Gao, Z; Yang, C; Hua, J; Huang, W; Ma, X; Wang, H; Dou, Z

    2008-02-01

    This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28-42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo. PMID:18001826

  20. Transferring color to single-band intensified night vision images

    NASA Astrophysics Data System (ADS)

    Toet, Alexander

    2004-08-01

    We present a method to give single band intensified nightvision imagery a natural day-time color appearance. For input, the method requires a true color RGB source image and a grayscale nightvision target image. The source and target image are both transformed into a perceptually decorrelated color space. In this color space a best matching source pixel is determined for each pixel of the target image. The matching criterion uses the first order statistics of the luminance distribution in a small window around the source and target pixels. Once a best matching source pixel is found, its chromaticity values are assigned (transferred) to the target pixel while the original luminance value of the target pixel is retained. The only requirement of the method is that the compositions of the source and target scenes resemble each other.

  1. Developmental changes in epithelial transport characteristics of preimplantation rabbit blastocysts.

    PubMed Central

    Benos, D J

    1981-01-01

    1. Transepithelial sucrose and urea permeability coefficients were measured in different-aged preimplantation rabbit blastocysts. Sucrose permeability remained constant from 4 to 6 days after fertilization (approximately 1 X 10(-8) cm s-1), but thereafter increased. Urea permeability was very low throughout this period (5 X 10(-7) cm s-1) but a phloretin-sensitive component to urea influx appeared between day 6 and day 7 post coitum (p.c.). 2. Glucose uptake was small (0.2-0.5 nmol cm-2 s-1 and independent of external sodium throughout the preimplantation period. 3. Methionine uptake was 10 times greater than glucose uptake, and was strongly dependent upon the presence of external sodium in 5 and 6 day p.c. blastocysts. The sodium dependence of methionine uptake was lost by the seventh day after fertilization. 4. The transepithelial electrical resistance was always less than 35 omega cm2 in 4 day p.c. blastocysts, and averaged 1758 omega cm2 in 6 day p.c. blastocysts. 5. The transepithelial influx of 140 La averaged 0.46 +/- 0.11 nmol cm-2 h-1 (n = 6) in 4 day p.c. blastocysts. The 5 day p.c. embryos, on the other hand were impermeable to lanthanum (n = 10). 6. These results indicate that the epithelium of the preimplantation rabbit blastocyst undergoes dramatic changes in transport characteristics with development. The blastocyst can be characterized as a 'leaky' transporting epithelium during the early blastocyst period, and as a 'tight' epithelium during the mid to late stages of the preimplantation period. PMID:7320864

  2. Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling.

    PubMed

    Na, Jie; Lykke-Andersen, Karin; Torres Padilla, Maria Elena; Zernicka-Goetz, Magdalena

    2007-02-01

    Wnt signaling is essential for the regulation of cell polarity and cell fate in the early embryogenesis of many animal species. Multiple Wnt genes and its pathway members are expressed in the mouse early embryo, raising the question whether they play any roles in preimplantation development. Dishevelled is an important transducer of divergent Wnt pathways. Here we show that three of the mouse Dishevelled proteins are not only expressed in oocytes and during preimplantation development, but also display distinct spatio-temporal localization. Interestingly, as embryos reach blastocyst stage, Dishevelled 2 becomes increasingly associated with cell membrane in trophectoderm cells, while at E4.5, Dishevelled 3 is highly enriched in the cytoplasm of ICM cells. These changes are coincident with an increase in the active form of beta-catenin, p120catenin transcription and decrease of Kaiso expression, indicating an upregulation of Wnt signaling activity before implantation. When Dishevelled-GFP fusion proteins are overexpressed in single blastomeres of the 4-cell stage embryo, the progeny of this cell show reduction in cell adhesiveness and a rounded shape at the blastocyst stage. This suggests that perturbing Dvl function interferes with cell-cell adhesion through the non-canonical Wnt pathway in blastocysts. PMID:17005174

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

    PubMed

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

    2015-12-01

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

  4. The Prevalence of Chromosomal Deletions Relating to Developmental Delay and/or Intellectual Disability in Human Euploid Blastocysts

    PubMed Central

    He, Wenyin; Sun, Xiaofang; Liu, Lian; Li, Man; Jin, Hua; Wang, Wei-Hua

    2014-01-01

    Chromosomal anomalies in human embryos produced by in vitro fertilization are very common, which include numerical (aneuploidy) and structural (deletion, duplication or others) anomalies. Our previous study indicated that chromosomal deletion(s) is the most common structural anomaly accounting for approximately 8% of euploid blastocysts. It is still unknown if these deletions in human euploid blastocysts have clinical significance. In this study, we analyzed 15 previously diagnosed euploid blastocysts that had chromosomal deletion(s) using Agilent oligonucleotide DNA microarray platform and localized the gene location in each deletion. Then, we used OMIM gene map and phenotype database to investigate if these deletions are related with some important genes that cause genetic diseases, especially developmental delay or intellectual disability. As results, we found that the detectable chromosomal deletion size with Agilent microarray is above 2.38 Mb, while the deletions observed in human blastocysts are between 11.6 to 103 Mb. With OMIM gene map and phenotype database information, we found that deletions can result in loss of 81-464 genes. Out of these genes, 34–149 genes are related with known genetic problems. Furthermore, we found that 5 out of 15 samples lost genes in the deleted region, which were related to developmental delay and/or intellectual disability. In conclusion, our data indicates that all human euploid blastocysts with chromosomal deletion(s) are abnormal and transfer of these embryos may cause birth defects and/or developmental and intellectual disabilities. Therefore, the embryos with chromosomal deletion revealed by DNA microarray should not be transferred to the patients, or further gene map and/or phenotype seeking is necessary before making a final decision. PMID:24409323

  5. 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). PMID:20708232

  6. Controlling energy transfer between multiple dopants within a single nanoparticle

    PubMed Central

    DiMaio, Jeffrey R.; Sabatier, Clment; Kokuoz, Baris; Ballato, John

    2008-01-01

    Complex core-shell architectures are implemented within LaF3 nanoparticles to allow for a tailored degree of energy transfer (ET) between different rare earth dopants. By constraining specific dopants to individual shells, their relative distance to one another can be carefully controlled. Core-shell LaF3 nanoparticles doped with Tb3+ and Eu3+ and consisting of up to four layers were synthesized with an outer diameter of ?10 nm. It is found that by varying the thicknesses of an undoped layer between a Tb3+-doped layer and a Eu3+-doped layer, the degree of ET can be engineered to allow for zero, partial, or total ET from a donor ion to an acceptor ion. More specifically, the ratio of the intensities of the 541-nm Tb3+ and 590 nm Eu3+ peaks was tailored from <0.2 to ?2.4 without changing the overall composition of the particles but only by changing the internal structure. Further, the emission spectrum of a blend of singly doped nanoparticles is shown to be equivalent to the spectra of co-doped particles when a core-shell configuration that restricts ET is used. Beyond simply controlling ET, which can be limiting when designing materials for optical applications, this approach can be used to obtain truly engineered spectral features from nanoparticles and composites made from them. Further, it allows for a single excitation source to yield multiple discrete emissions from numerous lanthanide dopants that heretofore would have been quenched in a more conventional active optical material. PMID:18250307

  7. 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.

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

    SciTech Connect

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

    1987-05-01

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

  9. Rabbit blastocysts accumulate (/sup 3/H)prostaglandins in vitro

    SciTech Connect

    Jones, M.A.; Harper, M.J.

    1984-08-01

    Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with /sup 3/H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of (/sup 3/H)PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of (/sup 3/H)PGE2 and (/sup 3/H)PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the (/sup 3/H)PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. The authors conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation.

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

    PubMed

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

    2016-03-01

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

  11. 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. PMID:25496989

  12. Differential expression of oxygen-regulated genes in bovine blastocysts.

    PubMed

    Harvey, A J; Navarrete Santos, A; Kirstein, M; Kind, K L; Fischer, B; Thompson, J G

    2007-03-01

    Low oxygen conditions (2%) during post-compaction culture of bovine blastocysts improve embryo quality, which is associated with a small yet significant increase in the expression of glucose transporter 1 (GLUT-1), suggesting a role of oxygen in embryo development mediated through oxygen-sensitive gene expression. However, bovine embryos to at least the blastocyst stage lack a key regulator of oxygen-sensitive gene expression, hypoxia-inducible factor 1alpha (HIF1alpha). A second, less well-characterized protein (HIF2alpha) is, however, detectable from the 8-cell stage of development. Here we use differential display to determine additional gene targets in bovine embryos in response to low oxygen conditions. While development to the blastocyst stage was unaffected by the oxygen concentration used during post-compaction culture, differential display identified oxygen-regulation of myotrophin and anaphase promoting complex 1 expression, with significantly lower levels observed following culture under 20% oxygen than 2% oxygen. These results further support the hypothesis that the level of gene expression of specific transcripts by bovine embryos alters in response to changes in the oxygen environment post-compaction. Specifically, we have identified two oxygen-sensitive genes that are potentially regulated by HIF2 in the bovine blastocyst. PMID:16998843

  13. Coherent single-electron transfer in coupled quantum dots

    NASA Astrophysics Data System (ADS)

    Fountoulakis, Antonios; Terzis, Andreas F.; Paspalakis, Emmanuel

    2009-10-01

    We theoretically investigate the coherent transfer of one electron between the ground states of a double coupled quantum dot structure. The coherent transfer of the electron is externally controlled by applied electromagnetic fields with on- or close-resonance driving frequencies and various shapes and duration. We derive the analytical expressions for the parameters of the external fields by approximating the quantum dot system as a three-level ?-type system. The analytical solutions are compared with numerical results and good agreement is found. The control methods developed here are applicable in symmetric and asymmetric quantum dot nanostructures.

  14. Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification.

    PubMed

    Morat, Roser; Castillo-Martn, Mriam; Yeste, Marc; Bonet, Sergi

    2014-12-01

    The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24h. Re-expansion rates were recorded at 3 and 24h and total cell number and apoptotic cells were determined at 24h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification-warming procedures and length of in vitro culture, as expanding and hatching-hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential. PMID:25469448

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

    PubMed

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

    2016-03-15

    Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts. PMID:26639642

  16. 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.

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

    PubMed

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

    2005-04-01

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

  18. 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 applied in cryopreservation cycles. MAIN RESULTS AND THE ROLE OF CHANCE Patient and cycle characteristics were similar in the pre- and post-intervention groups [mean (SD) female age: 39.6 ± 2.1 and 39.4 ± 2.2 years; range 36–44] as assessed by logistic regression. A total of 1609 versus 574 oocyte retrievals, 937 versus 350 embryo warming and 138 versus 27 oocyte warming cycles were performed in the pre- and post-intervention periods, respectively, resulting in 1854 and 508 embryo transfers, respectively. In the post-intervention period, 289 cycles were blastocyst stage with (n = 182) or without PGS (n = 107). A mean (SD) number of 2.9 ± 1.1 (range 1–4) and 1.4 ± 0.8 (range 1–3) embryos were transferred pre- and post-intervention, respectively (P < 0.01) and similar cumulative clinical pregnancy rates per transfer and per cycle were obtained: 26.8, 30.9% and 29.7, 26.3%, respectively. The total DR per oocyte retrieval cycle (21.0 and 20.4% pre- and post-intervention, respectively) defined as efficacy was not affected by the intervention [odds ratio (OR) = 0.8, 95% confidence interval (CI) = 0.7–1.1; P = 0.23]. However, a significantly increased live birth rate per transferred embryo (defined as efficiency) was observed in the post-intervention group 17.0 versus 10.6% (P < 0.01). Multiple DRs decreased from 21.0 in the preintervention to 6.8% in the post-intervention group (OR = 0.3. 95% CI = 0.1–0.7; P < 0.01). LIMITATIONS, REASONS FOR CAUTION In this study, the suitability of SET was assessed in individual women on the basis of both clinical and embryological prognostic factors and was not standardized. For the described eSET strategy coupled with an enhanced embryo selection policy, an optimized culture system, cryopreservation and aneuploidy screening programme is necessary. WIDER IMPLICATIONS OF THE FINDINGS Owing to the increased maternal morbidity and perinatal complications related to multiple pregnancies, it is recommended to extend the eSET policy to the AMA population. As shown in this study, enhanced embryo selection procedures might allow a reduction in the number of embryos transferred and the number of transfers to be performed without affecting the total efficacy of the treatment but increasing efficiency and safety. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER None. PMID:26150408

  19. Wdr74 Is Required for Blastocyst Formation in the Mouse

    PubMed Central

    Holston, Olivia; Adams, Danielle; Mager, Jesse

    2011-01-01

    Preimplantation is a dynamic developmental period during which a combination of maternal and zygotic factors program the early embryo resulting in lineage specification and implantation. A reverse genetic RNAi screen in mouse embryos identified the WD Repeat Domain 74 gene (Wdr74) as being required for these critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse. PMID:21799883

  20. Automatic segmentation of trophectoderm in microscopic images of human blastocysts.

    PubMed

    Singh, Amarjot; Au, Jason; Saeedi, Parvaneh; Havelock, Jon

    2015-01-01

    Accurate assessment of embryos viability is an extremely important task in the optimization of in vitro fertilization treatment outcome. One of the common ways of assessing the quality of a human embryo is grading it on its fifth day of development based on morphological quality of its three main components (Trophectoderm, Inner Cell Mass, and the level of expansion or the thickness of its Zona Pellucida). In this study, we propose a fully automatic method for segmentation and measurement of TE region of blastocysts (day-5 human embryos). Here, we eliminate the inhomogeneities of the blastocysts surface using the Retinex theory and further apply a level-set algorithm to segment the TE regions. We have tested our method on a dataset of 85 images and have been able to achieve a segmentation accuracy of 84.6% for grade A, 89.0% for grade B, and 91.7% for grade C embryos. PMID:25216475

  1. Transfer-free batch fabrication of single layer graphene transistors.

    PubMed

    Levendorf, Mark P; Ruiz-Vargas, Carlos S; Garg, Shivank; Park, Jiwoong

    2009-12-01

    Full integration of graphene into conventional device circuitry would require a reproducible large scale graphene synthesis that is compatible with conventional thin film technology. We report the synthesis of large scale single layer graphene directly onto an evaporated copper film. A novel fabrication method was used to directly pattern these graphene sheets into devices by simply removing the underlying copper film. Raman and conductance measurements show that the mechanical and electrical properties of our single layer graphene are uniform over a large area, ( Ferrari, A. C. et al. Phys. Rev. Lett. 2006, 97, 187401.) which leads to a high device yield and successful fabrication of ultra long (>0.5 mm) graphene channels. Our graphene based devices present excellent electrical properties including a promising carrier mobility of 700 cm(2)/V.s and current saturation characteristics similar to devices based on exfoliated graphene ( Meric, I.. et al. Nat Nanotechnol. 2008, 3, 654-659). PMID:19860406

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Gigahertz single-hole transfer in Si tunable-barrier pumps

    NASA Astrophysics Data System (ADS)

    Yamahata, Gento; Karasawa, Takeshi; Fujiwara, Akira

    2015-01-01

    We report high-speed single-hole (SH) transfer using Si tunable-barrier pumps comprising p-type metal-oxide-semiconductor field-effect transistors. A clear SH-transfer-current plateau with the current level of about 160 pA was observed when a clock signal having a frequency of 1 GHz was applied to one of the gates. Temperature dependence measurements of the transfer current reveal that the transfer probability is dominated by non-equilibrium SH escape by thermal hopping from the electrically formed charge island. The lower bound of the relative error rate for the 1-GHz transfer is about 10-3 at a temperature of about 17 K. In addition, we investigate the frequency dependence of the transfer, where we discuss possible sources causing the change in the error rate. These results pave the way for accurate manipulation of SHs and its application to metrological current standards.

  7. 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.

  8. Spatially Mapping Energy Transfer from Single Plasmonic Particles to Semiconductor Substrates via STEM/EELS.

    PubMed

    Li, Guoliang; Cherqui, Charles; Bigelow, Nicholas W; Duscher, Gerd; Straney, Patrick J; Millstone, Jill E; Masiello, David J; Camden, Jon P

    2015-05-13

    Energy transfer from plasmonic nanoparticles to semiconductors can expand the available spectrum of solar energy-harvesting devices. Here, we spatially and spectrally resolve the interaction between single Ag nanocubes with insulating and semiconducting substrates using electron energy-loss spectroscopy, electrodynamics simulations, and extended plasmon hybridization theory. Our results illustrate a new way to characterize plasmon-semiconductor energy transfer at the nanoscale and bear impact upon the design of next-generation solar energy-harvesting devices. PMID:25845028

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    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.

  10. 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

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

    PubMed

    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

  12. Transferred large area single crystal MoS2 field effect transistors

    NASA Astrophysics Data System (ADS)

    Lee, Choong Hee; McCulloch, William; Lee, Edwin W.; Ma, Lu; Krishnamoorthy, Sriram; Hwang, Jinwoo; Wu, Yiying; Rajan, Siddharth

    2015-11-01

    Transfer of epitaxial, two-dimensional (2D) MoS2 on sapphire grown via synthetic approaches is a prerequisite for practical device applications. We report centimeter-scale, single crystal, synthesized MoS2 field effect transistors (FETs) transferred onto SiO2/Si substrates, with a field-effect mobility of 4.5 cm2 V-1 s-1, which is among the highest mobility values reported for the transferred large-area MoS2 transistors. We demonstrate simple and clean transfer of large-area MoS2 films using deionized water, which can effectively avoid chemical contamination. The transfer method reported here allows standard i-line stepper lithography process to realize multiple devices over the entire film area.

  13. Single and double charge transfer of He(2+) ions with molecules at near-thermal energies

    NASA Technical Reports Server (NTRS)

    Tosh, R. E.; Johnsen, R.

    1993-01-01

    Rate coefficients were measured for charge-transfer reactions of He(2+) ions with H2, N2, O2, CO, CO2, and H2O. The experiments were carried out using a selected-ion drift-tube mass spectrometer. Total rate coefficients are found to be very large and are generally close to the limiting Langevin capture rate coefficients or the corresponding ADO-model (Su and Bowers, 1973) coefficients. The product-ion spectra indicate that both single and double charge transfer and possibly transfer ionization occur in these reactions.

  14. Current quantization due to single-electron transfer in Si-wire charge-coupled devices

    NASA Astrophysics Data System (ADS)

    Fujiwara, Akira; Zimmerman, Neil M.; Ono, Yukinori; Takahashi, Yasuo

    2004-02-01

    We observe a quantized current due to single-electron transfer in a small charge-coupled device, which consists of a narrow Si-wire channel with fine gates; the gate is used to form a tunable barrier potential. By modulating two barrier potentials under the fine gates with phase-shifted pulse voltages, quantized numbers of electrons are injected into and extracted from the charge island sandwiched by the two barriers. Current plateaus due to single-electron transfer are clearly observed at 20 K with frequencies up to 100 MHz and a current level of 16 pA.

  15. Time-resolved energy transfer from single chloride-terminated nanocrystals to graphene

    SciTech Connect

    Ajayi, O. A. E-mail: cww2104@columbia.edu; Wong, C. W. E-mail: cww2104@columbia.edu; Anderson, N. C.; Wolcott, A.; Owen, J. S.; Cotlet, M.; Petrone, N.; Hone, J.; Gu, T.; Gesuele, F.

    2014-04-28

    We examine the time-resolved resonance energy transfer of excitons from single n-butyl amine-bound, chloride-terminated nanocrystals to two-dimensional graphene through time-correlated single photon counting. The radiative biexponential lifetime kinetics and blinking statistics of the individual surface-modified nanocrystal elucidate the non-radiative decay channels. Blinking modification as well as a 4 reduction in spontaneous emission were observed with the short chloride and n-butylamine ligands, probing the energy transfer pathways for the development of graphene-nanocrystal nanophotonic devices.

  16. Planar embryos have poor prognosis in terms of blastocyst formation and implantation.

    PubMed

    Ebner, T; Maurer, M; Shebl, O; Moser, M; Mayer, R B; Duba, H C; Tews, G

    2012-09-01

    Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include embryos that are characterized by a particular planar constellation of four blastomeres with presumed incomplete cleavage. Since little is known on the developmental fate of such conceptuses, within a 10-month period all consecutive patients were screened for day-2 planar embryos. A total of 64/2070 embryos with suboptimal blastomere configuration were detected (3.1%). In conventional IVF, planar embryos were significantly less frequent (0.7%) as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular sperm extraction (5.4%; P<0.01). Interestingly, embryos with a cleavage anomaly showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (P<0.001) and blastocyst quality (P=NS) was higher in tetrahedral embryos. There was a significant increase in implantation rate if tetrahedral embryos could be transferred compared with when planar embryos had to be transferred (P<0.01). It may be postulated that, in planar embryos, the mitotic spindle might have been affected, e.g. sperm centrosome composition or function, which in turn might have led to the observed cleavage anomaly. Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include more planar embryos that are characterized by a particular flat constellation of four blastomeres with presumed premature cleavage (like a tetrafoliate clover). Since little is known on the developmental fate of such embryos within a 10-month study period, all consecutive patients were screened for the presence of day-2 planar embryos (study group). A total of 64 (out of 2070) embryos with abnormal blastomere configuration were detected (3.1%). Interestingly, in conventional IVF (0.7%), the presence of planar embryos was significantly less frequent as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular biopsy (5.4%; P<0.01). Embryos from the study group showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (survival to day 5 of preimplantation development) was higher in the normally cleaved control group (P<0.001) and so was blastocyst quality; however, the latter parameter did not reach level of significance. This was also reflected in a significantly higher implantation rate in the control group (P<0.01). Based on present data, it may be postulated that, in planar embryos, the mitotic spindle (which involves the sperm centrosome) might have been affected, which in turn might have led to an incomplete cleavage. PMID:22796233

  17. Distinct differences in global gene expression profiles in non-implanted blastocysts and blastocysts resulting in live birth.

    PubMed

    Kirkegaard, Kirstine; Villesen, Palle; Jensen, Jacob Malte; Hindkjr, Johnny Juhl; Klvraa, Steen; Ingerslev, Hans Jakob; Lykke-Hartmann, Karin

    2015-10-25

    Results from animal models points towards the existence of a gene expression profile that is distinguishably different in viable embryos compared with non-viable embryos. Knowledge of human embryo transcripts is however limited, in particular with regard to how gene expression is related to clinical outcome. The purpose of the present study was therefore to determine the global gene expression profiles of human blastocysts. Next Generation Sequencing was used to identify genes that were differentially expressed in non-implanted embryos and embryos resulting in live birth. Three trophectoderm biopsies were obtained from morphologically high quality blastocysts resulting in live birth and three biopsies were obtained from non-implanting blastocysts of a comparable morphology. Total RNA was extracted from all samples followed by complete transcriptome sequencing. Using a set of filtering criteria, we obtained a list of 181 genes that were differentially expressed between trophectoderm biopsies from embryos resulting in either live birth or no implantation (negative hCG), respectively. We found that 37 of the 181 genes displayed significantly differential expression (p<0.05), e.g. EFNB1, CYTL1 and TEX26 and TESK1, MSL1 and EVI5 in trophectoderm biopsies associated with live birth and non-implanting, respectively. Out of the 181 genes, almost 80% (145 genes) were up-regulated in biopsies from un-implanted embryos, whereas only 20% (36 genes) showed an up-regulation in the samples from embryos resulting in live birth. Our findings suggest the presence of molecular differences visually undetectable between implanted and non-implanted embryos, and represent a proof of principle study. PMID:26117173

  18. Proton transfer and photoluminescence intermittency of single emitters in dyed crystals.

    PubMed

    Riley, Erin A; Hess, Chelsea M; Pioquinto, Jan Rey L; Kaminsky, Werner; Kahr, Bart; Reid, Philip J

    2013-04-25

    The role of proton transfer in the photoluminescence intermittency (PI) of single molecules of violamine R (VR) overgrown in potassium acid phthalate (KAP) crystals is evaluated in comparisons of protonated (KAP) and deuterated (DKAP) mixed crystals between 23 and 60 C. The PI is analyzed by the construction of cumulative distribution functions that are statistically compared. We find that the on- and off-interval duration distributions change with isotopic substitution consistent with proton transfer contributing to the PI of VR. The on- and off-interval duration distributions have distinct temperature dependencies consistent with different mechanisms for dark state production and decay. Additional evidence for proton-transfer is provided by distributions of single molecule emission-energy maxima that reflect emission from protonated and deprotonated VR. A mechanism for the PI of KAP is presented, where the dark state is assigned to formation of the colorless, leuco form of VR, formed by proton transfer from VR to the KAP lattice, and decay of the dark state involves ring-opening promoted by proton transfer from KAP to VR. The distributed kinetics for dark-state production and decay are modeled using a log-normal distribution for the PI data in preference to a power-law previously assumed. A discussion of the log-normal distribution with regards to PI and proton transfer is presented. PMID:22913588

  19. Embryo Aggregation Promotes Derivation Efficiency of Outgrowths from Porcine Blastocysts

    PubMed Central

    Lee, Sang-Goo; Park, Jin-Kyu; Choi, Kwang-Hwan; Son, Hye-Young; Lee, Chang-Kyu

    2015-01-01

    Porcine embryonic stem cells (pESCs) have become an advantageous experimental tool for developing therapeutic applications and producing transgenic animals. However, despite numerous reports of putative pESC lines, deriving validated pESC lines from embryos produced in vitro remains difficult. Here, we report that embryo aggregation was useful for deriving pESCs from in vitro-produced embryos. Blastocysts derived from embryo aggregation formed a larger number of colonies and maintained cell culture stability. Our derived cell lines demonstrated expression of pluripotent markers (alkaline phosphatase, Oct4, Sox2, and Nanog), an ability to form embryoid bodies, and the capacity to differentiate into the three germ layers. A cytogenetic analysis of these cells revealed that all lines derived from aggregated blastocysts had normal female and male karyotypes. These results demonstrate that embryo aggregation could be a useful technique to improve the efficiency of deriving ESCs from in vitro-fertilized pig embryos, studying early development, and deriving pluripotent ESCs in vitro in other mammals. PMID:26580280

  20. Human Endometrial CD98 Is Essential for Blastocyst Adhesion

    PubMed Central

    Domnguez, Francisco; Simn, Carlos; Quionero, Alicia; Ramrez, Miguel ngel; Gonzlez-Muoz, Elena; Burghardt, Hans; Cervero, Ana; Martnez, Sebastin; Pellicer, Antonio; Palacn, Manuel; Snchez-Madrid, Francisco; Yez-M, Mara

    2010-01-01

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

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

    PubMed Central

    Itoh, Yuichiro; Arnold, Arthur P.

    2014-01-01

    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. PMID:24817096

  2. 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.; Salm, 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 ?cmp=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.

  3. 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.

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

    PubMed

    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 θ_{cm}^{p}=70°. The longitudinal transfer K_{LL}, 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. PMID:26550716

  5. 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.

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

    DOE PAGESBeta

    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

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

    PubMed

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

    2015-10-28

    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. PMID:26400227

  8. 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. PMID:26731547

  9. 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

  10. The roles of wetting liquid in the transfer process of single layer graphene onto arbitrary substrates.

    PubMed

    Kim, Ju Hun; Yi, Junghwa; Jin, Hyeong Ki; Kim, Un Jeong; Park, Wanjun

    2013-11-01

    Wet transfer is crucial for most device structures of the proposed applications employing single layer graphene in order to take advantage of the unique physical, chemical, bio-chemical and electrical properties of the graphene. However, transfer methodologies that can be used to obtain continuous film without voids, wrinkles and cracks are limited although film perfectness critically depends on the relative surface tension of wetting liquids on the substrate. We report the importance of wetting liquid in the transfer process with a systematic study on the parameters governing film integrity in single layer graphene grown via chemical vapor deposition. Two different suspension liquids (in terms of polar character) are tested for adequacy of transfer onto SiO2 and hexamethyldisiloxane (HMDS). We found that the relative surface tension of the wetting liquid on the surfaces of the substrate is related to transfer quality. In addition, dimethyl sulfoxide (DMSO) is introduced as a good suspension liquid to HMDS, a mechanically flexible substrate. PMID:24245262

  11. Turbulent single-phase and two-phase semi-confined jet impingement heat transfer predictions

    SciTech Connect

    Wheeler, J.P.; Neti, S.

    1999-07-01

    Turbulent single-phase, and two-phase (air and water droplets) jet impingement cases are calculated and analyzed for a variety of configurations and input and boundary conditions including jet Reynolds number (Re{sub w}), non-dimensional distance to target wall (H/W), inlet turbulence intensity, droplet size and injection velocity, and loading ratio. A finite control volume method is used with a two-layer turbulence model for solving the continuous phase flow parameters, and a trajectory approach is used to make two-phase flow calculations. For the single-phase cases examined, a secondary rise in Nusselt number was seen for higher H/W values or larger Reynolds numbers. This secondary peak diminishes and shifts to higher x/2H as H/W is decreased, consistent with the empirical data. Significant enhancement to heat transfer (up to over 3 times that of the corresponding single-phase case) was observed with the introduction of droplets. The significant contributors to the enhanced heat transfer were determined to be the heat-sink provided for the continuous phase, increased continuous phase mass flow rate due to vapor generation, and momentum-induced changes in the flow-field. Wall-to-droplet heat transfer and lift forces were not significant for the set of conditions examined. A reduction in loading ratio substantially reduced heat transfer, and variation of initial injection velocity for small droplets did not have an effect due to the relatively short aerodynamic response time.

  12. Interferometric measurement of the temporal transfer function of a single-shot picosecond streak camera.

    PubMed

    Louradour, F; Vampouille, M; Reynaud, F; Martinelli, G

    1993-05-01

    Temporal beats between two separate pulsed lasers of different adjustable frequency are used as a means of measuring the temporal transfer function of a single-shot picosecond streak camera. We describe two parallel pulse generators that use tunable distributed-feedback dye lasers. These lasers produce two pulses of different frequency that interfere at the camera. The temporal fringe contrast at the camera output provides a direct measure of its transfer function. Fringe spacing combined with spectroscopic measure of the laser wavelengths provides a calibration of the temporal scale. PMID:19802249

  13. 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.

  14. Non-Markovian theory for the waiting time distributions of single electron transfers.

    PubMed

    Welack, Sven; Yan, YiJing

    2009-09-21

    We derive a non-Markovian theory for waiting time distributions of consecutive single electron transfer events. The presented microscopic Pauli rate equation formalism couples the open electrodes to the many-body system, allowing to take finite bias and temperature into consideration. Numerical results reveal transient oscillations of distinct system frequencies due to memory in the waiting time distributions. Memory effects can be approximated by an expansion in non-Markovian corrections. This method is employed to calculate memory landscapes displaying preservation of memory over multiple consecutive electron transfers. PMID:19778104

  15. Gating of single molecule junction conductance by charge transfer complex formation.

    PubMed

    Vezzoli, Andrea; Grace, Iain; Brooke, Carly; Wang, Kun; Lambert, Colin J; Xu, Bingqian; Nichols, Richard J; Higgins, Simon J

    2015-12-01

    The solid-state structures of organic charge transfer (CT) salts are critical in determining their mode of charge transport, and hence their unusual electrical properties, which range from semiconducting through metallic to superconducting. In contrast, using both theory and experiment, we show here that the conductance of metal |single molecule| metal junctions involving aromatic donor moieties (dialkylterthiophene, dialkylbenzene) increase by over an order of magnitude upon formation of charge transfer (CT) complexes with tetracyanoethylene (TCNE). This enhancement occurs because CT complex formation creates a new resonance in the transmission function, close to the metal contact Fermi energy, that is a signal of room-temperature quantum interference. PMID:26510687

  16. 78 FR 41070 - Notice of Single-Case Deviation from Competition Requirements: Transfer of Grantee Request for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-09

    ... Competition Requirements: Transfer of Grantee Request for the Detroit Healthy Start Program, Detroit, MI...). ACTION: Notice of Single-Case Deviation from Competition Requirements: Transfer of Grantee Request for... a grantee transfer without competition for the Detroit Michigan Healthy Start program to...

  17. 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.

  18. Transfer doping of single isolated nanodiamonds, studied by scanning probe microscopy techniques.

    PubMed

    Bolker, Asaf; Saguy, Cecile; Kalish, Rafi

    2014-09-26

    The transfer doping of diamond surfaces has been applied in various novel two-dimensional electronic devices. Its extension to nanodiamonds (ND) is essential for ND-based applications in many fields. In particular, understanding the influence of the crystallite size on transfer doping is desirable. Here, we report the results of a detailed study of the electronic energetic band structure of single, isolated transfer-doped nanodiamonds with nanometric resolution using a combination of scanning tunneling spectroscopy and Kelvin force microscopy measurements. The results show how the band gap, the valence band maximum, the electron affinity and the work function all depend on the ND's size and nanoparticle surface properties. The present analysis, which combines information from both scanning tunneling spectroscopy and Kelvin force microscopy, should be applicable to any nanoparticle or surface that can be measured with scanning probe techniques. PMID:25181280

  19. 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.

  20. Passive techniques for the enhancement of convective heat transfer in single phase duct flow

    NASA Astrophysics Data System (ADS)

    Rainieri, S.; Bozzoli, F.; Cattani, L.

    2014-11-01

    This review presents the main results of the experimental campaign on passive techniques for the enhancement of forced convective single phase heat transfer in ducts, performed in the last years at the Laboratory of the Industrial Engineering Department of the University of Parma by the Applied Physics research group. The research was mainly focused on two passive techniques, widely adopted for the thermal processing of medium and high viscosity fluids, based on wall corrugation and on wall curvature. The innovative compound heat transfer enhancement technique that couples together the effect of wall curvature and of wall corrugation has been investigated as well. The research has been mainly focused on understanding the causal relationship between the heat transfer surface modification and the convection enhancement phenomenon, by accounting the effect of the fluid Prandtl number. The pressure loss penalties were also evaluated. The principal results are presented and discussed.

  1. Boiling heat transfer on single phosphor bronze and copper mesh microstructures

    NASA Astrophysics Data System (ADS)

    Orman, ?ukasz J.

    2014-03-01

    The paper presents experimental results of boiling heat transfer of distilled water and ethyl alcohol on surfaces covered with single layers of wire mesh structures made of phosphor bronze and copper. For each material two kinds of structures have been considered (higher and lower) in order to determine the impact of the height of the structure on boiling heat transfer. The wire diameter of the copper meshes was 0,25 mm and 0,32 mm, while of the bronze meshes: 0,20 mm and 0,25 mm. The structures had the same mesh aperture (distance between the wires - 0,50 mm for copper and 0,40 for bronze) but different wire diameter and, consequently, different height of the layers. The tests have been performed under ambient pressure in the pool boiling mode. The obtained results indicate a visible impact of the layer height on the boiling heat transfer performance of the analysed microstructures.

  2. Partial nitrogen atom transfer: a new synthetic tool to design single-molecule magnets.

    PubMed

    Ding, Mei; Rouzires, Mathieu; Losovyj, Yaroslav; Pink, Maren; Clrac, Rodolphe; Smith, Jeremy M

    2015-09-21

    Incomplete nitrogen atom transfer from the iron(IV) nitride complex PhB(MesIm)3Fe?N to the vanadium(III) complex V(Mes)3(THF) quantitatively provides the bimetallic complex PhB(MesIm)3Fe-N?V(Mes)3. Structural and spectroscopic characterizations reveal that the nitride ligand forms a linear bridge between V(V) and high-spin Fe(II) metal ions, confirming that atom transfer is accompanied by electron transfer. In the presence of an applied dc field, the complex displays slow relaxation of the magnetization, revealing its single-molecule magnet properties with an estimation of the energy barrier at about 10 K. This complex establishes a synthetic principle for the assembly of paramagnetic complexes bridged by nitride ligands. PMID:26322964

  3. 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.

  4. 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

  5. Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

    PubMed

    Gmez, M C; Serrano, M A; Pope, C Earle; Jenkins, J A; Biancardi, M N; Lpez, M; Dumas, C; Galiguis, J; Dresser, B L

    2010-09-01

    The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines. PMID:20708127

  6. Screening Single Phase Laminar Convective Heat Transfer of Nanofluids in a Micro-tube

    NASA Astrophysics Data System (ADS)

    Bitaraf Haghighi, Ehsan; Anwar, Zahid; Lumbreras, Itziar; Aliakbar Mirmohammadi, Seyed; Behi, Mohammadreza; Khodabandeh, Rahmatollah; Palm, Bjrn

    2012-11-01

    Nano scale solid particles dispersed in base fluids are a new class of engineered colloidal solutions called nanofluids. Several studies reported enhancement of heat transfer by using nanofluids. This article reports convective single-phase heat transfer coefficients in an open 30 cm long, 0.50 mm internal diameter stainless steel test section. The setup is used for screening single phase laminar convective heat transfer with water and three different nanofluids: water based Al2O3, ZrO2, and TiO2 (all with 9 wt% of particles). A syringe pump with adjustable pumping speed is used to inject fluids into the test section. Thirteen T-type thermocouples are attached on the outer surface of the test section to record the local wall temperatures. Furthermore, two T-type thermocouples are used to measure inlet and outlet fluid temperatures. A DC power supply is used to heat up the test section and a differential pressure transducer is used to measure the pressure drop across the tube. Furthermore, the effective thermal conductivities of these nanofluids are measured using the Transient Plane Source (TPS) method at a temperature range of 20 - 50C. The experimental average values of heat transfer coefficients for nanofluids are compared with water. Enhancement in heat transfer of nanofluids is observed only when compared at constant Reynolds number (Due to higher viscosity for nanofluids, higher velocity or mass flow rate is required for nanofluids to reach the same Reynolds number). The other methods of comparison: equal mass flow rate, volume flow rate, pressure drop and pumping power did not show any augmentation of the heat transfer coefficient for the tested nanofluids compared to water.

  7. A boiling heat transfer surface for creating a single stream of vapor bubbles.

    NASA Astrophysics Data System (ADS)

    Douglas, Zachary W.; Smith, Marc K.; Glezer, Ari

    2006-11-01

    The high heat transfer rate characteristic of boiling is limited by the vapor-bubble removal rate and a critical transition to film boiling. External forces, such as acoustic waves, can be used to enhance vapor-bubble removal and improve heat transfer. In order to explore such enhanced vapor-bubble removal processes, a boiling heat transfer surface has been designed to control the location, growth, and detachment of a single stream of vapor bubbles. The device consists of an insulating annulus surrounding a thermally conductive pin 1 mm in radius. The upper surfaces of the annulus and the pin have a thin polished electro-plated copper coating. When heated from below, the pin provides a thermal conduit that creates a local hot spot on the copper surface. The majority of all bubble nucleations occur within 3 mm of the center of the pin. A thin hydrophobic coating centered on the hot spot encourages the formation of a single vapor bubble that grows to a size determined by the radius of the hydrophobic coating. When the bubble detaches from the surface, a new vapor bubble forms in the same location. High-speed video and bubble-size and thermal measurements will be presented to document the characteristics and performance of this heat transfer surface.

  8. 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.

  9. Natural convection heat transfer from a single and multiple heated thin cylinders in water

    NASA Astrophysics Data System (ADS)

    Khalifa, Abdul Jabbar N.; Hussien, Zaid Ali

    2015-11-01

    Natural convection heat transfer from a single heated cylinder and three heated cylinders in an assembly of nine cylinders immersed in water is investigated at different heat flux values. The thin cylinders are assembled in three by three array of square pitch having a pitch to diameter ratio of 2. Empirical correlations for the overall Nusselt number versus average modified Rayleigh number are obtained and compared to the literature's correlations.

  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 electrochemical cell. For example, we are able to use this technique to track electroluminescence of single Au NPs, and the electrodeposition of individual Ag NPs in-situ. These metallic NPs are useful to enhance light harvesting in organic photovoltaic systems. The scattering at the surface of an indium tin oxide (ITO) working electrode was measured during a potential sweep. Utilizing Mie scattering theory and high resolution scanning electron microscopy (SEM), the scattering data were used to calculate current-potential curves depicting the electrodeposition of individual Ag NPs. The oxidation of individual presynthesized and electrodeposited Ag NPs was also investigated using fluorescence and DFS microscopies. Our work has produced 1 US provisional patent, 15 published manuscripts, 1 submitted and two additional in-writing manuscripts. 5 graduate students, 1 postdoctoral student, 1 visiting professor, and two undergraduate students have received research training in the area of electrochemistry and optical spectroscopy under support of this award.

  11. 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

  12. Single lanthanide-doped oxide nanoparticles as donors in fluorescence resonance energy transfer experiments.

    PubMed

    Casanova, Didier; Giaume, Domitille; Gacoin, Thierry; Boilot, Jean-Pierre; Alexandrou, Antigoni

    2006-10-01

    We used lanthanide-ion doped oxide nanoparticles, Y(0.6)Eu(0.4)VO(4), as donors in fluorescent resonance energy transfer (FRET) experiments. The choice of these nanoparticles allows us to combine the advantages of the lanthanide-ion emission, in particular the long lifetime and the large Stokes shift between absorption and emission, with the detectability of the nanoparticles at the single-particle level. Using cyanine 5 (Cy5) organic molecules as acceptors, we demonstrated FRET down to the single-nanoparticle level. We showed that, due to the long donor lifetime, unambiguous and precise FRET measurements can be performed in solution even in the presence of large free acceptor concentrations. Highly efficient energy transfer was obtained for a large number of acceptor molecules per donor nanoparticle. We determined FRET efficiencies as a function of Cy5 concentration which are in good agreement with a multiple acceptor-multiple donor calculation. On the basis of the donor emission recovery due to acceptor photobleaching, we demonstrated energy transfer from single-nanoparticle donors in fluorescence microscopy experiments. PMID:17004778

  13. 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.

  14. 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 ?m 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.

  15. 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

  16. 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.

  17. Gating of single molecule junction conductance by charge transfer complex formation

    NASA Astrophysics Data System (ADS)

    Vezzoli, Andrea; Grace, Iain; Brooke, Carly; Wang, Kun; Lambert, Colin J.; Xu, Bingqian; Nichols, Richard J.; Higgins, Simon J.

    2015-11-01

    The solid-state structures of organic charge transfer (CT) salts are critical in determining their mode of charge transport, and hence their unusual electrical properties, which range from semiconducting through metallic to superconducting. In contrast, using both theory and experiment, we show here that the conductance of metal |single molecule| metal junctions involving aromatic donor moieties (dialkylterthiophene, dialkylbenzene) increase by over an order of magnitude upon formation of charge transfer (CT) complexes with tetracyanoethylene (TCNE). This enhancement occurs because CT complex formation creates a new resonance in the transmission function, close to the metal contact Fermi energy, that is a signal of room-temperature quantum interference.The solid-state structures of organic charge transfer (CT) salts are critical in determining their mode of charge transport, and hence their unusual electrical properties, which range from semiconducting through metallic to superconducting. In contrast, using both theory and experiment, we show here that the conductance of metal |single molecule| metal junctions involving aromatic donor moieties (dialkylterthiophene, dialkylbenzene) increase by over an order of magnitude upon formation of charge transfer (CT) complexes with tetracyanoethylene (TCNE). This enhancement occurs because CT complex formation creates a new resonance in the transmission function, close to the metal contact Fermi energy, that is a signal of room-temperature quantum interference. Electronic supplementary information (ESI) available: Synthesis of 1c; experimental details of conductance measurements, formation of charge transfer complexes of 1c and 2 in solution; further details of theoretical methods. See DOI: 10.1039/c5nr04420k

  18. Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein

    NASA Astrophysics Data System (ADS)

    Ha, Taekjip; Ting, Alice Y.; Liang, Joy; Chemla, Daniel S.; Schultz, Peter G.; Weiss, Shimon; Deniz, Ashok A.

    1999-08-01

    Biological molecules together with available labeling chemistries provide an ideal setting to investigate the interaction between two closely spaced dye molecules. The photo-excitation of a donor molecule can be non-radiatively transferred to a near-by acceptor molecule via the induced-dipole-induced-dipole interaction in a distance-dependent manner. In this work, we further elaborate on single-molecule fluorescence resonance energy transfer measurements between two dye molecules attached to a single protein - staphylococcal nuclease molecules [T. Ha, A.Y. Ting, J. Liang, W.B. Caldwell, A.A. Deniz, D.S. Chemla, P.G. Schultz, S. Weiss, Proc. Natl. Acad. Sci. USA 96 (1999) 893-898]. Temporal fluctuations in the energy transfer signal include: (1) reversible transitions to dark states; (2) irreversible photodestruction; (3) intersystem crossing to and from the triplet state; (4) spectral fluctuations; (5) rotational dynamics of the dyes; and (6) distance changes between the two dyes. To extract biologically relevant information from such measurements, an experimental strategy and data analysis schemes are developed. First, abrupt photophysical events, such as (1)-(3) are identified and removed from the data. The remaining slow, gradual fluctuations in the energy transfer signal are due to spectral shifts, rotational dynamics and distance changes of the dyes. Direct measurements of each dye's spectral fluctuation and rotational dynamics indicate that these, by themselves, cannot fully account for the observed energy transfer fluctuations. It is therefore concluded that inter-dye distance changes must be present as well. The distance and orientational dynamics are shown to be dependent on the binding of the active-site inhibitor (deoxythymidine diphosphate) to the protein. The inhibitor most probably affects the protein's stability and the dye-protein interaction, possibly by amplifying the motion of the linker arm between the fluorophore and the protein.

  19. Blinking photoluminescence properties of single TiO2 nanodiscs: interfacial electron transfer dynamics.

    PubMed

    Jeon, Ki-Seok; Oh, Seung-Do; Suh, Yung Doug; Yoshikawa, Hiroyuki; Masuhara, Hiroshi; Yoon, Minjoong

    2009-01-21

    Blinking photoluminescence was observed in single TiO2 nanodiscs (NDs) by using a laser scanning confocal microscope (LSCM)-coupled steady-state and ps-time-resolved photoluminescence (PL) spectroscopic system, while it was not significantly observed for TiO2 quantum dots (QDs). Analysis of the PL blinking time trajectories revealed single-exponential kinetics with the average lifetimes of on-state (approximately 286 ms) and off-state (approximately 58 ms), implying the existence of inherent surface-trap sites which can be filled by photogenerated electron or hole. The PL spectra of single TiO2 NDs exhibited broad surface emissions with four decay times, which may be due to diffusion of the energies of electron or hole trap states related to surface structural changes by modification of TiO2 QDs. These results and the surface structural analysis (IR and XPS) suggests a simple model for the PL blinking of single TiO2 NDs that is based on repetitive interfacial electron transfer to the inherent surface trap sites (4Ti4+-OH) with Auger-assisted hole trapping in the multiple surface states as modified by the diffusive coordinate model and the surface-trap-filling model. Based on this blinking mechanism and kinetics, the rates of the interfacial electron transfer and the back electron transfer in TiO2 NDs were determined to be 18 ns and 58 ms, respectively, which are slow enough to keep the polarization of e-h pairs at the surface for efficient photocatalysis and photovoltaic activities. The present methodology and results may be applicable to obtain surface exciton dynamics of various photoelectronic semiconductor nanostructures. PMID:19283271

  20. Bisphenol a influences blastocyst implantation via regulating integrin ?3 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 ? (ER?) in the pregnant mice. In addition, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression levels of integrin ?3 and trophinin in the endometrium and blastocysts. Conclusion: BPA declines ER? expression in endometrium, and inhibits adhesion protein expression in endometrium and blastocysts, causing the adhesion failure of blastocyst implantation. PMID:26884915

  1. Doping single-walled carbon nanotubes through molecular charge-transfer: a theoretical study.

    PubMed

    Manna, Arun K; Pati, Swapan K

    2010-07-01

    We study the effect of the molecular charge transfer on the electronic structure of metallic (5,5) and semiconducting (8,0) single-walled carbon nanotubes (SWNTs) induced by surface adsorption of various organic donor-acceptor molecules of different affinities using ab initio density functional theory. Our results, obtained from first-principles spin-polarized calculations show that the adsorption of molecules with different affinities reflects the difference in interaction strength that measure the overall energy of adsorption. Moderate values of the binding energy of these surface adsorbed molecular charge-transfer complexes suggest that the nature of interaction is in the physisorption regime, and mainly governs by Coulombic forces. We also find that the large band gap of semiconducting (8,0) SWNT can be tuned through the surface adsorption of selective organic molecules which gives rise to mid-gap localized molecular levels near the Fermi energy with tuning of band gap region. Interestingly, we find that the metallic (5,5) SWNT and semiconducting (8,0) SWNT turn into semiconducting and metallic nanotubes respectively in presence of selective surface adsorbed molecules, corroborating recent experimental findings. We also suggest that these charge transfer effect can be probed through optical conductivity measurement, as the low-frequency profiles are affected by charge transfer. PMID:20648348

  2. Energy transfer in polymer electrophosphorescent light emitting devices with single and multiple doped luminescent layers

    NASA Astrophysics Data System (ADS)

    Kawamura, Yuichiro; Yanagida, Shozo; Forrest, Stephen R.

    2002-07-01

    We study energy transfer in efficient polymer electrophosphorescent organic light emitting diodes (PHOLEDs) using poly(9-vinylcarbazole) (PVK) host doped with one or more phosphorescent cyclometalated Ir(III) complexes. Single dopant double heterostructure PHOLEDs exhibited saturated color luminescence due to emissive triplet metal-to-ligand charge-transfer to ground state transitions of the Ir(III) dopants. Blue PHOLEDs, excited by an endothermic process from the host polymer, exhibited an emission maximum at a wavelength of lambdamax=474 nm, with an external quantum efficiency of eta]ext=1.3[plus-or-minus0.1% and luminous power efficiency of eta]p=0.8[plus-or-minus0.1 lm/W. The green PHOLED exhibited eta]ext=5.1[plus-or-minus0.1%, with etaext>2% for both red and yellow emission. Resonant energy transfer between green emitting fac-tris (2-phenylpyridyl)Ir(III) and red emitting bis2-(2')-benzothienyl-pyridinato- N,C3')(acetylacetonate)Ir(III was observed to nearly double the efficiency of red emission when both dopants were simultaneously blended in a PVK host. PHOLEDs containing a blend of red, yellow, and blue Ir(III) complex dopants produced white light emission with eta]ext=2.1[plus-or-minus0.1%. Our results suggest that deep lying energy states in the PVK conductive matrix may limit the energy transfer efficiency in phosphor doped polymer OLEDs.

  3. Doping single-walled carbon nanotubes through molecular charge-transfer: a theoretical study

    NASA Astrophysics Data System (ADS)

    Manna, Arun K.; Pati, Swapan K.

    2010-07-01

    We study the effect of the molecular charge transfer on the electronic structure of metallic (5,5) and semiconducting (8,0) single-walled carbon nanotubes (SWNTs) induced by surface adsorption of various organic donor-acceptor molecules of different affinities using ab initio density functional theory. Our results, obtained from first-principles spin-polarized calculations show that the adsorption of molecules with different affinities reflects the difference in interaction strength that measure the overall energy of adsorption. Moderate values of the binding energy of these surface adsorbed molecular charge-transfer complexes suggest that the nature of interaction is in the physisorption regime, and mainly governs by Coulombic forces. We also find that the large band gap of semiconducting (8,0) SWNT can be tuned through the surface adsorption of selective organic molecules which gives rise to mid-gap localized molecular levels near the Fermi energy with tuning of band gap region. Interestingly, we find that the metallic (5,5) SWNT and semiconducting (8,0) SWNT turn into semiconducting and metallic nanotubes respectively in presence of selective surface adsorbed molecules, corroborating recent experimental findings. We also suggest that these charge transfer effect can be probed through optical conductivity measurement, as the low-frequency profiles are affected by charge transfer.

  4. Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM.

    PubMed

    Vanderzwalmen, P; Ectors, F; Grobet, L; Prapas, Y; Panagiotidis, Y; Vanderzwalmen, S; Stecher, A; Frias, P; Liebermann, J; Zech, N H

    2009-11-01

    During embryo vitrification, it is advisable that cooling and storage should occur in a carrier device in which there is complete separation of the embryos from liquid nitrogen to ensure asepsis. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. Blastocysts originating from couples with male and/or female factor infertility (group 1) or from oocyte donors (group 2) or from in-vitro matured oocytes (group 3) were gradually exposed to increasing concentrations of dimethylsulphoxide/ethylene glycol (5/5%, 10/10% and 20/20%) before aseptic vitrification using a specially designed carrier (VitriSafe), a modification of the open hemi-straw plug device. A total of 120 aseptic vitrification/warming cycles were performed in group 1, 91 in group 2 and 22 in group 3. Survival rates before embryo transfer, ongoing pregnancy and implantation rates were as follows: for group 1, 73, 43 and 26%; for group 2, 88, 53 and 34%; and for group 3, 69, 50 and 38%, respectively. In spite of reduced cooling rates due to aseptic vitrification conditions, a three-step exposure to cryoprotectant solutions protects the embryos effectively from cryo-injuries and guaranties high survival rates. PMID:20021718

  5. Universally distributed single-copy genes indicate a constant rate of horizontal transfer.

    PubMed

    Creevey, Christopher J; Doerks, Tobias; Fitzpatrick, David A; Raes, Jeroen; Bork, Peer

    2011-01-01

    Single copy genes, universally distributed across the three domains of life and encoding mostly ancient parts of the translation machinery, are thought to be only rarely subjected to horizontal gene transfer (HGT). Indeed it has been proposed to have occurred in only a few genes and implies a rare, probably not advantageous event in which an ortholog displaces the original gene and has to function in a foreign context (orthologous gene displacement, OGD). Here, we have utilised an automatic method to identify HGT based on a conservative statistical approach capable of robustly assigning both donors and acceptors. Applied to 40 universally single copy genes we found that as many as 68 HGTs (implying OGDs) have occurred in these genes with a rate of 1.7 per family since the last universal common ancestor (LUCA). We examined a number of factors that have been claimed to be fundamental to HGT in general and tested their validity in the subset of universally distributed single copy genes. We found that differing functional constraints impact rates of OGD and the more evolutionarily distant the donor and acceptor, the less likely an OGD is to occur. Furthermore, species with larger genomes are more likely to be subjected to OGD. Most importantly, regardless of the trends above, the number of OGDs increases linearly with time, indicating a neutral, constant rate. This suggests that levels of HGT above this rate may be indicative of positively selected transfers that may allow niche adaptation or bestow other benefits to the recipient organism. PMID:21850220

  6. Single-Molecule Interfacial Electron Transfer Dynamics at Dye-Sensitized TiO2 Nanoparticles

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; Wang, Yuanmin; He, Yufan

    2009-03-01

    Interfacial electron transfer dynamics is important for environmental and catalytic reactions. Extensive ensemble-averaged studies have indicated inhomogeneous and complex dynamics of interfacial ET reaction. To characterize the inhomogeniety and the complex mechanism, we have applied single-molecule spectroscopy and correlated AFM/STM imaging to study the Interfacial ET dynamics of dye molecules adsorbed at the surface of TiO2 nanoparticles. The interfacial ET activity of individual dye molecules showed fluctuations and intermittency at time scale of milliseconds to seconds. The fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the dynamics. Furthermore, we have applied site-specific AFM-Raman spectroscopy on analyzing ET associated mode-specific vibrational reorganization energy barriers. Our experiments revealed site-to-site variations in the vibrational reorganization energy barriers in the interfacial ET systems. Our recent experiments on single-molecule metal-to-ligand electron transfer (3) and single-molecule STM manipulation will also be discussed.

  7. Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion

    PubMed Central

    Cuman, Carly; Van Sinderen, Michelle; Gantier, Michael P.; Rainczuk, Kate; Sorby, Kelli; Rombauts, Luk; Osianlis, Tiki; Dimitriadis, Evdokia

    2015-01-01

    Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure. PMID:26629549

  8. Single scattering solution for radiative transfer through Rayleigh and aerosol atmosphere

    NASA Technical Reports Server (NTRS)

    Otterman, J.

    1977-01-01

    A solution is presented to the radiative transfer of the solar irradiation through a turbid atmosphere, based on the single-scattering approximation, i.e., an assumption that a photon that underwent scattering either leaves the top of the atmosphere or strikes the surface. The solution depends on a special idealization of the scattering phase function of the aerosols. The equations developed are subsequently applied to analyze quantitatively the enhancement of the surface irradiation and the enhancement of the scattered radiant emittance as seen from above the atmosphere, caused by the surface reflectance and atmospheric back scattering. An order of magnitude error analysis is presented.

  9. COBRA-SFS predictions of single assembly spent fuel heat transfer data

    SciTech Connect

    Lombardo, N.J.; Michener, T.E.; Wheeler, C.L.; Rector, D.R.

    1986-04-01

    The study reported here is one of several efforts to evaluate and qualify the COBRA-SFS computer code for use in spent fuel storage system thermal analysis. The ability of COBRA-SFS to predict the thermal response of two single assembly spent fuel heat transfer tests was investigated through comparisons of predictions with experimental test data. From these comparisons, conclusions regarding the computational treatment of the physical phenomena occurring within a storage system can be made. This objective was successfully accomplished as reasonable agreement between predictions and data were obtained for the 21 individual test cases of the two experiments.

  10. 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.

  11. Distance versus energy fluctuations and electron transfer in single protein molecules.

    PubMed

    Tang, Jau; Lin, Sheng-Hsien

    2006-06-01

    Stochastic nature due to distance and energy fluctuations of single protein molecules involved in electron-transfer (ET) reactions is studied. Distance fluctuations have been assumed previously for causing the slow fluctuations in the ET rates between a donor-acceptor pair constrained to a native protein. Although the observed t(-1/2) power law can be derived using Langevin dynamics with a simple chain model, some discrepancies exist. The friction coefficient and the Rouse segment time constant deduced from experimental data are several orders of magnitude too large, even though the extracted force constant is reasonable. Therefore, questions are raised about the distance-fluctuation mechanism and the activationless ET hypothesis. As an alternative mechanism, we considered fluctuations in activation energy and analyzed the data from two different single protein experiments to determine spectral distribution of energy fluctuations. PMID:16906810

  12. Single crystalline BaTiO{sub 3} thin films synthesized using ion implantation induced layer transfer

    SciTech Connect

    Park, Young-Bae; Diest, Kenneth; Atwater, Harry A.

    2007-10-01

    Layer transfer of BaTiO{sub 3} thin films onto silicon-based substrates has been investigated. Hydrogen and helium ions were co-implanted to facilitate ion-implantation-induced layer transfer of films from BaTiO{sub 3} single crystals. From thermodynamic equilibrium calculations, we suggest that the dominant species during cavity nucleation and growth are H{sub 2}, H{sup +}, H{sub 2}O, Ba{sup 2+} and Ba-OH, and that the addition of hydrogen to the Ba-Ti-O system can effectively suppress volatile oxide formation during layer transfer and subsequent annealing. After ion implantation, BaTiO{sub 3} layers contain microstructural defects and hydrogen precipitates in the lattice, but after layer transfer, the single crystal is found to be stoichiometric. Using direct wafer bonding and layer splitting, single crystal BaTiO{sub 3} thin films were transferred onto amorphous Si{sub 3}N{sub 4} and Pt substrates. Micro-Raman spectroscopy indicated that the density of defects generated by ion implantation in BaTiO{sub 3} can be significantly reduced during post-transfer annealing, returning the transferred layer to its single crystal state. Characterization using piezoresponse force microscopy shows that the layer transferred thin films are ferroelectric, with domain structures and piezoresponse characteristics similar to that of bulk crystals.

  13. Enantioselective N-Heterocyclic Carbene-Catalyzed ?-Hydroxylation of Enals Using Nitroarenes: An Atom Transfer Reaction That Proceeds via Single Electron Transfer

    PubMed Central

    2015-01-01

    A novel oxidative N-heterocyclic carbene-catalyzed reaction pathway has been discovered. Alkyl and aryl enals undergo ?-hydroxylation via oxygen atom transfer from electron-deficient nitrobenzenes, followed by trapping of the resultant acyl azolium by the solvent. The proposed mechanism involves a single electron transfer event to initiate the reaction followed by radical recombination. This represents a profound mechanistic departure from the established two-electron disconnects in NHC catalysis. PMID:25302860

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

  15. In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer.

    PubMed

    Isobe, Tomohiro; Ikebata, Yoshihisa; Do, Lanh Thi Kim; Tanihara, Fuminori; Taniguchi, Masayasu; Otoi, Takeshige

    2015-07-01

    The optimization of single-embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick-up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen-thawed embryos after a direct transfer. When two-cell-stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen-thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen-thawed embryos after transfer to recipients. PMID:25488699

  16. Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy: potentials and challenges.

    PubMed

    Sekatskii, S K; Dukenbayev, K; Mensi, M; Mikhaylov, A G; Rostova, E; Smirnov, A; Suriyamurthy, N; Dietler, G

    2015-12-12

    A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal-dye molecules as donor-acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed. PMID:26407105

  17. Stress transfer in microdroplet tensile test: PVC-coated and uncoated Kevlar-29 single fiber

    NASA Astrophysics Data System (ADS)

    Zhenkun, Lei; Quan, Wang; Yilan, Kang; Wei, Qiu; Xuemin, Pan

    2010-11-01

    The single fiber/microdroplet tensile test is applied for evaluating the interfacial mechanics between a fiber and a resin substrate. It is used to investigate the influence of a polymer coating on a Kevlar-29 fiber surface, specifically the stress transfer between the fiber and epoxy resin in a microdroplet. Unlike usual tests, this new test ensures a symmetrical axial stress on the embedded fiber and reduces the stress singularity that appears at the embedded fiber entry. Using a homemade loading device, symmetrical tensile tests are performed on a Kevlar-29 fiber with or without polyvinylchloride (PVC) coating, the surface of which is in contact with two epoxy resin microdroplets during curing. Raman spectra on the embedded fiber are recorded by micro-Raman Spectroscopy under different strain levels. Then they are transformed to the distributions of fiber axis stress based on the relationship between stress and Raman shift. The Raman results reveal that the fiber axial stresses increase with the applied loads, and the antisymmetric interfacial shear stresses, obtained by a straightforward balance of shear-to-axial forces argument, lead to the appearance of shear stress concentrations at a distance to the embedded fiber entry. The load is transferred from the outer fiber to the embedded fiber in the epoxy microdroplet. As is observed by scanning electronic microscopy (SEM), the existence of a flexible polymer coating on the fiber surface reduces the stress transfer efficiency.

  18. Quick Cooling and Filling Through a Single Port for Cryogenic Transfer Operations

    NASA Technical Reports Server (NTRS)

    Jones, J. R.; Fesmire, James E.; MacDowell, L. G. (Technical Monitor)

    2001-01-01

    Improved technology for the efficient transfer of cryogens is needed for future on-orbit fueling and remote Lunar/Mars operations. The cooling and filling of a liquid nitrogen (LN2) test vessel through a single port were investigated in a series of experiments. A new 'in-space' transfer tube design concept was used to demonstrate the ability to quickly cool and load cryogens through a single feed-through connection. Three different fill tube configurations with three different diameters were tested. The tubes providing the quickest cooldown time and the quickest fill time for the test article tank were determined. The results demonstrated a clear trade-off between cooling time and filling time for the optimum tube design. This experimental study is intended to improve technology for future flight tank designs by reducing fill system size, complexity, heat leak rate, and operations time. These results may be applied to Space Shuttle Power Reactant Storage and Distribution (PRSD) System upgrades and other future applications. Further study and experimental analysis for optimization of the fill tube design are in progress.

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

    PubMed

    Cagnone, Gael L M; Sirard, Marc-Andr

    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. PMID:23426876

  20. The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage

    PubMed Central

    2013-01-01

    Background The Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220C/min is far lower than that reported with open vitrification systems such as the cryoloop (?15,000C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an open vitrification system routinely used in our laboratory. Methods Human embryos were vitrified at either the 810 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (23 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of?transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going. Conclusions The Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 810 cell stage as well as the blastocyst stage. Use of this type of closed sealed system that prevents direct contact between the embryos and liquid nitrogen reduces the potential risk of sample cross-contamination or infection. These preliminary data and live birth outcomes have paved the way toward transitioning to a closed vitrification system in our own IVF program. PMID:23672340

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

    PubMed

    Labont, 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. Ultrafast Redistribution of E. coli SSB Along Long Single-Stranded DNA via Intersegment Transfer

    PubMed Central

    Lee, Kyung Suk; Marciel, Amanda B.; Kozlov, Alexander G.; Schroeder, Charles M.; Lohman, Timothy M.; Ha, Taekjip

    2014-01-01

    Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure which can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt2/s, which is at least six hundred times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double stranded DNA. Our work suggests that SSB can also migrate via a long range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation. PMID:24792418

  3. 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

  4. A distribution-based method to resolve single-molecule Frster resonance energy transfer observations

    PubMed Central

    Backovi?, Mihailo; Price, E. Shane; Johnson, Carey K.; Ralston, John P.

    2011-01-01

    We introduce a new approach to analyze single-molecule Frster resonance energy transfer (FRET) data. The method recognizes that FRET efficiencies assumed by traditional ensemble methods are unobservable for single molecules. We propose instead a method to predict distributions of FRET parameters obtained directly from the data. Distributions of FRET rates, given the data, are precisely defined using Bayesian methods and increase the information derived from the data. Benchmark comparisons find that the response time of the new method outperforms traditional methods of averaging. Our approach makes no assumption about the number or distribution of underlying FRET states. The new method also yields information about joint parameter distributions going beyond the standard framework of FRET analysis. For example, the running distribution of FRET means contains more information than any conceivable single measure of FRET efficiency. The method is tested against simulated data and then applied to a pilot-study sample of calmodulin molecules immobilized in lipid vesicles, revealing evidence for multiple dynamical states. PMID:21495770

  5. 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.

  6. 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

  7. A single-tube assembly of DNA using the transfer-PCR (TPCR) platform.

    PubMed

    Erijman, Ariel; Shifman, Julia M; Peleg, Yoav

    2014-01-01

    DNA cloning is a basic methodology employed for multiple applications in all life-science disciplines. In order to facilitate DNA cloning we developed Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. TPCR reaction is most efficient within a narrow range of primer concentrations. Adaptation of the TPCR should facilitate, simplify, and significantly reduce time and costs for DNA assembly, as well as protein engineering studies. In the current publication we describe a detailed protocol for implementation of the TPCR method for DNA assembly. PMID:24395359

  8. Impact of a single base pair substitution on the charge transfer rate along short DNA hairpins.

    PubMed

    Renaud, Nicolas; Berlin, Yuri A; Ratner, Mark A

    2013-09-10

    Numerical studies of hole migration along short DNA hairpins were performed with a particular emphasis on the variations of the rate and quantum yield of the charge separation process with the location of a single guanine:cytosine (G:C) base pair. Our calculations show that the hole arrival rate increases as the position of the guanine:cytosine base pair shifts from the beginning to the end of the sequence. Although these results are in agreement with recent experimental findings, the mechanism governing the charge migration along these sequences is revisited here. Instead of the phenomenological two-step hopping mechanism via the guanine base, the charge propagation occurs through a delocalization of the hole density along the base pair stack. Furthermore, the variations of the charge transfer with the position of the guanine base are explained by the impact of the base pair substitutions on the delocalized conduction channels. PMID:23980166

  9. Single-Walled Carbon Nanotubes Alter Cytochrome C Electron Transfer and Modulate Mitochondrial Function

    PubMed Central

    Ma, Xiaowei; Zhang, Li-Hua; Wang, Li-Rong; Xue, Xue; Sun, Ji-Hong; Wu, Yan; Zou, Guozhang; Wu, Xia; Wang, Paul C.; Wamer, Wayne G.; Yin, Jun-Jie; Zheng, Kaiyuan; Liang, Xing-Jie

    2013-01-01

    Single-walled carbon nanotubes (SWCNTs) are broadly used for various biomedical applications such as drug delivery, in vivo imaging and cancer photothermal therapy due to their unique physiochemical properties. However, once they enter the cells, the effects of SWCNTs to the intracellular organelles and macromolecules are not comprehensively understood. Cytochrome c (Cyt c), as a key component of the electron transport chain in mitochondria, plays an essential role in cellular energy consumption, growth and differentiation. In this study, we found the mitochondrial membrane potential (MMP) and mitochondrial oxygen uptake were greatly decreased in human epithelial KB cells treated with SWCNTs, which accompanies the reduction of Cyt c. SWCNTs deoxidized Cyt c in a pH dependent manner as evidenced by the appearance of a 550 nm characteristic absorption peak, which intensity increased as pH increase. Circular dichroism measurement confirmed the pH-dependent conformational change, which facilitated closer association of SWCNTs with the heme pocket of Cyt c and thus expedited the reduction of Cyt c. The electron transfer of Cyt c is also disturbed by SWCNTs, as measured with electron spin resonance spectroscopy. In conclusion, the redox activity of Cyt c was affected by SWCNTs treatment due to attenuated electron transfer and conformational change of Cyt c, which consequently changed mitochondrial respiration of SWCNTs treated cells. This work is significant to SWCNTs research because it provided novel understanding to the disruption of SWCNTs to the mitochondria and has important implications for biomedical applications of SWCNTs. PMID:23171082

  10. Raman Spectroscopy of Charge Transfer Interactions Between Single Wall Carbon Nanotubes and [FeFe] Hydrogenase

    SciTech Connect

    Blackburn, J. L. Svedruzic, D.; McDonald, T. J.; Kim, Y. H.; King, P. W.; Heben, M. J.

    2008-01-01

    We report a Raman spectroscopy study of charge transfer interactions in complexes formed by single-walled carbon nanotubes (SWNTs) and [FeFe] hydrogenase I (CaHydI) from Clostridium acetobutylicum. The choice of Raman excitation wavelength and sample preparation conditions allows differences to be observed for complexes involving metallic (m) and semiconducting (s) species. Adsorbed CaHydI can reversibly inject electronic charge into the LUMOs of s-SWNTs, while charge can be injected and removed from m-SWNTs at lower potentials just above the Fermi energy. Time-dependent enzymatic assays demonstrated that the reduced and oxidized forms of CaHydI are deactivated by oxygen, but at rates that varied by an order of magnitude. The time evolution of the oxidative decay of the CaHydI activity reveals different time constants when complexed with m-SWNTs and s-SWNTs. The correlation of enzymatic assays with time-dependent Raman spectroscopy provides a novel method by which the charge transfer interactions may be investigated in the various SWNT-CaHydI complexes. Surprisingly, an oxidized form of CaHydI is apparently more resistant to oxygen deactivation when complexed to m-SWNTs rather than s-SWNTs.

  11. Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares.

    PubMed

    Mortensen, C J; Choi, Y-H; Ing, N H; Kraemer, D C; Vogelsang, M M; Hinrichs, Katrin

    2010-08-01

    Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 degrees C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 (HSPA1A) mRNA were determined by real-time RT-PCR in resulting blastocysts, and were compared to those for embryos derived in vivo from control or exercised mares. Exposure of oocytes to heat at the onset of in vitro maturation did not affect any measured end point. However, exposure to 42 degrees C late in maturation culture reduced rates of oocyte nuclear maturation for both the 2 h (43/105 (43%) compared to control 70/103 (68%); P < 0.01), and 4 h (47/106 (44%) compared to control 60/103 (59%); P < 0.05) groups. Additionally, late heat exposure reduced development to morulae and blastocyst stages after intracytoplasmic sperm injection (ICSI; 18/89 (20%) compared to control 43/128 (34%); P < 0.05). Seven days after oocyte heat exposure, resultant blastocysts had a higher abundance of HSPA1A gene transcripts, relative to those for 18S rRNA. In vitro-produced embryos and lower-quality in vivo-produced embryos had significantly higher relative HSPA1A mRNA (lower 18S rRNA) concentrations than did higher-quality in vivo-produced embryos. The authors concluded that equine oocytes were sensitive to heat during late in vitro maturation, and responded to thermal shock with an increased ratio of HSPA1A:18S gene expression that was measurable in the resulting blastocyst. Embryos produced in vitro (including controls) had increased levels of HSPA1A mRNA relative to 18S rRNA compared to in vivo-produced embryos, suggesting a response to environmental insult. PMID:20416934

  12. Analysis of photobleaching in single-molecule multicolor excitation and Frster resonance energy transfer measurements.

    PubMed

    Eggeling, Christian; Widengren, Jerker; Brand, Leif; Schaffer, Jrg; Felekyan, Suren; Seidel, Claus A M

    2006-03-01

    Dye photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Frster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation. PMID:16509620

  13. Ooplast transfer of triploid pronucleus zygote improve reconstructed human-goat embryonic development

    PubMed Central

    Yao, Ling; Wang, Pu; Liu, Jia; Chen, Jianquan; Tang, Hailiang; Sha, Hongying

    2014-01-01

    Poor development of the interspecies somatic cell nuclear transfer (iSCNT) embryos was due to nuclear-mitochondrial incompatibility. In humans, it has been known that ooplast transfer (OT) could support normal fertilization, the development of embryos and prevents the transmission of mtDNA disease. To investigate whether OT could support development of the iSCNT embryos, the ooplast of Triploid Pronucleus (3PN) zygote which would be discarded in IVF lab was transferred into the enucleated goat oocytes to construct humanized iSCNT embryos in our study. The results showed the 3PN-OT could significantly improve the early development of humanized iSCNT embryos. The percentage of blastocyst development of OT group was also higher than that of the control group. Interestingly, the morphology of some OT-iSCNT blastocysts was similar to normal human blastocysts in vitro fertilization, while the morphology of iSCNT blastocysts from control group was similar to goat blastocysts. Importantly, the pluripotent marker Oct4 of the OT-iSCNT blastocyst was expressed stronger than that of the control group. These results suggested that 3PN-OT could improve the developmental potency of human iSCNT embryos and would facilitate establishing ESCs from iSCNT blastocysts. PMID:25419417

  14. A transferable H2O interaction potential based on a single center multipole expansion: SCME.

    PubMed

    Wikfeldt, K T; Batista, E R; Vila, F D; Jnsson, H

    2013-10-21

    A transferable potential energy function for describing the interaction between water molecules is presented. The electrostatic interaction is described rigorously using a multipole expansion. Only one expansion center is used per molecule to avoid the introduction of monopoles. This single center approach turns out to converge and give close agreement with ab initio calculations when carried out up to and including the hexadecapole. Both dipole and quadrupole polarizability are included. All parameters in the electrostatic interaction as well as the dispersion interaction are taken from ab initio calculations or experimental measurements of a single water molecule. The repulsive part of the interaction is parametrized to fit ab initio calculations of small water clusters and experimental measurements of ice Ih. The parametrized potential function was then used to simulate liquid water and the results agree well with experiment, even better than simulations using some of the point charge potentials fitted to liquid water. The evaluation of the new interaction potential for condensed phases is fast because point charges are not present and the interaction can, to a good approximation, be truncated at a finite range. PMID:23949215

  15. Photobleaching-based quantitative analysis of fluorescence resonance energy transfer inside single living cell.

    PubMed

    Wang, Longxiang; Chen, Tongsheng; Qu, Junle; Wei, Xunbin

    2010-01-01

    The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ( 1 )) to selectively detect the fluorescence from donor, and a FRET channel (CH ( 2 )) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell. PMID:19588234

  16. 33 CFR 150.445 - When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water? 150.445 Section 150.445 Navigation and...: OPERATIONS Cargo Transfer Operations 150.445 When is oil in a single point mooring-oil transfer system...

  17. 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.

  18. Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferrioxamine.

    PubMed

    Harvey, A J; Kind, K L; Thompson, J G

    2007-07-01

    Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development. PMID:17329595

  19. 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

  20. Understanding Contemporary Transfer Pathways: A Multiple Case Analysis of Two to Four-Year Transfer in a Single State

    ERIC Educational Resources Information Center

    Cullop, Ashley M.

    2012-01-01

    Three quarters of community college students enter higher education with the goal of transferring to a four-year institution to pursue a bachelor's degree and as few as 11% succeed. Scholars cite four main reasons for why this gap in baccalaureate completion matters: (1) community colleges today are educating approximately one-half of all

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

    NASA Astrophysics Data System (ADS)

    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

    2012-02-01

    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 cm2 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.

  2. CORRELATION OF DEVELOPMENTAL DIFFERENCES OF NUCLEAR TRANSFER EMBRYOS CELLS TO THE METHYLATION PROFILES OF NUCLEAR TRANSFER DONOR CELLS IN SWINE

    PubMed Central

    Bonk, Aaron J.; Cheong, Hee-Tae; Li, Rongfeng; Lai, Liangxue; Hao, Yanhong; Liu, Zhonghua; Samuel, Melissa; Fergason, Emily A.; Whitworth, Kristin M.; Murphy, Clifton N.; Antoniou, Eric; Prather, Randall S.

    2008-01-01

    Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues. The methylation profiles of the donor cells were then analyzed with respect to their ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2, A7, and A8 had blastocyst rates of 11.7%a, 16.7%ab, and 20.0%b, respectively (ab P<0.05). Adult somatic cells included kidney, mammary (large), and mammary (small) also had different blastocyst rates (ab P<0.05) of 4.2% a, 10.7% ab, and 18.3% b, respectively. For clonal donor cells and for adult somatic cell groups the donor cells with the highest blastocyst rates also had methylation profiles with the lowest similarity to the methylation profiles of the in vivo-produced blastocysts. Conversely, the donor cells with the lowest blastocyst rates had methylation profiles with the highest similarity to the methylation profiles of the in vivo-produced blastocysts. Our findings show there is an inverse correlation to the similarity of the methylation profiles of the donor cells and the in vivo-produced embryos, and to the blastocyst rates following SCNT. PMID:17965590

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

    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.

  4. Single electron transfer mechanism of oxidative dechlorination of 4-chloroanisole on copper(II)-smectite

    SciTech Connect

    Govindaraj, N.; Mortland, M.M.; Boyd, S.A.

    1987-11-01

    4-Chloroanisole was found to react with Cu(II)-smectite forming a blue clay-organic complex. The presence of radical cation intermediates in the complex was confirmed by electron spin resonance and infrared spectroscopy. The radical cation intermediates were formed via an initial one electron oxidation of 4-chloroanisole by Cu(II)-smectite. Coupling of the radical cation of 4-chloroanisole (I) with a neutral 4-chloroanisole molecule gave a biphenyl radical cation (VI). Single electron transfer from the dimerized radical cation (VI) to Cu(II)-smectite resulted in the formation of a nonradical dication biphenyl intermediate (V). Reaction of the blue clay-organic complex with methanol resulted in the formation of the final dechlorinated dimeric product, viz., 4,4'-dimethoxybiphenyl. Chloride ion was recovered form the methanol extract. It is suggested that Cu(II)-smectite may be a useful catalyst in the oxidative polymerization and dechlorination of chlorinated aromatic toxicants. The products of these reactions should be significantly less toxic than the parent compounds. 15 references, 4 figures, 2 tables.

  5. 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.

  6. Current Status of Comprehensive Chromosome Screening for Elective Single-Embryo Transfer

    PubMed Central

    Wu, Ming-Yih; Chao, Kuang-Han; Chen, Chin-Der; Chang, Li-Jung; Chen, Shee-Uan; Yang, Yu-Shih

    2014-01-01

    Most in vitro fertilization (IVF) experts and infertility patients agree that the most ideal assisted reproductive technology (ART) outcome is to have a healthy, full-term singleton born. To this end, the most reliable policy is the single-embryo transfer (SET). However, unsatisfactory results in IVF may result from plenty of factors, in which aneuploidy associated with advanced maternal age is a major hurdle. Throughout the past few years, we have got a big leap in advancement of the genetic screening of embryos on aneuploidy, translocation, or mutations. This facilitates a higher success rate in IVF accompanied by the policy of elective SET (eSET). As the cost is lowering while the scale of genome characterization continues to be up over the recent years, the contemporary technologies on trophectoderm biopsy and freezing-thaw, comprehensive chromosome screening (CCS) with eSET appear to be getting more and more popular for modern IVF centers. Furthermore, evidence has showen that, by these avant-garde techniques (trophectoderm biopsy, vitrification, and CCS), older infertile women with the help of eSET may have an opportunity to increase the success of their live birth rates approaching those reported in younger infertility patients. PMID:24991216

  7. Single-collision studies of hot atom energy transfer and chemical reaction

    SciTech Connect

    Valentini, J.J. )

    1991-01-01

    This report discusses research in the collision dynamics of translationally hot atoms, with funding with DOE for the project Single-Collision Studies of Hot Atom Energy Transfer and Chemical Reaction,'' Grant Number DE-FG03-85ER13453. The work reported here was done during the period September 9, 1988 through October 31, 1991. During this period this DOE-funded work has been focused on several different efforts: (1) experimental studies of the state-to-state dynamics of the H + RH {yields} H{sub 2} R reactions where RH is CH{sub 4}, C{sub 2}H{sub 6}, or C{sub 3}H{sub 8}, (2) theoretical (quasiclassical trajectory) studies of hot hydrogen atom collision dynamics, (3) the development of photochemical sources of translationally hot molecular free radicals and characterization of the high resolution CARS spectroscopy of molecular free radicals, (4) the implementation of stimulated Raman excitation (SRE) techniques for the preparation of vibrationally state-selected molecular reactants.

  8. Single-collision studies of hot atom energy transfer and chemical reaction. Final report

    SciTech Connect

    Valentini, J.J.

    1991-12-31

    This report discusses research in the collision dynamics of translationally hot atoms, with funding with DOE for the project ``Single-Collision Studies of Hot Atom Energy Transfer and Chemical Reaction,`` Grant Number DE-FG03-85ER13453. The work reported here was done during the period September 9, 1988 through October 31, 1991. During this period this DOE-funded work has been focused on several different efforts: (1) experimental studies of the state-to-state dynamics of the H + RH {yields} H{sub 2} R reactions where RH is CH{sub 4}, C{sub 2}H{sub 6}, or C{sub 3}H{sub 8}, (2) theoretical (quasiclassical trajectory) studies of hot hydrogen atom collision dynamics, (3) the development of photochemical sources of translationally hot molecular free radicals and characterization of the high resolution CARS spectroscopy of molecular free radicals, (4) the implementation of stimulated Raman excitation (SRE) techniques for the preparation of vibrationally state-selected molecular reactants.

  9. 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

  10. 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

  11. Female bovine blastocysts are more prone to apoptosis than male ones.

    PubMed

    Ghys, Emmanuelle; Dallemagne, Matthew; De Troy, Delphine; Sauvegarde, Caroline; Errachid, Abdelmounaim; Donnay, Isabelle

    2016-03-01

    Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of invitro-produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid-based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts. PMID:26506912

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

    PubMed Central

    Wang, Hehai; Ding, Tianbing; Brown, Naoko; Yamamoto, Yasutoshi; Prince, Lawrence S.; Reese, Jeff; Paria, B. C.

    2008-01-01

    It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells. PMID:18423437

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

    PubMed

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

    2015-08-01

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

  15. Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst

    PubMed Central

    2012-01-01

    Background The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system. Results A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human. Conclusion Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast. PMID:23126590

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

    PubMed

    Hwang, Jae Yeon; Choi, Kwang-Hwan; Lee, Dong-Kyung; Kim, Seung-Hun; Kim, Eun Bae; Hyun, Sang-Hwan; Lee, Chang-Kyu

    2015-12-18

    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

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

    PubMed Central

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

    2015-01-01

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

  18. A multifunctional nanocomposite for luminescence resonance energy transfer-guided synergistic monitoring and therapy under single near infrared light.

    PubMed

    Zhang, Yu; Shen, Ting-Ting; Zhang, Hao-Li; Kirillov, Alexander M; Cai, Hui-Juan; Wu, Jiang; Liu, Wei-Sheng; Tang, Yu

    2016-04-01

    By utilizing a drug coordinated to Eu(3+) as a blocker of luminescence resonance energy transfer from the Eu(3+) complex to gold nanotriangles, we successfully implemented multiple functions into one nanocomposite; it operates under single near infrared light and is efficient for the temperature/luminescence monitoring of drug release and synergistic turning-on of photothermal chemotherapy. PMID:26912095

  19. Amination of 3-Substituted Benzofuran-2(3H)-ones Triggered by Single-Electron Transfer.

    PubMed

    Yang, Chen; Liu, Yang; Yang, Jin-Dong; Li, Yi-He; Li, Xin; Cheng, Jin-Pei

    2016-03-01

    An efficient amination reaction of 3-substituted benzofuran-2(3H)-ones promoted by cesium carbonate was developed. A putative mechanism involving a single-electron-transfer event was proposed, which represents a new reactivity for benzofuran-2(3H)-ones. PMID:26911719

  20. Mediated Electron Transfer at Vertically Aligned Single-Walled Carbon Nanotube Electrodes During Detection of DNA Hybridization.

    PubMed

    Wallen, Rachel; Gokarn, Nirmal; Bercea, Priscila; Grzincic, Elissa; Bandyopadhyay, Krisanu

    2015-12-01

    Vertically aligned single-walled carbon nanotube (VASWCNT) assemblies are generated on cysteamine and 2-mercaptoethanol (2-ME)-functionalized gold surfaces through amide bond formation between carboxylic groups generated at the end of acid-shortened single-walled carbon nanotubes (SWCNTs) and amine groups present on the gold surfaces. Atomic force microscopy (AFM) imaging confirms the vertical alignment mode of SWCNT attachment through significant changes in surface roughness compared to bare gold surfaces and the lack of any horizontally aligned SWCNTs present. These SWCNT assemblies are further modified with an amine-terminated single-stranded probe-DNA. Subsequent hybridization of the surface-bound probe-DNA in the presence of complementary strands in solution is followed using impedance measurements in the presence of Fe(CN)6 (3-/4-) as the redox probe in solution, which show changes in the interfacial electrochemical properties, specifically the charge-transfer resistance, due to hybridization. In addition, hybridization of the probe-DNA is also compared when it is attached directly to the gold surfaces without any intermediary SWCNTs. Contrary to our expectations, impedance measurements show a decrease in charge-transfer resistance with time due to hybridization with 300nM complementary DNA in solution with the probe-DNA attached to SWCNTs. In contrast, an increase in charge-transfer resistance is observed with time during hybridization when the probe-DNA is attached directly to the gold surfaces. The decrease in charge-transfer resistance during hybridization in the presence of VASWCNTs indicates an enhancement in the electron transfer process of the redox probe at the VASWCNT-modified electrode. The results suggest that VASWCNTs are acting as mediators of electron transfer, which facilitate the charge transfer of the redox probe at the electrode-solution interface. PMID:26105789

  1. Mediated Electron Transfer at Vertically Aligned Single-Walled Carbon Nanotube Electrodes During Detection of DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Wallen, Rachel; Gokarn, Nirmal; Bercea, Priscila; Grzincic, Elissa; Bandyopadhyay, Krisanu

    2015-06-01

    Vertically aligned single-walled carbon nanotube (VASWCNT) assemblies are generated on cysteamine and 2-mercaptoethanol (2-ME)-functionalized gold surfaces through amide bond formation between carboxylic groups generated at the end of acid-shortened single-walled carbon nanotubes (SWCNTs) and amine groups present on the gold surfaces. Atomic force microscopy (AFM) imaging confirms the vertical alignment mode of SWCNT attachment through significant changes in surface roughness compared to bare gold surfaces and the lack of any horizontally aligned SWCNTs present. These SWCNT assemblies are further modified with an amine-terminated single-stranded probe-DNA. Subsequent hybridization of the surface-bound probe-DNA in the presence of complementary strands in solution is followed using impedance measurements in the presence of Fe(CN)6 3-/4- as the redox probe in solution, which show changes in the interfacial electrochemical properties, specifically the charge-transfer resistance, due to hybridization. In addition, hybridization of the probe-DNA is also compared when it is attached directly to the gold surfaces without any intermediary SWCNTs. Contrary to our expectations, impedance measurements show a decrease in charge-transfer resistance with time due to hybridization with 300 nM complementary DNA in solution with the probe-DNA attached to SWCNTs. In contrast, an increase in charge-transfer resistance is observed with time during hybridization when the probe-DNA is attached directly to the gold surfaces. The decrease in charge-transfer resistance during hybridization in the presence of VASWCNTs indicates an enhancement in the electron transfer process of the redox probe at the VASWCNT-modified electrode. The results suggest that VASWCNTs are acting as mediators of electron transfer, which facilitate the charge transfer of the redox probe at the electrode-solution interface.

  2. 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

  3. Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

    PubMed Central

    THONGKITTIDILOK, Chommanart; THARASANIT, Theerawat; SONGSASEN, Nucharin; SANANMUANG, Thanida; BUARPUNG, Sirirak; TECHAKUMPHU, Mongkol

    2015-01-01

    This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages. PMID:25985792

  4. Single breath xenon polarization transfer contrast (SB-XTC): implementation and initial results in healthy humans

    PubMed Central

    Muradyan, Iga; Butler, James P; Dabaghyan, Mikayel; Hrovat, Mirko; Dregely, Isabel; Ruset, Iulian; Topulos, George P; Frederick, Eric; Hatabu, Hiroto; Hersman, William F; Patz, Samuel

    2012-01-01

    Purpose To implement and characterize a single-breath xenon transfer contrast (SB-XTC) method to assess the fractional diffusive gas transport F in the lung: to study the dependence of F and its uniformity as a function of lung volume; to estimate local alveolar surface area per unit gas volume SA/VGas from multiple diffusion time measurements of F; to evaluate the reproducibility of the measurements and the necessity of B1 correction in cases of centric and sequential encoding. Materials and Methods In SB-XTC three or four gradient echo images separated by inversion/saturation pulses were collected during a breath-hold in eight healthy volunteers, allowing the mapping of F (thus SA/VGas) and correction for other contributions such as T1 relaxation, RF depletion and B1 inhomogeneity from inherently registered data. Results Regional values of F and its distribution were obtained; both the mean value and heterogeneity of F increased with the decrease of lung volume. Higher values of F in the bases of the lungs in supine position were observed at lower volumes in all volunteers. Local SA/VGas (with a mean standard deviation of SA/VGas=8930cm-1) was estimated in vivo near functional residual capacity. Calibration of SB-XTC on phantoms highlighted the necessity for B1 corrections when k-space is traversed sequentially; with centric ordering B1 distribution correction is dispensable. Conclusion SB-XTC technique is implemented and validated for in vivo measurements of local SA/VGas. PMID:23011916

  5. Single-molecule Frster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I.

    PubMed

    Berezhna, Svitlana Y; Gill, Joshua P; Lamichhane, Rajan; Millar, David P

    2012-07-11

    Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule Frster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations--open, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3'-5' exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair. PMID:22650319

  6. dc-switchable and single-nanocrystal-addressable coherent population transfer

    NASA Astrophysics Data System (ADS)

    Gunceler, Deniz; Bulutay, Ceyhun

    2010-12-01

    Achieving coherent population transfer in the solid-state is challenging compared to atomic systems due to closely spaced electronic states and fast decoherence. Here, within an atomistic pseudopotential theory, we offer recipes for the stimulated Raman adiabatic passage in embedded silicon and germanium nanocrystals. The transfer efficiency spectra display characteristic Fano resonances. By exploiting the Stark effect, we predict that transfer can be switched off with a dc voltage. As the population transfer is highly sensitive to structural variations, with a choice of a sufficiently small two-photon detuning bandwidth, it can be harnessed for addressing individual nanocrystals within an ensemble.

  7. Regional changes in the binding of (/sup 3/H) concanavalin A to mouse blastocysts at implantation: an autoradiographic study

    SciTech Connect

    Carollo, J.R.; Weitlauf, H.M.

    1981-11-01

    Implanting and delayed-implanting mouse blastocysts were incubated in vitro with (3H) concanavalin A (Con A), and the distribution of binding on their surfaces was determined by light microscopic autoradiography. The density of binding was uniform on the trophectoderm of delayed-implanting embryos and was not changed on the polar surface of implanting embryos. However, binding was reduced on the proximal mural and distal mural trophectoderm of implanting blastocysts by 36% and 60%, respectively. These results suggest that there is a regional reduction in the density of mannose-like sugars on the surface of mouse blastocysts at the time of attachment and implantation.

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

    PubMed

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

    2016-03-01

    The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me2SO) for equilibration, and cooling in 16% EG + 16% Me2SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ∼24 °C/15 min and body 37 °C/3 min), and the replacement of Me2SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and re-expansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37 °C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me2SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (P < 0.05). The modified protocol of loading EG at 37 °C for 3 minutes has increased the embryo survival of the original protocol from 67% to 91% and the developmental rate from 57% to 83% at 5-day culture. These results were comparable to or better than those reported in human or other species, indicating that this optimized method is well suited to any commercial embryo transfer program in the dromedary camel. PMID:26603656

  9. Feasibility for a Single-Stage-to-Orbit Launch to a Geosynchronous Transfer Orbit by Pulse Laser Propulsion

    NASA Astrophysics Data System (ADS)

    Katsurayama, Hiroshi; Komurasaki, Kimiya; Arakawa, Yoshihiro

    An air-breathing pulse laser powered launcher has been proposed as an alternative to conventional chemical launch systems. The trajectory from the ground to a geosynchronous transfer orbit by pulse laser propulsion is calculated by modeling the thrust during pulsejet, ramjet and rocket flight modes, and the launch cost is estimated. The results show that the pulse laser powered launcher can transfer 0.085kg payload per 1MW beam power to a geosynchronous orbit, and the cost becomes quarter of existing systems if one can divide a single launch into 22,500 multiple launches.

  10. 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. PMID:26269424

  11. Two-photon-induced hot-electron transfer to a single molecule in a scanning tunneling microscope

    SciTech Connect

    Wu, S. W.; Ho, W.

    2010-08-15

    The junction of a scanning tunneling microscope (STM) operating in the tunneling regime was irradiated with femtosecond laser pulses. A photoexcited hot electron in the STM tip resonantly tunnels into an excited state of a single molecule on the surface, converting it from the neutral to the anion. The electron-transfer rate depends quadratically on the incident laser power, suggesting a two-photon excitation process. This nonlinear optical process is further confirmed by the polarization measurement. Spatial dependence of the electron-transfer rate exhibits atomic-scale variations. A two-pulse correlation experiment reveals the ultrafast dynamic nature of photoinduced charging process in the STM junction. Results from these experiments are important for understanding photoinduced interfacial charge transfer in many nanoscale inorganic-organic structures.

  12. Oocyte recovery, in vitro insemination, and transfer into the oviduct after its microsurgical repair at a single laparotomy.

    PubMed

    Tesark, J; Pilka, L; Dvork, M; Trvnk, P

    1983-04-01

    A method combining recovery of a preovulatory oocyte, its in vitro insemination, and transfer into the oviduct with tubal microsurgery at a single laparotomy is described. Of four women in whom tubal microsurgery was clinically indicated and the accompanying tubal transfer of an oocyte inseminated in vitro was performed, two became pregnant. It is not clear whether fertilization occurred in vitro or in vivo. One of the pregnancies miscarried in the fifth week after surgery; the other pregnancy continued without any complications. A normal male infant was subsequently delivered at term. The proposed method is discussed as a possible alternative to in vitro fertilization and embryo transfer in some cases of infertility and as a suitable supplemental procedure in some cases in which tubal microsurgery is indicated. PMID:6832403

  13. Modulation of energy/electron transfer in gold nanoclusters by single walled carbon nanotubes and further consequences

    NASA Astrophysics Data System (ADS)

    Das, Tarasankar; Maity, Arnab; Mondal, Somen; Purkayastha, Pradipta

    2015-04-01

    Semiconductor or metallic character in single-walled carbon nanotubes (SWCNTs) is developed because of their chirality and diameter. Depending upon the extent of these characters in a particular sample of SWCNT, various electronic and mechanical applications are formulated. In this work we used protein protected red emitting gold nanoclusters (AuNCs) to enhance the metallic character in SWCNTs through electron transfer induced by photonic excitation. The AuNCs have been synthesized following a known protocol that generates Au+ protected Au0 clusters. Normal and carboxylic acid functionalized SWCNTs were obtained commercially for usage in the experiments. The non-functionalized SWCNTs facilitate intersystem electron transfer while the functionalized ones defer the phenomenon, which, in turn, affects the metallic character in the nanotubes. Steady state and time resolved fluorescence spectroscopy prove the dynamics and electrochemistry supports the intersystem electron transfer process.

  14. Local field effects in the energy transfer between a chromophore and a carbon nanotube: a single-nanocompound investigation.

    PubMed

    Roquelet, Cyrielle; Vialla, Fabien; Diederichs, Carole; Roussignol, Philippe; Delalande, Claude; Deleporte, Emmanuelle; Lauret, Jean-Sbastien; Voisin, Christophe

    2012-10-23

    Energy transfer in noncovalently bound porphyrin/carbon nanotube compounds is investigated at the single-nanocompound scale. Excitation spectroscopy of the luminescence of the nanotube shows two resonances arising from intrinsic excitation of the nanotube and from energy transfer from the porphyrin. Polarization diagrams show that both resonances are highly anisotropic, with a preferred direction along the tube axis. The energy transfer is thus strongly anisotropic despite the almost isotropic absorption of porphyrins. We account for this result by local field effects induced by the large optical polarizability of nanotubes. We show that the local field correction extends over several nanometers outside the nanotubes and drives the overall optical response of functionalized nanotubes. PMID:23005601

  15. 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.

  16. Evaluation method of single blow experiment for the determination of heat transfer coefficient and dispersive Peclet number

    NASA Astrophysics Data System (ADS)

    Roetzel, Wilfried; Na Ranong, Chakkrit

    2015-12-01

    An evaluation method is developed for single blow experiments with liquids on heat exchangers. The method is based on the unity Mach number dispersion model. The evaluation of one experiment yields merely one equation for the two unknowns, the number of transfer units and the dispersive Peclet number. Calculations on an example confirm that one single blow test alone cannot provide reliable values of the unknowns. A second test with a liquid of differing heat capacity is required, or a tracer experiment for the measurement of the Peclet number. A modified method is developed for gases. One experiment yields the effective number of transfer units and approximate values of the two unknowns. The numerical evaluation of calculated experiments demonstrates the applicability of the evaluation methods.

  17. [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

  18. 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

  19. 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. PMID:26771201

  20. Substrate-controlled Rh(ii)-catalyzed single-electron-transfer (SET): divergent synthesis of fused indoles.

    PubMed

    Chen, Kai; Zhu, Zi-Zhong; Liu, Jia-Xin; Tang, Xiang-Ying; Wei, Yin; Shi, Min

    2016-01-01

    Rh(ii)-catalyzed diversified ring expansions controlled by single-electron-transfer (SET) have been disclosed in this communication, producing a series of indole-fused azetidines and 1H-carbazoles or related derivatives in moderate to good yields via Rh2(III,II) nitrene radical intermediates. The direction of ring expansion branches according to different ring sizes of methylenecycloalkanes. PMID:26548476

  1. The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis.

    PubMed Central

    Massip, A; Mulnard, J; Vanderzwalmen, P; Hanzen, C; Ectors, F

    1982-01-01

    The behaviour of the cow blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: continuous expansion followed by hatching; discontinuous expansion interrupted by few contractions and followed by hatching; discontinuous expansion interrupted by several rapid contractions without hatching. This demonstrated that the pulsatile activity of the blastocyst is not a necessary condition of hatching but also that only a moderate pulsatile activity is compatible with normal hatching. The time of hatching in vitro corresponded approximately with the time of zona loss in vivo (9-10 days). Rupture of the zona occurred at any point of the trophoblast layer. Hatching by herniation through a reduced opening of the zona was occasionally observed. The behavior of the embryos from a particular animal was very similar but differences were noted between embryos from different animals. Images Fig. 3 PMID:7076563

  2. 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.

  3. Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts

    PubMed Central

    Zhou, Dewang; Ren, Jin-Xiang; Ryan, Thomas M.; Higgins, N. Patrick; Townes, Tim M.

    2004-01-01

    The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes. PMID:15356288

  4. From blastocyst to gastrula: gene regulatory networks of embryonic stem cells and early mouse embryogenesis

    PubMed Central

    Parfitt, David-Emlyn; Shen, Michael M.

    2014-01-01

    To date, many regulatory genes and signalling events coordinating mammalian development from blastocyst to gastrulation stages have been identified by mutational analyses and reverse-genetic approaches, typically on a gene-by-gene basis. More recent studies have applied bioinformatic approaches to generate regulatory network models of gene interactions on a genome-wide scale. Such models have provided insights into the gene networks regulating pluripotency in embryonic and epiblast stem cells, as well as cell-lineage determination in vivo. Here, we review how regulatory networks constructed for different stem cell types relate to corresponding networks in vivo and provide insights into understanding the molecular regulation of the blastocystgastrula transition. PMID:25349451

  5. The Dnmt3b splice variant is specifically expressed in in vitro-manipulated blastocysts and their derivative ES cells.

    PubMed

    Horii, Takuro; Suetake, Isao; Yanagisawa, Eikichi; Morita, Sumiyo; Kimura, Mika; Nagao, Yasumitsu; Imai, Hiroshi; Tajima, Shoji; Hatada, Izuho

    2011-10-01

    Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos. PMID:21666347

  6. 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.

  7. 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

  8. Pulsatile activity and hatching of in vitro produced cow blastocysts: effects of serum supplementation.

    PubMed

    van Heule, A; van Langendonckt, A; Donnay, I; Dessy, F; Massip, A

    2001-04-01

    The effect of serum added to a modified SOF medium on pulsatile activity and hatching of in vitro produced cow blastocysts was investigated by time-lapse cinematography. Embryos were generated from abattoir material and cultured in mSOF without serum or with 10% FCS added at 42h pi. Addition of serum significantly increases pulsatile activity before zona rupture and reduces the time of hatching. Pulsatile activity does not seem to be involved in the hatching process. PMID:11778742

  9. Targeted Organ Generation Using Mixl1-Inducible Mouse Pluripotent Stem Cells in Blastocyst Complementation

    PubMed Central

    Kobayashi, Toshihiro; Kato-Itoh, Megumi

    2015-01-01

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

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

    PubMed

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

    2015-01-15

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

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

    PubMed Central

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

    2014-01-01

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

  12. Ovarian Stimulation by Exogenous Gonadotropin Decreases the Implantation Rate and Expression of Mouse Blastocysts Integrins

    PubMed Central

    Fayazi, Mehri; Beigi Boroujeni, Mandana; Salehnia, Mojdeh; Khansarinejad, Behzad

    2014-01-01

    Background: Integrins are heterodimeric glycoprotein receptors that regulate the interaction of cells with extracellular matrix and may have a critical role in implantation. The aim of this study was to investigate the effect of ovulation induction on the expression of ?4, ?v, ?1, and ?3 integrins in mouse blastocyst at the time of implantation. Methods: The ovarian stimulated and non-stimulated pregnant mice were sacrificed on the morning of 5th day of pregnancy. The blastocysts were collected, and the expression of ?v, ?4, ?1, and ?3 integrins was examined using real-time RT-PCR and immunocytochemical techniques, then their ovarian hormones were analyzed at the same time. The implantation sites in uterine horns of other pregnant mice in both groups were determined under a stereomicroscope on the 7th day of pregnancy. Results: The results showed that the expression of ?v, ?1, and ?3 integrins in both mRNA and protein levels was significantly lower in the ovarian stimulated group than the control group, and the maximum ratio of expression was belonged to ?1 molecule (P>0.05). Conclusion: The implantation rate in superovulated mice was significantly lower than control mice. It was suggested that ovulation induction decreased the expression of ?v, ?1, and ?3 integrins of mouse blastocysts. PMID:24375157

  13. CXADR is required for AJ and TJ assembly during porcine blastocyst formation.

    PubMed

    Kwon, Jeong-Woo; Kim, Nam-Hyung; Choi, Inchul

    2016-04-01

    Coxsackie virus and adenovirus receptor (CXADR) is a member of the immunoglobulin superfamily as well as a member of the junctional adhesion molecule family of adhesion receptor. In human pre-implantation embryos, CXADR was detected and co-localized with tight junction (TJ) proteins on the membrane of the trophectoderm. However, its physiological roles were not elucidated in terms of blastocyst formation. Here, we reported expression patterns and biological functions of CXADR in porcine pre-implantation embryos. The transcripts of CXADR were detected at all stages of pre-implantation. Particularly, its expression dramatically increased and preferentially localized at the edge of cell-cell contacts, rather than in the nucleus from the eight-cell stage onwards. CXADR expression was knocked down (KD) by microinjecting double-stranded RNA into one-cell parthenotes. The vast majority of CXADR KD embryos failed to develop to the blastocyst stage, and a few developed KD blastocysts did not expand fully. Analysis of adherens junction (AJ)- and TJ-associated genes/proteins using qRT-PCR, immunocytochemistry and assessment of TJ permeability using FITC-dextran uptake assay revealed that the developmental failure and relatively small cavities are attributed to the defects of TJ assembly. In summary, CXADR is necessary for the AJ and TJ assembly/biogenesis during pre-implantation development. PMID:26729920

  14. WNT3 and membrane-associated ?-catenin regulate trophectoderm lineage differentiation in human blastocysts.

    PubMed

    Krivega, M; Essahib, W; Van de Velde, H

    2015-09-01

    WNT/?-catenin signaling has been described as a crucial regulator of embryonic stem cells and embryogenesis. However, little is known on its role during human preimplantation embryo development, besides the RNA expression of its multiple players. In this study, we performed ?-catenin loss- and gain-of-function studies on human preimplantation embryos by adding either Cardamonin or GSK3 inhibitor, 1-Azakenpaullone, to the embryo culture medium from the cleavage until blastocyst stages (Days 3-5/6). ?-Catenin was displayed in the cortical region underneath the membrane during all stages, but it only showed nuclear localization at cleavage stages after stabilization with 1-Azakenpaullone. We did not observe any effects on the inner cell mass markers NANOG, POU5F1, SOX2 and SALL4 in these functional experiments. However, both ?-catenin degradation and stabilization caused inhibition of the trophectoderm (TE) fate, illustrated by KRT18 and GATA3 RNA, and CDX2 protein expression. Based on the TE-specific WNT3 protein expression in blastocysts, we postulated that this protein may be an upstream regulator for the observed membrane ?-catenin function. The addition of either WNT3 or 1-Azakenpaullone to the culture medium promoted EOMES expression specific for trophoblast development. In both studies, the canonical WNT pathway target gene, TCF1, was not affected. Therefore, we conclude that WNT3 and membrane-associated ?-catenin promote progenitor trophoblast development in human blastocysts. These results have important implications in assisted reproduction and stem cell biology. PMID:26108805

  15. 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?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. PMID:22618890

  16. Expression of heparan sulfate proteoglycan (perlecan) in the mouse blastocyst is regulated during normal and delayed implantation.

    PubMed

    Smith, S E; French, M M; Julian, J; Paria, B C; Dey, S K; Carson, D D

    1997-04-01

    Previous studies have shown that expression of the heparan sulfate proteoglycan, perlecan, on the external trophectodermal cell surfaces of mouse blastocysts increases during acquisition of attachment competence. However, it is not clear if this change in perlecan protein expression also is reflected at the level of perlecan mRNA expression. In the present investigation, the spatial and temporal patterns of perlecan mRNA expression in the mouse embryo during the periimplantation period were examined by in situ hybridization and reverse transcriptase-polymerase chain reaction. In addition, a delayed implantation model was used to determine the expression of perlecan mRNA and protein in dormant and estrogen-activated hatched blastocysts. The results demonstrate that perlecan mRNA expression is low in morulae, but increases in Day 4 blastocysts, attaining maximal expression in Day 4.5 attachment-competent blastocysts. In contrast, perlecan mRNA is detected in both the dormant and estrogen-activated delayed blastocysts; however, within 12 hr of blastocyst activation by estrogen, both perlecan protein and heparan sulfate chain expression markedly increase. Taken together, these results suggest that during normal development perlecan mRNA expression increases with the acquisition of attachment competence. Moreover, perlecan protein expression also is attenuated during delayed implantation and appears to increase in response to nidatory estrogen, perhaps via the increased translation of preexisting perlecan mRNA. PMID:9142982

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

    PubMed

    Lpez-Cardona, Angela P; Fernndez-Gonzlez, Ral; Prez-Crespo, Miriam; Aln, 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. PMID:26224009

  18. Growth, transfer, structural, optical, and electrical properties of large-size transition-metal dichalcogenide monolayer single-crystals

    NASA Astrophysics Data System (ADS)

    Yang, Zheng; Hsu, Bo; Xiao, Jiao; Poulos, George; YANG Research Group Team

    2015-03-01

    We report growth, transfer process, as well as structural, optical, and electrical properties of large-size and high-quality two-dimensional transition-metal dichalcogenide MX2 (M=Mo, W; X=S, Se) single-crystalline triangular-shape nanosheets composed of one to a few monolayers. A vapor-trapping enhanced chemical vapor deposition approach was employed for the MX2 monlayer single crystal growth. The number of layers, crystallinity, and uniformity of the as-grown MX2 were characterized and confirmed by Raman and photoluminescence measurements. The MX2 monlayer single-crystal triangles show comparable size and uniformity to the state-of-the-art results reported as of now. The optical properties of the MX2 were studied based on the analysis of the photoluminescence results. The electrical properties including resistivity, mobility, carrier type and concentration, and contact resistance of the MX2 were characterized by both three-terminal field-effect transistor and Hall effect transport measurements. The Hall bar devices were fabricated by lithography and dry-etching of the as-grown single-crystalline MX2. The transfer process of the MX2 from growth substrate (SiO2-on-Si) to various substrates was successfully demonstrated. Acknowledgement to the funding support from Ignite Award.

  19. A viable foal obtained by equine somatic cell nuclear transfer using oocytes recovered from immature follicles of live mares.

    PubMed

    Choi, Young-Ho; Norris, Jody D; Velez, Isabel C; Jacobson, Candace C; Hartman, David L; Hinrichs, Katrin

    2013-03-15

    The presence of heterogenous mitochondria from the host ooplast affects the acceptance of offspring obtained by somatic cell nuclear transfer. This might be avoided by obtaining oocytes from selected females, but is then complicated by low numbers of available oocytes. We examined the efficiency of equine somatic cell nuclear transfer using oocytes recovered by transvaginal aspiration of immature follicles from 11 mares. Use of metaphase I oocytes as cytoplasts and of scriptaid (a histone deacetylase inhibitor) treatment during oocyte activation were evaluated to determine if these approaches would increase blastocyst production. In experiment 1, blastocyst development was 0/14 for metaphase I oocytes and 4/103 (4%) for metaphase II oocytes. Three blastocysts were transferred to recipient mares, resulting in two pregnancies and one live foal, which died shortly after birth. In experiment 2, blastocyst development was 2/47 (4%) for control oocytes and 1/83 (1%) for scriptaid-treated oocytes. No foals were born from two blastocysts transferred in the control group. The blastocyst from the scriptaid treatment resulted in birth of a live foal. In conclusion, this is apparently the first report of production of a viable cloned foal from oocytes collected from immature follicles of live mares, supporting the possibility of cloning using oocytes from selected mares. PMID:23312717

  20. Cloned calves derived from somatic cell nuclear transfer embryos cultured in chemically defined medium or modified synthetic oviduct fluid

    PubMed Central

    Hong, So Gun

    2011-01-01

    Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures. PMID:21368567

  1. [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 52.18.3%. 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

  2. Single-excitation dual-color coherent lasing by tuning resonance energy transfer processes in porous structured nanowires.

    PubMed

    Wang, Zhaona; Shi, Xiaoyu; Yu, Ruomeng; Wei, Sujun; Chang, Qing; Wang, Yanrong; Liu, Dahe; Wang, Zhong Lin

    2015-10-01

    Single-excitation dual-color coherent lasing was achieved in a mixed random system of a binary dye and the suspension of gold-silver porous nanowires with plenty of nanogaps. This greatly enhanced the local electromagnetic field in the visible range and guaranteed a low threshold and high Q factor (>10?000) operator for simultaneous dual-color lasing. By tuning the resonance energy transfer process in the stimulated emission, triple output modes (single chartreuse lasing, chartreuse and red dual-color lasing, and single red coherent lasing) were easily obtained. This triple-mode coherent random lasing introduces a new approach to designing multi-functional micro-optoelectronic devices for multi-color speckle-free imaging and interference. PMID:26349545

  3. Single-excitation dual-color coherent lasing by tuning resonance energy transfer processes in porous structured nanowires

    NASA Astrophysics Data System (ADS)

    Wang, Zhaona; Shi, Xiaoyu; Yu, Ruomeng; Wei, Sujun; Chang, Qing; Wang, Yanrong; Liu, Dahe; Wang, Zhong Lin

    2015-09-01

    Single-excitation dual-color coherent lasing was achieved in a mixed random system of a binary dye and the suspension of gold-silver porous nanowires with plenty of nanogaps. This greatly enhanced the local electromagnetic field in the visible range and guaranteed a low threshold and high Q factor (>10 000) operator for simultaneous dual-color lasing. By tuning the resonance energy transfer process in the stimulated emission, triple output modes (single chartreuse lasing, chartreuse and red dual-color lasing, and single red coherent lasing) were easily obtained. This triple-mode coherent random lasing introduces a new approach to designing multi-functional micro-optoelectronic devices for multi-color speckle-free imaging and interference.Single-excitation dual-color coherent lasing was achieved in a mixed random system of a binary dye and the suspension of gold-silver porous nanowires with plenty of nanogaps. This greatly enhanced the local electromagnetic field in the visible range and guaranteed a low threshold and high Q factor (>10 000) operator for simultaneous dual-color lasing. By tuning the resonance energy transfer process in the stimulated emission, triple output modes (single chartreuse lasing, chartreuse and red dual-color lasing, and single red coherent lasing) were easily obtained. This triple-mode coherent random lasing introduces a new approach to designing multi-functional micro-optoelectronic devices for multi-color speckle-free imaging and interference. Electronic supplementary information (ESI) available: Materials and methods, Fig. S1-6. See DOI: 10.1039/c5nr03349g

  4. 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)

  5. Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer.

    PubMed

    Deng, Wei; Yang, Dongshan; Zhao, Bentian; Ouyang, Zhen; Song, Jun; Fan, Nana; Liu, Zhaoming; Zhao, Yu; Wu, Qinghong; Nashun, Bayaer; Tang, Jiangjing; Wu, Zhenfang; Gu, Weiwang; Lai, Liangxue

    2011-01-01

    Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer. PMID:21603633

  6. The Effects of Perioperative Analgesia on Litter Size in Crl:CD1(ICR) Mice Undergoing Embryo Transfer

    PubMed Central

    Goulding, David R; Myers, Page H; Goulding, Eugenia H; Blankenship, Terry L; Grant, Mary F; Forsythe, Diane B

    2010-01-01

    The objective of this study was to evaluate the effect on litter size of 2 analgesics used perioperatively during mouse embryo transfer surgery. Day 2.5 pseudopregnant CD1 mice (n = 96) were divided equally into 2 analgesic treatment groups and a saline control group. Each mouse received a single, subcutaneous dose of buprenorphine hydrochloride (0.1 mg/kg), flunixin meglumine (2.5 mg/kg), or saline immediately after induction of anesthesia with 2.5% isoflurane. Each mouse then was prepared for aseptic surgery. Blastocysts had previously been collected from C57BL/6NCrl female mice that were synchronized and superovulated by using pregnant mare serum gonadotropin and human chorionic gonadotropin and mated with C57BL/6NTac male mice 3.5 d before collection. Viable blastocysts were pooled, and 8 were selected arbitrarily and transplanted into the right uterine horn of each pseudopregnant CD1 mouse. Mice were monitored throughout pregnancy, and the number of pups at birth was documented. No statistically significant difference was found between the 3 groups. These results indicate that perioperative analgesic treatment with buprenorphine or flunixin in the CD1 mouse undergoing embryo transfer is not associated with increased embryonic loss. PMID:20819387

  7. A DFT-based theoretical study on the photophysics of 4-hydroxyacridine: single-water-mediated excited state proton transfer.

    PubMed

    Paul, Bijan Kumar; Mahanta, Subrata; Singh, Rupashree Balia; Guchhait, Nikhil

    2010-02-25

    Study of intra- and intermolecular hydrogen-bonding interaction and excited state proton transfer reaction has been carried out in 4-hydroxyacridine (4-HA) and its hydrated clusters theoretically. Density functional theory [B3LYP/6-311++G(d,p)] has been exploited to calculate structural parameters and relative energies of different conformers of 4-HA and its hydrates. The substantial impact of solvent reaction field on hydrogen-bond energies, conformational equilibrium, and tautomerization reaction in aqueous medium have been realized by employing Onsager and PCM reaction field methods, and the stability of the conformers of 4-HA is found to be profusely modulated by the electrostatic influence of the solvent. A deeper insight into the nature of H-bonding in 4-HA and its hydrated clusters has been achieved under the provision of natural bond orbital and atoms in molecule analysis. Elucidation of potential energy curves for proton transfer reaction reveals that an intrinsic and two-water-molecule-assisted proton transfer (PT) reaction in 4-HA is hindered by high energy barrier in the S(1) surface, whereas single-water-assisted PT reaction is practically rendered barrierless. At the same time, the appreciably high barrier height of the ground state potential energy curve in all the cases unambiguously rules out the possibility of ground state proton transfer reaction. PMID:20121061

  8. Density functional theory based calculations of the transfer integral in a redox-active single-molecule junction

    NASA Astrophysics Data System (ADS)

    Kastlunger, Georg; Stadler, Robert

    2014-03-01

    There are various quantum chemical approaches for an ab initio description of transfer integrals within the framework of Marcus theory in the context of electron transfer reactions. In our paper, we aim to calculate transfer integrals in redox-active single molecule junctions, where we focus on the coherent tunneling limit with the metal leads taking the position of donor and acceptor and the molecule acting as a transport mediating bridge. This setup allows us to derive a conductance, which can be directly compared with recent results from a nonequilibrium Green's function approach. Compared with purely molecular systems we face additional challenges due to the metallic nature of the leads, which rules out some of the common techniques, and due to their periodicity, which requires k-space integration. We present three different methods, all based on density functional theory, for calculating the transfer integral under these constraints, which we benchmark on molecular test systems from the relevant literature. We also discuss many-body effects and apply all three techniques to a junction with a Ruthenium complex in different oxidation states.

  9. 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.

  10. One-step transfer or exchange of arbitrary multipartite quantum states with a single-qubit coupler

    NASA Astrophysics Data System (ADS)

    Yang, Chui-Ping; Su, Qi-Ping; Zheng, Shi-Biao; Han, Siyuan

    2015-08-01

    The transfer or exchange of multipartite quantum states is critical to the realization of large-scale quantum information processing and quantum communication. In this work, we demonstrate that by using a single quantum two-level systema qubitas a coupler, arbitrary multipartite quantum states (either entangled or separable) can be transferred or exchanged simultaneously between two sets of qubits. During the entire process, the coupler remains unexcited minimizing the effect of coupler decoherence on the process. This feature allows one to use qubits with rapid frequency tunability and a large range of frequency tuning, such as phase qubits, as couplers. Our findings offer the potential to significantly reduce the resources needed to construct and operate large-scale quantum information networks consisting of many multiqubit registers, memory cells, and processing units.

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

    PubMed

    Ochota, Małgorzata; Pasieka, Anna; Niżański, Wojciech

    2016-03-15

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

  12. Probing electron transfer mechanisms in Shewanella oneidensis MR-1 using a nanoelectrode platform and single-cell imaging

    PubMed Central

    Jiang, Xiaocheng; Hu, Jinsong; Fitzgerald, Lisa A.; Biffinger, Justin C.; Xie, Ping; Ringeisen, Bradley R.; Lieber, Charles M.

    2010-01-01

    Microbial fuel cells (MFCs) represent a promising approach for sustainable energy production as they generate electricity directly from metabolism of organic substrates without the need for catalysts. However, the mechanisms of electron transfer between microbes and electrodes, which could ultimately limit power extraction, remain controversial. Here we demonstrate optically transparent nanoelectrodes as a platform to investigate extracellular electron transfer in Shewanella oneidensis MR-1, where an array of nanoholes precludes or single window allows for direct microbe-electrode contacts. Following addition of cells, short-circuit current measurements showed similar amplitude and temporal response for both electrode configurations, while in situ optical imaging demonstrates that the measured currents were uncorrelated with the cell number on the electrodes. High-resolution imaging showed the presence of thin, 4- to 5-nm diameter filaments emanating from cell bodies, although these filaments do not appear correlated with current generation. Both types of electrodes yielded similar currents at longer times in dense cell layers and exhibited a rapid drop in current upon removal of diffusible mediators. Reintroduction of the original cell-free media yielded a rapid increase in current to ?80% of original level, whereas imaging showed that the positions of >70% of cells remained unchanged during solution exchange. Together, these measurements show that electron transfer occurs predominantly by mediated mechanism in this model system. Last, simultaneous measurements of current and cell positions showed that cell motility and electron transfer were inversely correlated. The ability to control and image cell/electrode interactions down to the single-cell level provide a powerful approach for advancing our fundamental understanding of MFCs. PMID:20837546

  13. The oxidation of sulfur dioxide by single and double oxygen transfer paths.

    PubMed

    Troiani, Anna; Rosi, Marzio; Salvitti, Chiara; de Petris, Giulia

    2014-09-15

    The oxidation of SO2 by nonmetal oxoanions in the gas phase is investigated in an experimental and theoretical study of the structure of the species involved and the reaction kinetics and mechanism. SO3 , SO3(.-) and SO4(.-) are efficiently produced by reaction of On XO(-) anions (X=Cl, Br, and I; n=0 and 1) with SO2 ; XO(-) ions mainly react to give SO3 by oxygen-atom transfer, whereas OXO(-) ions mainly give SO3(.-) by oxygen-anion transfer. On descending the halogen group from chlorine to iodine, the SO3 /SO3(.-) ratio decreases and increases for reactions involving XO(-) and OXO(-) anions, respectively, whereas the formation of SO4(.-) is particularly significant with OIO(-). Kinetic factors play a major role in the reactions of On XO(-), depending on the halogen atom and its oxidation state. PMID:24953698

  14. Radiative, conductive and convective heat-transfers in a single Monte Carlo algorithm

    NASA Astrophysics Data System (ADS)

    Fournier, Richard; Blanco, Stéphane; Eymet, Vincent; El Hafi, Mouna; Spiesser, Christophe

    2016-01-01

    It was recently shown that null-collision algorithms could lead to grid-free radiative- transfer Monte Carlo algorithms that immediately benefit of computer-graphics tools for an efficient handling of complex geometries [1, 2]. We here explore the idea of extending the approach to heat transfer problems combining radiation, conduction and convection. This is possible as soon as the model can be given the form of a second-kind Fredholm equation. In the following pages, we show that this is quite straightforward at the stationnary limit in the linear case. The oral presentation will provide corresponding simulation examples. Perspectives will then be drawn concerning the extension to non-stationnary cases and non-linear coupling.

  15. 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

    2015-01-01

    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. PMID:25868011

  16. Inner-sphere electron-transfer single iodide mechanism for dye regeneration in dye-sensitized solar cells.

    PubMed

    Jeon, Jiwon; Goddard, William A; Kim, Hyungjun

    2013-02-20

    During the regeneration of the oxidized dye in dye-sensitized solar cells, the redox couple of I(-)/I(3)(-) reduces the photo-oxidized dye. The simplest mechanism would be a direct charge-transfer mechanism from I(-) to D(+) [D(+) + I(-) ? D(0) + I], called the single iodide process (SIP). However, this is an unfavorable equilibrium because the redox potential of I()/I(-) is 1.224 V vs SHE, which is 0.13 V higher than that of the dye. This led to the postulation of the two iodide process (TIP) [(D(+)I(-)) + I(-) ? (DI(2)(-)) ? D(0) + I(2)(-))] for a sufficiently high reducing power, but TIP is not consistent with either the recent experimental data suggesting the first-order kinetics or recent time-resolved spectroscopic measurements. To resolve this conundrum, we used quantum mechanics including Poisson-Boltzmann solvation to examine the electron-transfer process between I(-) and D(+) for the Ru(dcb)(2)NCS(2) or N3 dye. We find that I(-) is attracted to the oxidized dye, positioning I(-) next to the NCS. At this equilibrium position, the I(-) electron is already 40% transferred to the NCS, showing that the redox potential of I(-) is well matched with the dye. This matching of the redox potential occurs because I(-) is partially desolvated as it positions itself for the inner-sphere electron transfer (ISET). The previous analyses all assumed an outer-sphere electron-transfer process. Thus our ISET-SIP model is consistent with the known redox potentials and with recent experimental reports. With the ISET-SIP mechanism, one can start to consider how to enhance the dye regeneration kinetics by redesigning ligands to maximize the interaction with iodide. PMID:23384053

  17. Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency.

    PubMed

    Liao, Hung-Fu; Mo, Chu-Fan; Wu, Shinn-Chih; Cheng, Dai-Han; Yu, Chih-Yun; Chang, Kai-Wei; Kao, Tzu-Hao; Lu, Chia-Wei; Pinskaya, Marina; Morillon, Antonin; Lin, Shih-Shun; Cheng, Winston T K; Bourc'his, Dborah; Bestor, Timothy; Sung, Li-Ying; Lin, Shau-Ping

    2015-10-01

    Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming. PMID:26159833

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

    Energy Science and Technology Software Center (ESTSC)

    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

  19. 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.

  20. 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.

  1. 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

    Pea Alvarez, Miriam; Mayorga Burrezo, Paula; Kertesz, Miklos; Iwamoto, Takahiro; Yamago, Shigeru; Xia, Jianlong; Jasti, Ramesh; Lpez Navarrete, Juan T; Taravillo, Mercedes; Baonza, Valentn 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

  2. 33 CFR 150.445 - When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water? 150.445 Section 150.445 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DEEPWATER PORTS DEEPWATER PORTS: OPERATIONS Cargo Transfer Operations §...

  3. 33 CFR 150.445 - When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water? 150.445 Section 150.445 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DEEPWATER PORTS DEEPWATER PORTS: OPERATIONS Cargo Transfer Operations §...

  4. 33 CFR 150.445 - When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false When is oil in a single point mooring-oil transfer system (SPM-OTS) displaced with water? 150.445 Section 150.445 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DEEPWATER PORTS DEEPWATER PORTS: OPERATIONS Cargo Transfer Operations §...

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

    PubMed Central

    Pongsuthirak, Pallop; Songveeratham, Sorramon

    2015-01-01

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

  6. Accumulation of advanced glycation end products in the rabbit blastocyst under maternal diabetes.

    PubMed

    Haucke, Elisa; Navarrete Santos, Alexander; Simm, Andreas; Henning, Christian; Glomb, Marcus A; Grke, Jacqueline; Schindler, Maria; Fischer, Bernd; Navarrete Santos, Anne

    2014-08-01

    Diabetes mellitus (DM) during pregnancy is one of the leading causes of perinatal morbidity and birth defects. The mechanism by which maternal hyperglycemia, the major teratogenic factor, induces embryonic malformations remains unclear. Advanced glycation end products (AGEs) are known to accumulate during the course of DM and contribute to the development of diabetic complications. Employing a diabetic rabbit model, we investigated the influence of maternal hyperglycemia during the preimplantation period on AGE formation (pentosidine, argpyrimidine, and N(?)-carboxymethyllysine (CML)) in the reproductive tract and the embryo itself. As a consequence of type 1 DM, the AGE levels in blood plasma increased up to 50%, correlating closely with an AGE accumulation in the endometrium of diabetic females. Embryos from diabetic mothers had increased protein-bound CML levels and showed enhanced fluorescent signals for AGE-specific fluorescence in the blastocyst cavity fluid (BCF). The quantification of CML by HPLC-mass spectrometry (MS/MS) showed a higher amount of soluble CML in the BCF of blastocysts from diabetic rabbits (0.260.05??mol/l) compared with controls (0.180.02??mol/l). The high amount of AGEs in blastocysts from diabetic mothers correlates positively with an increased AGER (receptor for AGE (RAGE)) mRNA expression. Our study gives alarming insights into the consequences of poorly controlled maternal diabetes for AGE formation in the embryo. Maternal hyperglycemia during the preimplantation period is correlated with an increase in AGE formation in the uterine environment and the embryo itself. This may influence the development of the embryo through increased AGE-mediated cellular stress by RAGEs. PMID:24821834

  7. Derivation of Human Embryonic Stem Cell Lines from Vitrified Human Blastocysts.

    PubMed

    Bradley, Cara K; Schaft, Julia; Roy, Tammie K; Dumevska, Biljana; Peura, Teija T

    2016-01-01

    Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have become excess to clinical needs in assisted reproduction programs. They provide cellular models for embryonic development and disease, and are thought to be useful for future cell replacement therapies and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines. This includes blastocyst cryopreservation using a highly efficient vitrification protocol, the production and use of fibroblast feeder cells, embryo plating and passaging of resulting cellular outgrowths, and cryopreservation of putative stem cells lines. PMID:24961221

  8. 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.

  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. 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.

  11. 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

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

    PubMed

    Bagal, 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. PMID:23221832

  13. Effects of Trichostatin A on In vitro Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer

    PubMed Central

    Jeong, Yeon Ik; Park, Chi Hun; Kim, Huen Suk; Jeong, Yeon Woo; Lee, Jong Yun; Park, Sun Woo; Lee, Se Yeong; Hyun, Sang Hwan; Kim, Yeun Wook; Shin, Taeyoung; Hwang, Woo Suk

    2013-01-01

    Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9?114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT. PMID:25049758

  14. Effects of Trichostatin A on In vitro Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer.

    PubMed

    Jeong, Yeon Ik; Park, Chi Hun; Kim, Huen Suk; Jeong, Yeon Woo; Lee, Jong Yun; Park, Sun Woo; Lee, Se Yeong; Hyun, Sang Hwan; Kim, Yeun Wook; Shin, Taeyoung; Hwang, Woo Suk

    2013-12-01

    Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9?114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT. PMID:25049758

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

    PubMed Central

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

    2014-01-01

    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 )/NPB(700 )/mCP:Firpic-x%:Ir(ppy)3-0.5%:Ir(piq)3-y%(300 )/TPBi(300 )/Liq(20 )/Al(1200 ). 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). PMID:25388087

  16. 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

    2014-01-01

    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 )/NPB(700 )/mCP:Firpic-x%:Ir(ppy)3-0.5%:Ir(piq)3-y%(300 )/TPBi(300 )/Liq(20 )/Al(1200 ). 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). PMID:25388087

  17. 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 )/NPB(700 )/mCP:Firpic-x%:Ir(ppy)3-0.5%:Ir(piq)3-y%(300 )/TPBi(300 )/Liq(20 )/Al(1200 ). 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).

  18. Enhancement of adoptive T cell transfer with single low dose pretreatment of doxorubicin or paclitaxel in mice

    PubMed Central

    Chuang, Hui-Yen; Chang, Ya-Fang; Hwang, Jeng-Jong

    2015-01-01

    Ex vivo expansion of CD8+ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8+ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8+ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8+ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2×106 CD8+ T-cells and achieved the same level of tumor growth inhibition as that of 5×106 CD8+ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-β, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8+ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8+ T-cells which also stay longer. PMID:26683520

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Effect of the Pr3+ ? Gd3+ energy transfer in multicomponent garnet single crystal scintillators

    NASA Astrophysics Data System (ADS)

    Babin, V.; Nikl, M.; Kamada, K.; Beitlerova, A.; Yoshikawa, A.

    2013-09-01

    Luminescence processes in the undoped and Pr3+-doped (Gd,RE)3(Ga,Al)5O12, RE = Lu,Y, multicomponent garnets are studied by time-resolved photoluminescence spectroscopy. Energy transfer processes between Pr3+ and Gd3+ causing significant deterioration of the scintillation performance are considered in detail. As is shown in current work, an overlap of the 5d1-3H4 emission transition of Pr3+ and 8S-6Px absorption transition of Gd3+ results in unwanted depletion of Pr3+ 5d1 excited state and is further intensified by the concentration quenching in the Gd3+-sublattice. This process explains a drastic decrease of light yield in Pr3+-doped Gd3+-containing multicomponent garnets observed in a previous work.

  4. Solvent-mediated tautomerization of purine: single to quadruple proton transfer

    NASA Astrophysics Data System (ADS)

    Ahn, Doo-Sik; Lee, Sungyul; Kim, Bongsoo

    2004-06-01

    We present calculations for the structures and the tautomerization reaction of purine and purine - (H 2O) n ( n=1-3) clusters. We find two pathways (via the carbene and the sp 3-type intermediate) for the 9 ? 7 tautomerization of bare purine. The barrier heights for the 9 ? 3 and 9 ? 7 tautomerization of bare purine are calculated to be large (60-70 kcal/mol). Hydrogen bonding with the water molecule(s), however, dramatically lowers the 9 ? 3 barrier by the concerted multiple proton transfer mechanism, favoring the formation of the conformer 3(H)- relative to the 7(H)-purine in the microsolvated environment, in contrast to the gas phase or the aqueous solution.

  5. Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique.

    PubMed

    Booth, P J; Tan, S J; Reipurth, R; Holm, P; Callesen, H

    2001-01-01

    Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development of the system was based on results using parthenogenetic and in vitro fertilized zygotes in order to (a) select the optimal primary activation agent that induced the lowest lysis rate but highest parthenogenetic blastocyst yield, (b) evaluate the quantity and quality of zona-free blastocysts produced in WOWs, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20.8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1%) blastocysts per successfully fused and surviving reconstructed embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1%) recipients pregnant at 5 months after transfer. These results suggest that the zona-free nuclear transfer technique generates blastocysts of equivalent quantity and quality compared to conventional micromanipulation methods, requires less technical expertise, is less time consuming and can double the daily output of reconstructed embryos (even after taking into consideration the rejection of the half oocytes containing the metaphase plate). PMID:11945223

  6. Probing Single-Molecule T4 Lysozyme Conformational Dynamics by Intramolecular Fluorescence Energy Transfer

    SciTech Connect

    Chen, Yu; Hu, Dehong; Vorpagel, Erich R.; Lu, H PETER.

    2003-07-16

    We demonstrate the use of single-molecule spectroscopy to study enzyme conformational motions of T4 lysozyme under hydrolysis reaction of the polysaccharide walls of E. Coli B cells.By attaching a donoracceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, the hinge-bending motions of the enzyme are measured by monitoring the donor-acceptor emission intensity as a function of time. The overall enzymatic reaction rate constants are found to vary widely from molecule to molecule. The dominant contribution to this static inhomogeneity is attributed to enzyme searching for reactive sites on the substrate.

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

    PubMed Central

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

    2015-01-01

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

  8. Manipulation of a single Mn spin using excitation transfer between two coupled CdTe/ZnTe quantum dots

    NASA Astrophysics Data System (ADS)

    Goryca, Mateusz

    2010-02-01

    A semiconductor quantum dot (QD) containing a single Mn atom is a promising system from the point of view of future information processing and storage devices. An efficient optical read-out of the single Mn spin state in a CdTe/ZnTe quantum dot, as well as studies of dynamics of this state, were recently reported by L. Besombes and co-workers. However, to construct the building blocks of future memory devices basing on single magnetic atoms the ability to control a single spin is still needed. This work is focused on the advancement in writing and storing of information on the Mn spin state. We demonstrate optical writing of information on the spin state of a single Mn ion embedded in a CdTe QD and we test the storage time in the range of a few tenths of a millisecond. A spin-conserving excitation transfer between two coupled QDs is used as a tool for optical manipulation of the Mn spin. Excitons resonantly created in a dot without magnetic atom by circularly polarized light tunnel to the dot with the Mn ion in a few picoseconds. Then they act on the Mn ion via the sp-d exchange interaction and orient its spin. The orientation is much more efficient in presence of a magnetic field of about 1T, due to suppression of fast spin relaxation channels. Dynamics of the Mn spin under polarized excitation as well as the information storage time on the Mn spin was measured in a time-resolved experiment, in which the intensity and polarization of excitation were modulated. Observed dynamics can be described with a simple rate equation model. The storage time was enhanced by the magnetic field and reached about half a millisecond at 1T.

  9. The interfering effects of superovulation and vitrification upon some important epigenetic biomarkers in mouse blastocyst.

    PubMed

    Bakhtari, Azizollah; Rahmani, Hamid-Reza; Bonakdar, Elham; Jafarpour, Farnoosh; Asgari, Vajiheh; Hosseini, Sayyed-Mortaza; Hajian, Mehdi; Edriss, Mohammad-Ali; Nasr-Esfahani, Mohammad-Hossein

    2014-12-01

    Appropriate epigenetic changes in preimplantation embryos are critical for embryonic development and successful pregnancy. The aim of this study was to evaluate the effects of some assisted reproductive techniques (ARTs) on a panel of epigenetic biomarkers by immunofluorescence staining at blastocyst stage. For this purpose, four treatment groups were designed: control (C), superovulation (S), superovulation+in vitro culture (SI), and superovulation+vitrification+in vitro culture (SVI). Results showed that vitrification decreased the developmental competence of embryos cultured in vitro (P<0.05). Semi-quantitative analysis revealed that vitrification decreased the fluorescence intensity of global DNA methylation in the inner cell mass (ICM), in SVI Group in comparison to C group (P<0.05). Superovulation, elevated the level of H3K9acetylation of trophectoderm (TE) in comparison to C and SI groups (P<0.05). Furthermore, ARTs manipulations influenced H3K9acetylation in the ICM (P<0.05). The fluorescence intensity of H4K12acetylation in TE for SVI group was higher than C and S (P<0.05). For H3K4tri-methylation, S group had higher fluorescence intensity in the ICM in comparison to SI and SVI (P<0.05). Finally, in vitro culture decreased Pou5f1 protein signal in comparison to in vivo-derived embryos at blastocyst stage (P<0.05). In conclusion, ART manipulations may have important influences on multiple epigenetic biomarkers. PMID:25307438

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

    PubMed Central

    Naturil-Alfonso, Carmen; Saenz-de-Juano, Mara dels Desamparats; Pearanda, David S.

    2012-01-01

    Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. PMID:23251477

  11. 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

  12. Lysophosphatidic Acid Signaling in Late Cleavage and Blastocyst Stage Bovine Embryos

    PubMed Central

    Torres, Ana Catarina; Boruszewska, Dorota; Batista, Mariana; Kowalczyk-Zieba, Ilona; Sinderewicz, Emilia; Saulnier-Blache, Jean Sebastian; Woclawek-Potocka, Izabela; Lopes-da-Costa, Luis

    2014-01-01

    Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues. In the cow, LPA is involved in luteal and early pregnancy maintenance. Here, we evaluated the presence and role of LPA in bovine early embryonic development. In relevant aspects, bovine embryos reflect more closely the scenario occurring in human embryos than the mouse model. Transcription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX and cPLA2) and of LPA receptors (LPAR14) were detected in Days 5 and 8 in vitro produced embryos. Embryonic LPA production into culture medium was also detected at both stages of development. Supplementation of culture medium with LPA (10?5?M) between Days 2 and 8 had no effect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis (PTGS2, PGES, and PGFS) and steroidogenesis (3?HSD). However, LPA treatment affected transcription levels of embryo quality markers, decreasing BAX (apoptotic) and increasing BCL2 (antiapoptotic) and IGF2R (growth marker) gene transcription levels. Blastocyst transcription of OCT4 (pluripotency marker) was not affected by LPA stimulation. In conclusion, LPA is an early bovine embryonic autocrine/paracrine signaling mediator, and LPA action may be relevant in early embryo-maternal interactions leading to embryonic survival. PMID:24833815

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

    PubMed

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

    2016-01-01

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

  14. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    NASA Astrophysics Data System (ADS)

    Osychenko, A. A.; Zalesskii, A. D.; Krivokharchenko, A. S.; Zhakhbazyan, A. K.; Ryabova, A. V.; Nadtochenko, V. A.

    2015-05-01

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated.

  15. Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies.

    PubMed

    Kopra, Kari; Hrm, Harri

    2015-12-25

    The increased number of therapeutic targets has led to a growing need for screening methods enabling possible inhibitor compound selection. Information for new therapeutic targets has been found mostly from sequencing of the human genome but this knowledge cannot be directly converted into clinically relevant drug molecules. After target identification, the multistep drug development process takes many years and hundreds of millions of dollars are spent without certainty of the outcome. The first and the most critical step in the drug development process is hit selection. The optimal high throughput screening method should provide the highest possible number of true positive hits for further studies and lead discovery. The result should be achieved with low material consumption in a rapid and automated process. Radioactive label based methods are sensitive, but due to the problems arising from the radioactivity, luminescence-based methods have become increasingly popular in screening. In this review, the time-resolved luminescence based quenching resonance energy transfer (QRET) technique is discussed for primary screening. PMID:25721971

  16. 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.

  17. Monozygotic twins with discordant karyotypes following preimplantation genetic screening and single embryo transfer: case report

    PubMed Central

    Gaillyova, Renata; Travnik, Pavel; Oracova, Eva; Vesela, Katerina; Hromadova, Lenka; Vesely, Jan; Musilova, Petra; Rubes, Jiri; Kadlecova, Jitka; Slamova, Iva; Makaturova, Eva; Vranova, Vladimira

    2010-01-01

    Purpose To report a case of monozygotic monochorial diamniotic twins with discordant karyotypes. Methods and results The pregnancy was achieved following a treatment cycle with intracytoplasmic sperm injection (ICSI) and preimplantation genetic screening (PGS) for chromosomes X, Y, 13, 16, 18, 21, 22. One embryo euploid for studied chromosomes was transferred. Prenatal ultrasonography revealed monozygotic twins. One fetus had growth retardation, multiple organ abnormalities and polyhydramnion. The other twin had normal ultrasound appearance. Delivery on week29 of gestation resulted in the birth of two females, a stillborn twin with karyotype 45,XX,-13[12]/46,XX,r(13)[3] and a healthy twin with normal karyotype. Conclusions The discordance in the twins karyotypes originated from a mosaic embryo. Structural chromosomal abnormality of the affected twin could not be revealed using standard PGS investigation. Embryo splitting occurred probably due to apoptotic process in an early stage of embryo development. Apoptosis represents one of the possible mechanisms which can explain the embryo twinning process globally. PMID:20700760

  18. 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.

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

    PubMed Central

    KANG, Hoin; PARK, Jong Im; ROH, Sangho

    2015-01-01

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

    PubMed

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

    2016-02-01

    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

  1. In situ superexchange electron transfer through a single molecule: A rectifying effect

    PubMed Central

    Kornyshev, Alexei A.; Kuznetsov, Alexander M.; Ulstrup, Jens

    2006-01-01

    An increasingly comprehensive body of literature is being devoted to single-molecule bridge-mediated electronic nanojunctions, prompted by their prospective applications in molecular electronics and single-molecule analysis. These junctions may operate in gas phase or electrolyte solution (in situ). For biomolecules, the latter is much closer to their native environment. Convenient target molecules are aromatic molecules, peptides, oligonucleotides, transition metal complexes, and, broadly, molecules with repetitive units, for which the conducting orbitals are energetically well below electronic levels of the solvent. A key feature for these junctions is rectification in the currentvoltage relation. A common view is that asymmetric molecules or asymmetric links to the electrodes are needed to acquire rectification. However, as we show here, this requirement could be different in situ, where a structurally symmetric system can provide rectification because of the Debye screening of the electric field in the nanogap if the screening length is smaller than the bridge length. The Galvani potentials of each electrode can be varied independently and lead to a transistor effect. We explore this behavior for the superexchange mechanism of electron transport, appropriate for a wide class of molecules. We also include the effect of conformational fluctuations on the lowest unoccupied molecular orbital (LUMO) energy levels; that gives rise to non-Arrhenius temperature dependence of the conductance, affected by the molecule length. Our study offers an analytical formula for the currentvoltage characteristics that demonstrates all these features. A detailed physical interpretation of the results is given with a discussion of reported experimental data. PMID:16641101

  2. A single gold nanorod as a plasmon resonance energy transfer based nanosensor for high-sensitivity Cu(II) detection.

    PubMed

    Jing, Chao; Shi, Lei; Liu, Xiaoyuan; Long, Yi-Tao

    2014-12-21

    Plasmon resonance energy transfer (PRET) has been widely applied in the detection of bio-recognition, heavy metal ions and cellular reactions with high sensitivity, based on the overlap between the plasmon resonance scattering band of nanoparticles and the absorption band of the surface-modified chromophore molecules. Previous sensors based on PRET were all implemented on gold nanospheres with scattering light in the range of 530 to 600 nm. In this work, a PRET-based nanosensor was developed on a thiol-di(2-picolyl)amine-modified single gold nanorod for the detection of Cu(2+) ions in aqueous solution with high sensitivity and selectivity. Compared to nanospheres, gold nanorods with tunable and wide plasmon resonance bands from the near-infrared to the infrared region exhibit promising potential for development as sensing probes. PMID:25338009

  3. Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1

    PubMed Central

    Margittai, M.; Widengren, J.; Schweinberger, E.; Schrder, G. F.; Felekyan, S.; Haustein, E.; Knig, M.; Fasshauer, D.; Grubmller, H.; Jahn, R.; Seidel, C. A. M.

    2003-01-01

    Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state. PMID:14668446

  4. Single-Nanoparticle Collision Events: Tunneling Electron Transfer on a Titanium Dioxide Passivated n-Silicon Electrode.

    PubMed

    Ahn, Hyun S; Bard, Allen J

    2015-11-01

    Single-nanoparticle collisions were observed on an n-type silicon electrode (600??m diameter) passivated by a thin layer of amorphous TiO2, where the current steps occurred by tunneling electron transfer. The observed collision frequency was in reasonable agreement with that predicted from theory. The isolated electrode, after a collision experiment, with a Pt/TiO2/n-Si architecture was shown to retain the photoelectrochemical properties of n-Si without photocorrosion or current decay. The Pt/TiO2/n-Si electrode produced 19?mA?cm(-2) of photocurrent density under 100?mW?cm(-2) irradiation from a xenon lamp during oxygen evolution without current fading for over 12?h. PMID:26377039

  5. 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.

  6. Optical horn antennas for efficiently transferring photons from a quantum emitter to a single-mode optical fiber.

    PubMed

    Grosjean, T; Mivelle, M; Burr, G W; Baida, F I

    2013-01-28

    We theoretically demonstrate highly efficient optical coupling between a single quantum emitter and a monomode optical fiber over remarkably broad spectral ranges by extending the concept of horn antenna to optics. The optical horn antenna directs the radiation from the emitter toward the optical fiber and efficiently phase-matches the photon emission with the fiber mode. Numerical results show that an optical horn antenna can funnel up to 85% of the radiation from a dipolar source within an emission cone semi-angle as small as 7 degrees (antenna directivity of 300). It is also shown that 50% of the emitted power from the dipolar source can be collected and coupled to an SMF-28 fiber mode over spectral ranges larger than 1000 nm, with a maximum energy transfer reaching 70 %. This approach may open new perspectives in quantum optics and sensing. PMID:23389160

  7. Multiple Reflection Effect on Spin-Transfer Torque Dynamics in Spin Valves with a Single or Dual Polarizer

    NASA Astrophysics Data System (ADS)

    Zhu, Weiwei; Zhang, Zongzhi; Zhang, Jianwei; Liu, Yaowen

    2015-04-01

    In this paper, spin-dependent multiple reflection effect on spin-transfer torque (STT) has been theoretically and numerically studied in a spin valve nanopillar with a single or dual spin-polarizer. By using a scattering matrix method, we formulate an analytical expression of STT that contains the multiple interfacial reflection effect. It is found that the multiple reflections could enhance the STT efficiency and reduce the critical switching current. The STT efficiency depends on the spin polarization of both the free layer and polarizer. In the nanopillars with a dual spin polarizer, the multiple reflections would cause an asymmetric frequency dependence on the applied current, albeit exactly the same parameters are used in all three ferromagnetic layers, indicating that the frequency in the negative current varies much faster than that in the positive case.

  8. Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer.

    PubMed

    Zeng, Yining; Liu, Hsiao-Wei; Landes, Christy F; Kim, Yoen Joo; Ma, Xiaojing; Zhu, Yongjin; Musier-Forsyth, Karin; Barbara, Paul F

    2007-07-31

    Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method. In this approach, an immobilized hairpin is exposed to a multistep programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer-only solutions. The sequence controllably "drags" single immobilized TAR hairpins among the kinetic stable states of the reaction mechanism; i.e., reactants, intermediates, and products. This single-molecule spectroscopy method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages along the reaction sequence, giving access to previously inaccessible kinetic processes and rate constants. By employing target oligonucleotides for specific TAR regions, we kinetically trap and investigate structural models for putative nucleation complexes for the annealing process. The new results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes. This includes information on the ability of NC to chaperone "reverse annealing" in single-strand transfer and the first observation of partially annealed, conformational substates in the annealing mechanism. PMID:17578926

  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. A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

    PubMed Central

    Park, Sang Kyu

    2014-01-01

    Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest. PMID:23820223

  11. 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.

  12. Analysis of Fluorescence Lifetime and Energy Transfer Efficiency in Single-Molecule Photon Trajectories of Fast-Folding Proteins.

    PubMed

    Chung, Hoi Sung; Louis, John M; Gopich, Irina V

    2016-02-01

    In single-molecule Frster resonance energy transfer (FRET) spectroscopy, the dynamics of molecular processes are usually determined by analyzing the fluorescence intensity of donor and acceptor dyes. Since FRET efficiency is related to fluorescence lifetimes, additional information can be extracted by analyzing fluorescence intensity and lifetime together. For fast processes where individual states are not well separated in a trajectory, it is not easy to obtain the lifetime information. Here, we present analysis methods to utilize fluorescence lifetime information from single-molecule FRET experiments, and apply these methods to three fast-folding, two-state proteins. By constructing 2D FRET efficiency-lifetime histograms, the correlation can be visualized between the FRET efficiency and fluorescence lifetimes in the presence of the submicrosecond to millisecond dynamics. We extend the previously developed method for analyzing delay times of donor photons to include acceptor delay times. To determine the kinetics and lifetime parameters accurately, we used a maximum likelihood method. We found that acceptor blinking can lead to inaccurate parameters in the donor delay time analysis. This problem can be solved by incorporating acceptor blinking into a model. While the analysis of acceptor delay times is not affected by acceptor blinking, it is more sensitive to the shape of the delay time distribution resulting from a broad conformational distribution in the unfolded state. PMID:26812046

  13. Na -dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

    SciTech Connect

    Van Winkle, L.J.; Christensen, H.N.; Campione, A.L.

    1985-10-05

    Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na -dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo (3,2,1)octane-3-carboxylic acid (BCO) by blastocysts was largely Na dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na -dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo (2,2,1)heptane-2-carboxylic acid and L-valine appeared to inhibit Na -dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, the authors conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system.

  14. 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

  15. 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.

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

    PubMed Central

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

    2015-01-01

    Objective Obese women experience worse reproductive outcomes compared to normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay. The objective of this study was to use a genetic mouse model of obesity in order to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. Methods New inbred, polygenic Type 2 diabetic TallyHO mice and age matched control C57BL/6 mice were superovulated to obtain morulae or blastocysts stage embryos which were cultured in human tubal fluid media. Deoxyglucose uptake was performed on insulin-stimulated individual blastocysts. Apoptosis was detected by confocal microscopy using TUNEL assay and Topro-3 nuclear dye. Embryos were scored for %TUNEL positive/total nuclei. AMPK activation, TNF? expression, and adiponectin expression were analyzed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by Bodipy. Finally all measured parameters were compared between TallyHO mice in morulaes cultured to blastocyst embryos in either human tubal fluid (HTF) media or HTF with 25ug/ml metformin added. Results TallyHo mice developed whole body abnormal insulin tolerance, decreased litter number and increased NEFA. Blastocysts demonstrated increased apoptosis, decreased insulin sensitivity, and decreased activation of AMP activated protein-kinase (AMPK). As a possible cause of the insulin resistance/abnormal P-AMPK, we found that Tumor necrosis Factor (TNF?) expression and lipid accumulation as detected by BODIPY were increased in TallyHO blastocysts and adiponectin was decreased. Culturing TallyHO morulae with the AMPK activator, metformin lead to a reversal of all abnormal findings, including increased p-AMPK, improved insulin-stimulated glucose uptake and normalization of lipid accumulation. Conclusions Women with obesity and insulin resistance experience poor pregnancy outcomes. Previously we have shown in mouse models of insulin resistance that AMPK activity is decreased and that activators of AMPK reverse the poor embryo outcomes. Here, we show for the first time using a genetically altered obese model, not a diet-induced model, that metformin reverses many of the adverse effects of obesity at the level of the blastocyst. Expanding on this we determine that activation of AMPK via metformin reduces lipid droplet accumulation, presumably by eliminating the inhibitory effects of TNF?, resulting in normalization of fatty acid oxidation and HADH2 activity. Metformin exposure in vitro was able to partially reversing these effects, at the level of the blastocyst and thus may be effective in preventing the adverse effects of obesity on pregnancy and reproductive outcomes. PMID:25472042

  17. Single dose pharmacokinetics, pharmacodynamics, tolerability and safety of BAY 605521, a potent inhibitor of cholesteryl ester transfer protein

    PubMed Central

    Boettcher, Michael-Friedrich; Heinig, Roland; Schmeck, Carsten; Kohlsdorfer, Christian; Ludwig, Matthias; Schaefer, Anja; Gelfert-Peukert, Sabine; Wensing, Georg; Weber, Olaf

    2012-01-01

    AIMS To determine pharmacokinetics (PK), pharmacodynamics (PD), tolerability and safety of BAY 605521, a potent inhibitor of cholesteryl ester transfer protein (CETP). METHODS The first in man (FIM) study investigated the safety, tolerability, pharmacodynamics and pharmacokinetics in healthy male subjects following administration of single oral doses. The study was performed using a randomized, single-blind, placebo-controlled, single dose-escalation design. Thirty-eight young healthy male subjects (aged 2045 years) received an oral dose of 5, 12.5, 25 or 50 mg BAY 605521 (n = 28) or were treated with a placebo (n = 10). RESULTS In all four dose steps, only one adverse event (25 mg; mild skin rash) was considered drug related. Clinical laboratory parameters showed no clinically relevant changes. A clear dose-dependent CETP inhibition could be demonstrated starting at a dose of 5 mg. At a dose of 25 mg, a CETP inhibition >50% over 18 h was observed. After 50 mg, CETP inhibition >50% lasted more than 50 h. Twenty-four h after administration mean HDL-C-values showed a nearly dose-proportional increase. Following administration of 50 mg, a significant HDL-C increase of about 30% relative to baseline values was found. BAY 605521 was slowly absorbed reaching maximum concentrations in plasma after 4 to 6 h. The disposition in plasma was multi-exponential with an estimated mean terminal half-life of 76 to 144 h. CONCLUSIONS BAY 605521 was clinically safe and well tolerated. No effects on heart rate, blood pressure and ECG recordings were observed during the study. A clear pharmacodynamic effect on CETP inhibition and HDL could be demonstrated. PMID:21838789

  18. 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

  19. 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 embryos. PMID:25180972

  20. Effects of vitrification on the expression of pluripotency, apoptotic and stress genes in in vitro-produced porcine blastocysts.

    PubMed

    Castillo-Martn, Miriam; Yeste, Marc; Pericuesta, Eva; Morat, Roser; Gutirrez-Adn, Alfonso; Bonet, Sergi

    2014-04-01

    The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX:BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.90.4% vs 11.92.0%) and peroxide levels (80.42.6 vs 97.23.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r=-0.561; HSPA1A, r=0.604) and peroxide levels (POU5F1, r=-0.590; HSPA1A, r=0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression. PMID:25322142

  1. Protocols for nuclear transfer in mice.

    PubMed

    Gao, Shaorong

    2006-01-01

    Cloning by nuclear transfer in mammals has revealed the remarkable ability of an oocyte to reprogram transferred cell nuclei and induce them to recapitulate the developmental program. This chapter summarizes the method used since 1998 for mouse cloning, which differs from that for large animal cloning. A Piezo-drill micromanipulator allows direct injection of nuclei into enucleated mice oocytes instead of the electrofusion method used for introducing a nucleus into an enucleated oocyte of a large animal. After activation, reconstructed embryos are allowed to develop to the morulae or blastocyst stage before transfer into surrogate mothers. PMID:16761716

  2. Chirality transfer from a single chiral molecule to 2D superstructures in alaninol on the Cu(100) surface.

    PubMed

    Contini, G; Gori, P; Ronci, F; Zema, N; Colonna, S; Aschi, M; Palma, A; Turchini, S; Catone, D; Cricenti, A; Prosperi, T

    2011-06-21

    The formation of 2D chiral monolayers obtained by self-assembly of chiral molecules on surfaces has been widely reported in the literature. Control of chirality transfer from a single molecule to surface superstructures is a challenging and important aspect for tailoring the properties of 2D nanostructures. However, despite the wealth of investigations performed in recent years, how chiral transfer takes place on a large scale still remains an open question. In this paper we report a coupling of scanning tunneling microscopy and low energy electron diffraction measurements with an original theoretical approach, combining molecular dynamics and essential dynamics with density functional theory, to investigate self-assembled chiral structures formed when alaninol adsorbs on Cu(100). The peculiarity of this system is related to the formation of tetrameric molecular structures which constitute the building blocks of the self-assembled chiral monolayer. Such characteristics make alaninol/Cu(100) a good candidate to reveal chiral expression changes. We find that the deposition of alaninol enantiomers results in the formation of isolated tetramers that are aligned along the directions of the substrate at low coverage or when geometrical confinement prevents long-range order. Conversely, a rotation of 14 with respect to the Cu(100) unit vectors is observed when small clusters of tetramers are formed. An insight to the process leading to a 2D globally chiral surface has been obtained by monitoring molecular assemblies as they grow from the early stages of adsorption, suggesting that the distinctive orientation of the self-assembled monolayer originates from a balance of cooperating forces which start acting only when tetramers pack together to form small clusters. PMID:21604683

  3. Imaging in vivo extracellular pH with a single paramagnetic chemical exchange saturation transfer magnetic resonance imaging contrast agent.

    PubMed

    Liu, Guanshu; Li, Yuguo; Sheth, Vipul R; Pagel, Mark D

    2012-02-01

    The measurement of extracellular pH (pHe) has potential utility for cancer diagnoses and for assessing the therapeutic effects of pH-dependent therapies. A single magnetic resonance imaging (MRI) contrast agent that is detected through paramagnetic chemical exchange saturation transfer (PARACEST) was designed to measure tumor pH(e) throughout the range of physiologic pH and with magnetic resonance saturation powers that are not harmful to a mouse model of cancer. The chemical characterization and modeling of the contrast agent Yb(3+)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-o-aminoanilide (Yb-DO3A-oAA) suggested that the aryl amine of the agent forms an intramolecular hydrogen bond with a proximal carboxylate ligand, which was essential for generating a practical chemical exchange saturation transfer (CEST) effect from an amine. A ratio of CEST effects from the aryl amine and amide was linearly correlated with pH throughout the physiologic pH range. The pH calibration was used to produce a parametric pH map of a subcutaneous flank tumor on a mouse model of MCF-7 mammary carcinoma. Although refinements in the in vivo CEST MRI methodology may improve the accuracy of pHe measurements, this study demonstrated that the PARACEST contrast agent can be used to generate parametric pH maps of in vivo tumors with saturation power levels that are not harmful to a mouse model of cancer. PMID:22418027

  4. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer

    PubMed Central

    Bcker, Wolfgang; Yin, Zhanhai; Drosse, Inga; Haasters, Florian; Rossmann, Oliver; Wierer, Matthias; Popov, Cvetan; Locher, Melanie; Mutschler, Wolf; Docheva, Denitsa; Schieker, Matthias

    2008-01-01

    Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chon-drogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications. PMID:18318690

  5. 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

  6. 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.

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

    NASA Astrophysics Data System (ADS)

    Filatov, Michael

    2014-09-01

    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 S0 state and a wave-like dependence of the S1 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 S0 and S1 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 S0 and S1 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 S0 and S1 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+-A- 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.

  8. Description of electron transfer in the ground and excited states of organic donoracceptor 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 donoracceptor (DA) 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 donoracceptor 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 donoracceptor 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 donoracceptor 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.

  9. Collapse of a Long Axis: Single-Molecule Förster Resonance Energy Transfer and Serpin Equilibrium Unfolding

    PubMed Central

    2015-01-01

    The energy required for mechanical inhibition of target proteases is stored in the native structure of inhibitory serpins and accessed by serpin structural remodeling. The overall serpin fold is ellipsoidal with one long and two short axes. Most of the structural remodeling required for function occurs along the long axis, while expansion of the short axes is associated with misfolded, inactive forms. This suggests that ellipticity, as typified by the long axis, may be important for both function and folding. Placement of donor and acceptor fluorophores approximately along the long axis or one of the short axes allows single-pair Förster resonance energy transfer (spFRET) to report on both unfolding transitions and the time-averaged shape of different conformations. Equilibrium unfolding and refolding studies of the well-characterized inhibitory serpin α1-antitrypsin reveal that the long axis collapses in the folding intermediates while the monitored short axis expands. These energetically distinct intermediates are thus more spherical than the native state. Our spFRET studies agree with other equilibrium unfolding studies that found that the region around one of the β strands, s5A, which helps define the long axis and must move for functionally required loop insertion, unfolds at low denaturant concentrations. This supports a connection between functionally important structural lability and unfolding in the inhibitory serpins. PMID:24749911

  10. 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 watergas 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 ?s, which is of the right magnitude for substrate trapping of sensitized catalyst intermediates. PMID:24961370

  11. 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.

  12. 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 ?s, which is of the right magnitude for substrate trapping of sensitized catalyst intermediates. PMID:24961370

  13. Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors.

    PubMed

    Miyake, Noriko; Miyake, Koichi; Yamamoto, Motoko; Hirai, Yukihiko; Shimada, Takashi

    2011-05-10

    Central nervous system (CNS) disorders are important targets for gene therapy; however, delivery of therapeutic proteins and/or genes to the brain remains a major challenge due to the difficulty of efficiently delivering viral vectors across the blood-brain barrier (BBB). In the present work, we tested the ability of several single-stranded adeno-associated viral (ssAAV) serotypes to deliver transgenes to the brain and spinal cord in neonatal mice. We injected ssAAV vectors encoding GFP (serotype-1, -8, -9 and -10: 1.510(11) vector genomes each) into the jugular vein of neonatal mice and assessed GFP expression immunohistochemically. Strong GFP signals were detected in both the brain and spinal cord after injection of any of these serotypes. ssAAV serotype-9 mediated gene transfer was the most efficient. GFP expression was detected throughout the brain, including the cortex, cerebellum, olfactory bulb and brainstem and was sustained for at least 18months. Immunohistochemical staining showed that the GFP signals were detected in GFAP positive astrocytes, NeuN positive neurons, and Calbindin positive purkinje cells. Our data suggest that systemic neonatal injection of ssAAV is an effective strategy for delivering transgenes to target neuronal systems that are not accessible to viral vectors in adult animals. These vectors should prove highly useful for efficient and long-term overexpression or downregulation of genes in CNS and spinal cord and could be a useful means of treating genetic neurological diseases. PMID:21397590

  14. 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

  15. 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.

  16. 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, ?ukasz; G?bka, 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.

  17. IVF with planned single-embryo transfer versus IUI with ovarian stimulation in couples with unexplained subfertility: an economic analysis.

    PubMed

    van Rumste, Minouche M E; Custers, Inge M; van Wely, Madelon; Koks, Carolien A; van Weering, Hans G I; Beckers, Nicole G M; Scheffer, Gabrielle J; Broekmans, Frank J M; Hompes, Peter G A; Mochtar, Monique H; van der Veen, Fulco; Mol, Ben W J

    2014-03-01

    Couples with unexplained subfertility are often treated with intrauterine insemination (IUI) with ovarian stimulation, which carries the risk of multiple pregnancies. An explorative randomized controlled trial was performed comparing one cycle of IVF with elective single-embryo transfer (eSET) versus three cycles of IUI-ovarian stimulation in couples with unexplained subfertility and a poor prognosis for natural conception, to assess the economic burden of the treatment modalities. The main outcome measures were ongoing pregnancy rates and costs. This study randomly assigned 58 couples to IVF-eSET and 58 couples to IUI-ovarian stimulation. The ongoing pregnancy rates were 24% in with IVF-eSET versus 21% with IUI-ovarian stimulation, with two and three multiple pregnancies, respectively. The mean cost per included couple was significantly different: €2781 with IVF-eSET and €1876 with IUI-ovarian stimulation (P<0.01). The additional costs per ongoing pregnancy were €2456 for IVF-eSET. In couples with unexplained subfertility, one cycle of IVF-eSET cost an additional €900 per couple compared with three cycles of IUI-ovarian stimulation, for no increase in ongoing pregnancy rates or decrease in multiple pregnancies. When IVF-eSET results in higher ongoing pregnancy rates, IVF would be the preferred treatment. Couples that have been trying to conceive unsuccessfully are often treated with intrauterine insemination (IUI) and medication to improve egg production (ovarian stimulation). This treatment carries the risk of multiple pregnancies like twins. We performed an explorative study among those couples that had a poor prognosis for natural conception. One cycle of IVF with transfer of one selected embryo (elective single-embryo transfer, eSET) was compared with three cycles of IUI-ovarian stimulation. The aim of this study was to assess the economic burden of both treatments. The Main outcome measures were number of good pregnancies above 12weeks and costs. We randomly assigned 58 couples to IVF-eSET and 58 couples to IUI-ovarian stimulation. The ongoing pregnancy rates were comparable: 24% with IVF-eSET versus 21% with IUI-ovarian stimulation. There were two multiple pregnancies with IVF-eSET and three multiple pregnancies with IUI-ovarian stimulation. The mean cost per included couple was significantly different, €2781 with IVF-eSET and €1876 with IUI-ovarian stimulation. The additional costs per ongoing pregnancy were €2456 for IVF-eSET. In couples with unexplained subfertility, one cycle of IVF-eSET costed an additional €900 per couple compared to three cycles of IUI-ovarian stimulation, for no increase in ongoing pregnancy rates or decrease in multiple pregnancies. We conclude that IUI-ovarian stimulation is the preferred treatment to start with. When IVF-eSET results in a higher ongoing pregnancy rate (>38%), IVF would be the preferred treatment. PMID:24456703

  18. Experimental and theoretical studies of gas-liquid mass transfer of a single bubble in a liquid-solid fluidized bed

    SciTech Connect

    Song, G.H.; Jean, R.H.; Fan, L.S. . Dept. of Chemical Engineering)

    1988-01-01

    Experiments are conducted using ozone as a tracer to study the interfacial gas-liquid mass transfer behavior of a single bubble in liquid and liquid-solid fluidized media. A spectrophotometric system is developed, which consists of an ultraviolet (UV) light source, optical fibers, narrow band interference filters, UV-visible photomultipliers, and computerized fast data acquisition. Bubble size, particle properties, and bed voidage are varied to examine the axial variation of the mass transfer rate. A theoretical model is developed for the prediction of the overall gas-liquid mass transfer coefficient of a single circular-cap in a two dimensional or spherical-cap bubble in a three dimensional liquid-solid fluidized bed. The penetration concept is used to account for the mass transfer rate across the bubble roof. Two cases are considered in the evaluation of the mass transfer rate across the bubble base. The prediction of the overall mass transfer coefficient based on the model is shown to agree reasonably well with the experimental results obtained in this study.

  19. DNA hairpins containing a diaminostilbene derivative as a photoinduced electron donor for probing the effects of single-base mismatches on excess electron transfer in DNA.

    PubMed

    Ito, Takeo; Hayashi, Aiko; Kondo, Akiko; Uchida, Tsukasa; Tanabe, Kazuhito; Yamada, Hisatsugu; Nishimoto, Sei-ichi

    2009-02-19

    To investigate the effects of local structural disorder induced by a single-base mismatch on excess electron transfer (EET) in DNA, a novel hairpin DNA containing diaminostilbene (DAS) as a photoinducible electron donor has been developed. It was clearly demonstrated that EET efficiency depends on the electron injection modes from the electron donors and redox properties of the mismatched bases. PMID:19170616

  20. 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.

  1. Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle.

    PubMed

    Mirshamsi, S M; Karamishabankareh, H; Ahmadi-Hamedani, M; Soltani, L; Hajarian, H; Abdolmohammadi, A R

    2013-01-30

    The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26?M of BCB for 90min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB-: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB-: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB-). After fertilization, the zygotes produced from OBCB+ and OBCB- oocytes were further stained with BCB for 10min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB-, OBCB-/ZBCB++, OBCB-/ZBCB+ and OBCB-/ZBCB-). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P<0.05) than ZBCB treatment (ZBCB-; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB-: 28.8%) was less (P<0.01) than single application of BCB test (ZBCB-: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB- zygotes (36.6%) were greater (P<0.05) than that from C oocytes (25.7%), OBCB- oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P>0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB- zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB-: 48.0%) was greater (P<0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB-: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test. PMID:23228698

  2. Live births following Karyomapping of human blastocysts: experience from clinical application of the method.

    PubMed

    Konstantinidis, Michalis; Prates, Renata; Goodall, N-Neka; Fischer, Jill; Tecson, Victoria; Lemma, Tsion; Chu, Bo; Jordan, Amy; Armenti, Erin; Wells, Dagan; Munné, Santiago

    2015-09-01

    The clinical application of a new, widely applicable method known as Karyomapping to carry out a total of 55 clinical cases of preimplantation genetic diagnosis (PGD) for single gene disorders is reported. Conventional polymerase chain reaction (PCR) testing was carried out in parallel to the new method for all cases. Clinical application of Karyomapping in this study resulted in three live births and nine clinical pregnancies out of 20 cases with a transfer. All in all, results presented in this study indicate that Karyomapping is a highly efficient, accurate and robust method for PGD of single gene disorders. Karyomapping can offer a more comprehensive assessment of the region of interest than conventional PCR analysis, allowing for more embryos to receive diagnosis (99.6% versus 96.8%), whereas its wide applicability reduces substantially the time that patients have to wait before starting their in vitro fertilization (IVF) cycle. Nonetheless, inclusion of elements of conventional PCR methodology, such as direct mutation detection, may be required in cases in which the gene of interest is in a region with reduced single nucleotide polymorphism (SNP) coverage (e.g. telomeric regions), when offering PGD for consanguineous couples, or in cases where no samples from additional family members are available. PMID:26206283

  3. Cu-O network dependence of optical charge-transfer gaps and spin-pair excitations in single-CuO sub 2 -layer compounds

    SciTech Connect

    Tokura, Y.; Koshihara, S.; Arima, T. ); Takagi, H.; Ishibashi, S.; Ido, T.; Uchida, S. )

    1990-06-01

    Spectra of optical conductivity and magnon Raman scattering have been investigated in single crystals of a parent family of cuprate superconductors with various types of Cu-O single-layer networks. The analysis of the spectra shows the systematic dependence of the charge-transfer gaps and covalent character of Cu-O bonds on the pattern of the Cu-O network, while the spin-exchange energy is rather convergent for all the single-CuO{sub 2}-sheet compounds.

  4. A High-yield Two-step Transfer Printing Method for Large-scale Fabrication of Organic Single-crystal Devices on Arbitrary Substrates

    NASA Astrophysics Data System (ADS)

    Deng, Wei; Zhang, Xiujuan; Pan, Huanhuan; Shang, Qixun; Wang, Jincheng; Zhang, Xiaohong; Zhang, Xiwei; Jie, Jiansheng

    2014-06-01

    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.

  5. 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

  6. 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

  7. Facile synthetic access to rhenium(II) complexes: activation of carbon-bromine bonds by single-electron transfer.

    PubMed

    Jiang, Yanfeng; Blacque, Olivier; Fox, Thomas; Frech, Christian M; Berke, Heinz

    2010-02-15

    The five-coordinated Re(I) hydride complexes [Re(Br)(H)(NO)(PR(3))(2)] (R=Cy 1 a, iPr 1 b) were reacted with benzylbromide, thereby affording the 17-electron mononuclear Re(II) hydride complexes [Re(Br)(2)(H)(NO)(PR(3))(2)] (R=Cy 3 a, iPr 3 b), which were characterized by EPR, cyclic voltammetry, and magnetic susceptibility measurements. In the case of dibromomethane or bromoform, the reaction of 1 afforded Re(II) hydrides 3 in addition to Re(I) carbene hydrides [Re(=CHR(1))(Br)(H)(NO)(PR(3))(2)] (R(1)=H 4, Br 5; R=Cy a, iPr b) in which the hydride ligand is positioned cis to the carbene ligand. For comparison, the dihydrogen Re(I) dibromide complexes [Re(Br)(2)(NO)(PR(3))(2)(eta(2)-H(2))] (R=Cy 2 a, iPr 2 b) were reacted with allyl- or benzylbromide, thereby affording the monophosphine Re(II) complex salts [R(3)PCH(2)R'][Re(Br)(4)(NO)(PR(3))] (R'=-CH=CH(2) 6, Ph 7). The reduction of Re(II) complexes has also been examined. Complex 3 a or 3 b can be reduced by zinc to afford 1 a or 1 b in high yield. Under catalytic conditions, this reaction enables homocoupling of benzylbromide (turnover frequency (TOF): 3 a 150, 3 b 134 h(-1)) or allylbromide (TOF: 3 a 575, 3 b 562 h(-1)). The reaction of 6 a and 6 b with zinc in acetonitrile affords in good yields the monophosphine Re(I) complexes [Re(Br)(2)(NO)(MeCN)(2)(PR(3))] (R=Cy 8 a, iPr 8 b), which showed high catalytic activity toward highly selective dehydrogenative silylation of styrenes (maximum TOF of 61 h(-1)). Single-electron transfer (SET) mechanisms were proposed for all these transformations. The molecular structures of 3 a, 6 a, 6 b, 7 a, 7 b, and 8 a were established by single-crystal X-ray diffraction studies. PMID:20066700

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

    PubMed Central

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

    2014-01-01

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

  9. Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction.

    PubMed

    Bi, Sai; Zhang, Zhipeng; Dong, Ying; Wang, Zonghua

    2014-10-17

    A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol-H2O2-horseradish peroxidase (HRP)-mimicking DNAzyme-fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X(⁎), YG and Y(⁎)) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3'-end of YG and fluorescein at 5'-end of Y(⁎). After the LCR, the resulting products of XYG/X(⁎)Y(⁎) with biotin-labeled X(⁎) were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol-H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis. PMID:25461149

  10. Influence of heat and mass transfer on the ignition and NO x formation in single droplet combustion

    NASA Astrophysics Data System (ADS)

    Moesl, Klaus G.; Schwing, Joachim E.; Fenninger, Wolfgang J.; Sattelmayer, Thomas

    2011-08-01

    The effect of heat and mass transfer on the ignition, and in a second step on the nitrogen oxide (NO x ) generation, of single burning droplets is examined in a numerical study. Spherical symmetry with no gravity and no forced convection is presumed; ambient temperature is set at 500 K, below the auto-ignition point. The essentials of a forced droplet ignition by an external energy source are introduced. Two methods are applied: heat introduction at a fixed radial position r and heat introduction at a fixed local equivalence ratio ? r . This study's distinctiveness compared to previous research is its focus on and its combination of partially pre-vaporized droplets and detailed chemistry, both being technically relevant in kerosene and diesel fuel combustion. The fuel of choice is n-decane (C10H22), and NO x production is studied exemplarily as a representative group of pollutant emissions. The conducted simulations show a decrease of NO x formation with an increase of the pre-vaporization rate Uppsi. This decrease is generally valid for both methods of heat introduction. However, results on flame stabilization and NO x production reveal a high sensitivity to parameters of the ignition model. The burning behavior during the initial stages is dominated by the ignition position. Extracting heat from the exhaust gas region of burning droplets shows no impact on the flame position nor on the relative NO x production. As a consequence, a well-founded modeling of the investigated droplet regime needs to resort to an iterative adaptation of the heat introduction parameters based on the findings of droplet burning and exhaust gas production.

  11. Polymerization Induced Self-Assembly of Alginate Based Amphiphilic Graft Copolymers Synthesized by Single Electron Transfer Living Radical Polymerization.

    PubMed

    Kapishon, Vitaliy; Whitney, Ralph A; Champagne, Pascale; Cunningham, Michael F; Neufeld, Ronald J

    2015-07-13

    Alginate-based amphiphilic graft copolymers were synthesized by single electron transfer living radical polymerization (SET-LRP), forming stable micelles during polymerization induced self-assembly (PISA). First, alginate macroinitiator was prepared by partial depolymerization of native alginate, solubility modification and attachment of initiator. Depolymerized low molecular weight alginate (∼12 000 g/mol) was modified with tetrabutylammonium, enabling miscibility in anhydrous organic solvents, followed by initiator attachment via esterification yielding a macroinitiator with a degree of substitution of 0.02, or 1-2 initiator groups per alginate chain. Then, methyl methacrylate was polymerized from the alginate macroinitiator in mixtures of water and methanol, forming poly(methyl methacrylate) grafts, prior to self-assembly, of ∼75 000 g/mol and polydispersity of 1.2. PISA of the amphiphilic graft-copolymer resulted in the formation of micelles with diameters of 50-300 nm characterized by light scattering and electron microscopy. As the first reported case of LRP from alginate, this work introduces a synthetic route to a preparation of alginate-based hybrid polymers with a precise macromolecular architecture and desired functionalities. The intended application is the preparation of micelles for drug delivery; however, LRP from alginate can also be applied in the field of biomaterials to the improvement of alginate-based hydrogel systems such as nano- and microhydrogel particles, islet encapsulation materials, hydrogel implants, and topical applications. Such modified alginates can also improve the function and application of native alginates in food and agricultural applications. PMID:26068280

  12. 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

  13. Studies of single- and two-phase heat transfer in a blocked four-rod bundle. Final report. [PWR

    SciTech Connect

    Drucker, M.; Dhir, V.K.

    1984-06-01

    Correlations have been developed for the heat transfer coefficient in the vicinity of flow blockages in rod bundles. The heat transfer data have been obtained on a four rod bundle with sleeve-type blockages for a Reynolds number range of 230 to 6900 and void fractions up to 0.15. Significant enhancement of the heat transfer coefficient has been observed downstream of the blockage.

  14. 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 Jimnez; Janssens, Stoffel D; Haenen, Ken; Schning, 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. PMID:22356595

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

    PubMed

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

    2015-03-01

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

  16. Human endometrial epithelial cells express ephrin A1: possible interaction between human blastocysts and endometrium via Eph-ephrin system.

    PubMed

    Fujiwara, Hiroshi; Yoshioka, Shinya; Tatsumi, Keiji; Kosaka, Kenzo; Satoh, Yukiyasu; Nishioka, Yoshihiro; Egawa, Miho; Higuchi, Toshihiro; Fujii, Shingo

    2002-12-01

    Eph receptor tyrosine kinases and their cell membrane-bound ligands, ephrins, are well known to function in cell-to-cell interaction and to play an important role in cell migration and adhesion during embryonic development in mammals. To investigate the involvement of the Eph-ephrin system in human embryo implantation, the expression of Eph receptors and ephrins was examined in human blastocysts and the endometrium. Immunohistochemical examination showed that ephrin A1 was expressed on human endometrial luminal and glandular epithelial cells in both the proliferative (cycle d 8-13; n = 8) and secretory (cycle d 18-24; n = 7) phases. RT-PCR analysis of isolated endometrial epithelial cells and stromal cells showed that ephrin A1 mRNA was predominantly expressed in endometrial epithelial cells. Northern blot analysis also confirmed the expression of ephrin A1 mRNA in the endometrium. In addition, nested RT-PCR analysis revealed the mRNA expression of Eph A1, one of the representative receptors for ephrin A1, in human blastocysts obtained from patients undergoing in vitro fertilization treatment. These findings indicate a possible interaction between human blastocysts and endometrial epithelial cells via the Eph-ephrin system. Because intracytoplasmic signals are induced by Eph receptors after ephrin stimulation, this system may be involved in the activation process of the human embryo during the implantation period. PMID:12466389

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

    PubMed Central

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

    2014-01-01

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

  18. Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

    SciTech Connect

    Sheth, Bhavwanti; Nowak, Rachael L.; Anderson, Rebecca; Kwong, Wing Yee; Papenbrock, Thomas; Fleming, Tom P.

    2008-11-01

    Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.

  19. Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition

    PubMed Central

    Jedrusik, Agnieszka; Cox, Andy; Wicher, Krzysztof; Glover, David M.; Zernicka-Goetz, Magdalena

    2015-01-01

    The first lineage segregation in the mouse embryo generates the inner cell mass (ICM), which gives rise to the pluripotent epiblast and therefore the future embryo, and the trophectoderm (TE), which will build the placenta. The TE lineage depends on the transcription factor Cdx2. However, when Cdx2 first starts to act remains unclear. Embryos with zygotic deletion of Cdx2 develop normally until the late blastocyst stage leading to the conclusion that Cdx2 is important for the maintenance but not specification of the TE. In contrast, down-regulation of Cdx2 transcripts from the early embryo stage results in defects in TE specification before the blastocyst stage. Here, to unambiguously address at which developmental stage Cdx2 becomes first required, we genetically deleted Cdx2 from the oocyte stage using a Zp3-Cre/loxP strategy. Careful assessment of a large cohort of Cdx2 maternal-zygotic null embryos, all individually filmed, examined and genotyped, reveals an earlier lethal phenotype than observed in Cdx2 zygotic null embryos that develop until the late blastocyst stage. The developmental failure of Cdx2 maternal-zygotic null embryos is associated with cell death and failure of TE specification, starting at the morula stage. These results indicate that Cdx2 is important for the correct specification of TE from the morula stage onwards and that both maternal and zygotic pools of Cdx2 are required for correct pre-implantation embryogenesis. PMID:25512302

  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; Andr, 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. Ultrafast optical switching at the telecommunication wavelength by single electron transfer between fluorene-containing redox polymer and its counter anion

    NASA Astrophysics Data System (ADS)

    Nagamura, Toshihiko; Nagai, Yasuyuki; Furube, Akihiro; Murata, Shigeo

    2004-10-01

    Ultrafast and large absorption change in the optical telecommunication wavelength region was achieved in solid films by making transitory expansion of a ?-electronic system with photoinduced electron transfer and reverse reactions between redox-active ion pairs. Femtosecond dynamics of absorption changes at about 500-1550 nm due to single electron transfer was studied for a poly(tetramethyleneoxide) film containing 2,7-bis(4-pyridiniumyl)fluorene unit as part of the main chain. It showed ON response in less than 100 fs and fastest OFF response in 240 fs at the telecommunication wavelength. This polymer film may be applied to tetrahertz all optical information processing in the telecommunication system.

  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 structural properties, they are typically more amenable to mechanistic analysis, and they are small and therefore require less material. Therefore, they have arguably greater potential as future efficient catalysts but must be efficiently coupled to accumulative charge separation. This Account discusses accumulative charge separation with focus on molecular and molecule-semiconductor hybrid systems. The coupling between charge separation and catalysis involves many challenges that are often overlooked, and they are not always apparent when studying water oxidation and fuel formation as separate half-reactions with sacrificial agents. Transition metal catalysts, as well as other multielectron donors and acceptors, cycle through many different states that may quench the excited sensitizer by nonproductive pathways. Examples where this has been shown, often with ultrafast rates, are reviewed. Strategies to avoid these competing energy-loss reactions and still obtain efficient coupling of charge separation to catalysis are discussed. This includes recent examples of dye-sensitized semiconductor devices with molecular catalysts and dyes that realize complete water splitting, albeit with limited efficiency. PMID:25675365

  3. Local heat transfer behavior and its impact on a single-row, annularly finned tube heat exchanger

    SciTech Connect

    Hu, X.; Jacobi, A.M. )

    1993-02-01

    Experimental studies of the local mass transfer characteristics of annularly finned tubes in crossflow are presented. Variations due to boundary layer development, forward-edge separation, the tube wake, horseshoe vortices, and tip vortices are discussed. In addition, regularly located local maxima in mass transfer rates associated with the horseshoe vortex system are found, and conjecture as to their mechanism is offered. Inferring heat transfer behavior from the mass transfer results, we find that the true fin efficiency is always less than that obtained with an assumed constant convective heat transfer coefficient. The difference is 3-7 percent for high-conductivity materials such as aluminum alloys, and 9-17 percent for low-conductivity materials such as mild steels. 26 refs., 7 refs., 2 tabs.

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

    PubMed

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

    2014-12-01

    Obese women experience worse reproductive outcomes than normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay of offspring. The aim of the present study was to use a genetic mouse model of obesity to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. New inbred, polygenic Type 2 diabetic TallyHO mice and age-matched control C57BL/6 mice were superovulated to obtain morula or blastocyst stage embryos that were cultured in human tubal fluid (HTF) medium. Deoxyglucose uptake was determined for individual insulin-stimulated blastocysts. Apoptosis was detected by confocal microscopy using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and Topro-3 nuclear dye. Embryos were scored for TUNEL-positive as a percentage of total nuclei. AMP-activated protein kinase (AMPK) activation, tumour necrosis factor (TNF)-α expression and adiponectin expression were analysed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by BODIPY. Comparisons were made between TallyHO morulae cultured to blastocyst embryos in either HTF medium or HTF medium with 25 μg mL(-1) metformin. TallyHO mice developed whole body abnormal insulin tolerance, had decreased litter sizes and increased non-esterified fatty acid levels. Blastocysts from TallyHO mice exhibited increased apoptosis, decreased insulin sensitivity and decreased AMPK. A possible cause for the insulin resistance and abnormal AMPK phosphorylation was the increased TNF-α expression and lipid accumulation, as detected by BODIPY, in TallyHO blastocysts and decreased adiponectin. Culturing TallyHO morulae with the AMPK activator metformin led to a reversal of all the abnormal findings, including increased AMPK phosphorylation, improved insulin-stimulated glucose uptake and normalisation of lipid accumulation. Women with obesity and insulin resistance experience poor pregnancy outcomes. Previously we have shown in mouse models of insulin resistance that AMPK activity is decreased and that activators of AMPK reverse poor embryo outcomes. Here, we show for the first time using a genetically altered obese model, not a diet-induced model, that metformin reverses many of the adverse effects of obesity at the level of the blastocyst. Expanding on this we determine that activation of AMPK via metformin reduces lipid droplet accumulation, presumably by eliminating the inhibitory effects of TNF-α, resulting in normalisation of fatty acid oxidation and HADH2 (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit) activity. Metformin exposure in vitro was able to partially reverse these effects, at the level of the blastocyst, and may thus be effective in preventing the adverse effects of obesity on pregnancy and reproductive outcomes. PMID:25472042

  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 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

  6. Determination of blade-to-coolant heat-transfer coefficients on a forced-convection, water-cooled, single-stage turbine

    NASA Technical Reports Server (NTRS)

    Freche, John C; Schum, Eugene F

    1951-01-01

    Blade-to-coolant convective heat-transfer coefficients were obtained on a forced-convection water-cooled single-stage turbine over a large laminar flow range and over a portion of the transition range between laminar and turbulent flow. The convective coefficients were correlated by the general relation for forced-convection heat transfer with laminar flow. Natural-convection heat transfer was negligible for this turbine over the Grashof number range investigated. Comparison of turbine data with stationary tube data for the laminar flow of heated liquids showed good agreement. Calculated average midspan blade temperatures using theoretical gas-to-blade coefficients and blade-to-coolant coefficients from stationary-tube data resulted in close agreement with experimental data.

  7. Transfer characteristics and low-frequency noise in single- and multi-layer MoS2 field-effect transistors

    NASA Astrophysics Data System (ADS)

    Sharma, Deepak; Motayed, Abhishek; Shah, Pankaj B.; Amani, Matin; Georgieva, Mariela; Glen Birdwell, A.; Dubey, Madan; Li, Qiliang; Davydov, Albert V.

    2015-10-01

    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 (MoS2) with different layer (L) counts. 4L to 6L devices showed highest ION-IOFF ratio (?108) whereas LFN was maximum for 1L device with normalized power spectral density (PSD) ?1.5 10-5 Hz-1. For devices with L ? 6, PSD was minimum (?2 10-8 Hz-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.

  8. Stable continuous-wave single-frequency Nd:YAG blue laser at 473 nm considering the influence of the energy-transfer upconversion.

    PubMed

    Wang, Yaoting; Liu, Jianli; Liu, Qin; Li, Yuanji; Zhang, Kuanshou

    2010-06-01

    We report a continuous-wave (cw) single frequency Nd:YAG blue laser at 473 nm end-pumped by a laser diode. A ring laser resonator was designed, the frequency doubling efficiency and the length of nonlinear crystal were optimized based on the investigation of the influence of the frequency doubling efficiency on the thermal lensing effect induced by energy-transfer upconversion. By intracavity frequency doubling with PPKTP crystal, an output power of 1 W all-solid-state cw blue laser of single-frequency operation was achieved. The stability of the blue output power was better than +/- 1.8% in the given four hours. PMID:20588326

  9. Heat Transfer Enhancement and Pressure-Loss Reduction for Fin-Surfaces of In-line Tube Bundle with a Single Front Row of Winglet Pairs

    NASA Astrophysics Data System (ADS)

    Kwak, Kyung-Min; Torii, Kahoru; Nishino, Koichi

    This paper proposes a novel technique that can augment heat transfer but nevertheless can reduce pressure-loss in a fin-tube heat exchanger with circular tubes in a relatively low Reynolds number flow, by deploying a single row of the delta winglet type of vortex generators beside the front row of the three-row tube bundles in an in-line arrangement. The winglets are placed with a heretofore-unused orientation for the purpose of augmentation of heat transfer. This orientation is called as common flow up configuration. The proposed device causes a significant separation delay, reduces form drag, and removes the zone of poor heat transfer from the near-wake of the tube. This enhancement strategy has been successfully verified by experiments in the proposed configuration. In the in-line tube bundle with three rows, the heat transfer was augmented by 10% to 20%, and yet the pressure loss was reduced by 8% to 15% for the Reynolds number (based on two times channel height) ranging from 350 to 2100, when the present winglets were built in a single front row.

  10. Chemo- and Enantioselective Addition and ?-Hydrogen Transfer Reduction of Carbonyl Compounds with Diethylzinc Reagent in One Pot Catalyzed by a Single Chiral Organometallic Catalyst.

    PubMed

    Huang, Huayin; Zong, Hua; Bian, Guangling; Song, Ling

    2015-12-18

    Using a single chiral phosphoramide-Zn(II) complex as the catalyst, the asymmetric ?-H transfer reduction of aromatic ?-trifluoromethyl ketones and enantioselective addition of aromatic aldehydes with Et2Zn in one pot were successfully realized, affording the corresponding additive products of secondary alcohols in high yields (up to 99%) with excellent enantioselectivities (up to 98% ee) and the reduction products of ?-trifluoromethyl alcohols in good to excellent yields with up to 77% ee. PMID:26579727

  11. Optically enhanced charge transfer between C60 and single-wall carbon nanotubes in hybrid electronic devices.

    PubMed

    Allen, Christopher S; Liu, Guoquan; Chen, Yabin; Robertson, Alex W; He, Kuang; Porfyrakis, Kyriakos; Zhang, Jin; Briggs, G Andrew D; Warner, Jamie H

    2014-01-01

    In this article we probe the nature of electronic interactions between the components of hybrid C60-carbon nanotube structures. Utilizing an aromatic mediator we selectively attach C60 molecules to carbon nanotube field-effect transistor devices. Structural characterization via atomic force and transmission electron microscopy confirm the selectivity of this attachment. Charge transfer from the carbon nanotube to the C60 molecules is evidenced by a blue shift of the Raman G(+) peak position and increased threshold voltage of the transistor transfer characteristics. We estimate this charge transfer to increase the device density of holes per unit length by up to 0.85 nm(-1) and demonstrate further optically enhanced charge transfer which increases the hole density by an additional 0.16 nm(-1). PMID:24241690

  12. Single-molecule conductance of a chemically modified, π-extended tetrathiafulvalene and its charge-transfer complex with F4TCNQ.

    PubMed

    García, Raúl; Herranz, M Ángeles; Leary, Edmund; González, M Teresa; Bollinger, Gabino Rubio; Bürkle, Marius; Zotti, Linda A; Asai, Yoshihiro; Pauly, Fabian; Cuevas, Juan Carlos; Agraït, Nicolás; Martín, Nazario

    2015-01-01

    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

  13. 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

  14. Long term costs and effects of reducing the number of twin pregnancies in IVF by single embryo transfer: the TwinSing study

    PubMed Central

    2010-01-01

    Background Pregnancies induced by in vitro fertilisation (IVF) often result in twin gestations, which are associated with both maternal and perinatal complications. An effective way to reduce the number of IVF twin pregnancies is to decrease the number of embryos transferred from two to one. The interpretation of current studies is limited because they used live birth as outcome measure and because they applied limited time horizons. So far, research on long-term outcomes of IVF twins and singletons is scarce and inconclusive. The objective of this study is to investigate the short (1-year) and long-term (5 and 18-year) costs and health outcomes of IVF singleton and twin children and to consider these in estimating the cost-effectiveness of single embryo transfer compared with double embryo transfer, from a societal and a healthcare perspective. Methods/Design A multi-centre cohort study will be performed, in which IVF singletons and IVF twin children born between 2003 and 2005 of whom parents received IVF treatment in one of the five participating Dutch IVF centres, will be compared. Data collection will focus on children at risk of health problems and children in whom health problems actually occurred. First year of life data will be collected in approximately 1,278 children (619 singletons and 659 twin children). Data up to the fifth year of life will be collected in approximately 488 children (200 singletons and 288 twin children). Outcome measures are health status, health-related quality of life and costs. Data will be obtained from hospital information systems, a parent questionnaire and existing registries. Furthermore, a prognostic model will be developed that reflects the short and long-term costs and health outcomes of IVF singleton and twin children. This model will be linked to a Markov model of the short-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies to enable the calculation of the long-term cost-effectiveness. Discussion This is, to our knowledge, the first study that investigates the long-term costs and health outcomes of IVF singleton and twin children and the long-term cost-effectiveness of single embryo transfer strategies versus double embryo transfer strategies. PMID:20961411

  15. The Influence of Single Nucleotide Polymorphism Microarray-Based Molecular Karyotype on Preimplantation Embryonic Development Potential

    PubMed Central

    Jin, Haixia; Liu, Yan; Guo, Yihong; Su, Yingchun; Sun, Yingpu

    2015-01-01

    In order to investigate the influence of the molecular karyotype based on single nucleotide polymorphism (SNP) microarray on embryonic development potential in preimplantation genetic diagnosis (PGD), we retrospectively analyzed the clinical data generated by PGD using embryos retrieved from parents with chromosome rearrangements in our center. In total, 929 embryos from 119 couples had exact diagnosis and development status. The blastocyst formation rate of balanced molecular karyotype embryos was 56.6% (276/488), which was significantly higher than that of genetic imbalanced embryos 24.5% (108/441) (P<0.001). No significant difference was detected in blastocyst formation rates in the groups of maternal age<30, 3035 and >35 respectively. Blastocyst formation rates of male and female embryos were 44.5% (183/411) and 38.8% (201/518) respectively, with no significant difference between them (P>0.05). The rates of balanced molecular karyotype embryos vary from groups of embryos with different cell numbers at 68 hours after insemination. The blastocyst formation rate of embryos with 68 cells (48.1%) was significantly higher than that of embryos with <6 cells (23.9%) and with >8 cells (42.9%) (P<0.05). As for the unbalanced embryos, there was no significant difference of the distribution of abnormal molecular karyotypes in the subgroup of the arrest, morula and blastocyst. Thus, we conclude that embryos with balanced molecular karyotype have significant higher development potential than those with imbalanced molecular karyotype whilst maternal age, embryo gender and types of abnormal molecular karyotype have no significant influence on blastocyst formation. Compared with embryos with <6 and >8 cells, embryos with 68 blastomeres have higher rate of balanced molecular karyotype and blastocyst formation. PMID:26381524

  16. Mid-luteal serum progesterone concentrations govern implantation rates for cryopreserved embryo transfers conducted under hormone replacement.

    PubMed

    Yovich, John L; Conceicao, Jason L; Stanger, James D; Hinchliffe, Peter M; Keane, Kevin N

    2015-08-01

    This study explores the relevance of mid-luteal serum hormonal concentrations in cryopreserved embryo transfer cycles conducted under hormone replacement therapy (HRT) control and which involved single-embryo transfer (SET) of 529 vitrified blastocysts. Widely ranging mid-luteal oestradiol and progesterone concentrations ensued from the unique HRT regimen. Oestradiol had no influence on clinical pregnancy or live birth rates, but an optimal progesterone range between 70 and 99 nmol/l (P < 0.005) was identified in this study. Concentrations of progesterone below 50 nmol/l and above 99 nmol/l were associated with decreased implantation rates. There was no clear interaction between oestradiol and progesterone concentrations but embryo quality grading did show a significant influence on outcomes (P < 0.001 and P = 0.002 for clinical pregnancy and live birth rates, respectively). Multiple comparison analysis showed that the progesterone effect was influential regardless of embryo grading, body mass index or the woman's age, either at vitrification or at cryopreserved embryo transfer. The results support the argument that careful monitoring of serum progesterone concentrations in HRT-cryopreserved embryo transfer is warranted and that further studies should explore pessary adjustments to optimize concentrations for individual women to enhance implantation rates. PMID:26099447

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

    PubMed

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

    2015-12-01

    Pregnancy rates from cryopreserved embryos remain lower than non-cryopreserved counterparts, even though these embryos appear morphologically normal. How epigenetic events, such as histone modifications, are affected by cryopreservation of embryos remains unknown. The current study evaluated the effect of conventional freezing/thawing of invitro produced bovine blastocyst embryos on histone modifications, H3K4me3 and H3K27me3. At day 7 of invitro culture, blastocyst stage embryos were either frozen by conventional freezing method (-0.5C/min in 1.5M ethylene glycol; F/T group) or remained in culture for an additional 18h (Ctrl). Frozen embryos were stored in liquid N2 for 14 days, thawed and placed in culture for 36h for recovery. Control and re-expanded frozen-thawed blastocysts from both groups were fixed in 4% paraformaldehyde and stored in PBS+0.1% triton-X at 4C. Immunofluorescence, utilizing antibodies against H3K4me3 and H3K27me3, was conducted and staining intensity was analyzed as percentage of total DNA. Day 7 blastocyst development rate was 35.55% (352/990) with blastocyst recovery at 54.23% (77/142) 36h post-thawing. Total cell numbers per blastocyst were not different amongst groups (117.812.49 and 116.114.69, F/T and Ctrl groups respectively). Global staining for the active mark, H3K4me3, was lower in F/T blastocysts compared to Ctrl (17.242.80% vs. 34.953.77%; P<0.01). However, staining for the inhibitory mark, H3K27me3, was nearly 2-fold higher in F/T blastocysts (40.413.83% vs. 21.293.92%; P<0.01). These results suggest that bovine blastocysts, subjected to conventional freezing methods, have altered histone modifications that may play a role in poor pregnancy rates. PMID:26408849

  18. 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. PMID:25785884

  19. Embryonic Stem Cells Derived from In Vivo or In Vitro-Generated Murine Blastocysts Display Similar Transcriptome and Differentiation Potential

    PubMed Central

    Simbulan, Rhodel K.; Di Santo, Marlea; Liu, Xiaowei; Lin, Wingka; Donjacour, Annemarie; Maltepe, Emin; Shenoy, Archana; Borini, Andrea; Rinaudo, Paolo

    2015-01-01

    The use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF) has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC) from blastocysts conceived after natural mating (mESCFB) or after IVF, using optimal (KSOM + 5% O2; mESCKAA) and suboptimal (Whittens Medium, + 20% O2, mESCWM) conditions. All three groups of embryos showed similar behavior during both derivation and differentiation into their respective mESC lines. Unsupervised hierarchical clustering of microarray data showed that blastocyst culture does not affect the transcriptome of derived mESCs. Transcriptomic changes previously observed in the inner cell mass (ICM) of embryos derived in the same conditions were not present in mESCs, regardless of method of conception or culture medium, suggesting that mESC do not fully maintain a memory of the events occurring prior to their derivation. We conclude that the fertilization method or culture media used to generate blastocysts does not affect differentiation potential, morphology and transcriptome of mESCs. PMID:25723476

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2014-01-01

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

  2. Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts.

    PubMed

    Harvey, A J; Kind, K L; Thompson, J G

    2004-01-01

    In cattle embryos, development to the blastocyst stage is improved in the presence of 10 micro;m 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1alpha and 2alpha (HIF1alpha, HIF2alpha) mRNA. Although HIF1alpha protein remained undetectable in bovine blastocysts, HIF2alpha protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 microm DNP improves the quality of bovine embryos, coincident with increased HIF2alpha protein localisation within ICM cells and increased HIFalpha mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2. PMID:15740689

  3. Addition of l-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos.

    PubMed

    Castillo-Martn, Miriam; Yeste, Marc; Soler, Albert; Morat, Roser; Bonet, Sergi

    2014-04-17

    The aim of the present study was to determine the effect of l-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification-warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without l-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without l-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with l-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with l-ascorbic acid. Thus, supplementing culture and/or vitrification media with l-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression. PMID:25322209

  4. Cryopreserved blastocysts have a lower implantation but an equal live birth rate as compared to fresh blastocysts of the same quality - a case-control study.

    PubMed

    Shebl, Omar; Ebner, Thomas; Sommergruber, Michael; Sir, Andreas; Tews, Gernot

    2009-01-01

    The purpose of this case-control study was to compare implantation, pregnancy, and live birth rate of cryopreserved embryos with that of fresh embryos. A total of 1,488 assisted reproductive technology (ART) trials including 188 cryopreserved embryo replacement trials were reviewed. After proofing the exclusion criteria, 112 patients undergoing a cryopreserved embryo replacement (study group) were matched with 112 patients undergoing a fresh embryo transfer (control group). Matching was done for age of patients, number, and quality of embryos (Day 5 embryos). Data concerning maternal characteristics, survival rate of warming procedure, implantation, pregnancy, ongoing pregnancy, and live birth rates were collected. A lower implantation rate was found for cryopreserved embryo replacement compared to transferring fresh embryos. Live birth rate was found to be equal in both groups. PMID:19562560

  5. 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. PMID:25586639

  6. Heat transfer characteristics from an array of thin strips pin fins due to their exposures to a single downward jet impingement

    NASA Astrophysics Data System (ADS)

    Eid, Eldesouki I.; Gomaa, Abdalla G.; Gomaa, Mohamed E.

    2011-02-01

    This paper investigates the heat transfer characteristics from thin strips pin fins due to their exposure to a single circular downward air jet impingement. Five aluminum specimens were considered; each one has a rectangular base of 84 mm 78 mm and it has an array of about 300 thin strips pin fins. A test rig consists mainly of air compressor; nozzle and protractor mechanism was setup. Experiments were done to find out the effects of attack angle, Reynolds number, nozzle-to-target spacing, lateral pitch and parallel pitch among the fins on the heat transfer characteristics. Empirical correlations were deduced to describe the experimental data. A CFD-numerical model was introduced to monitor the flow characteristics on a scale of more details than that possible in the experimental work. The comparison among the results of the present work and those by the literature shows about 50% improvement in heat transfer characteristics rather than the single jet impingement onto flat plates, cylindrical surfaces, ribbed walls and multiple jets impingement onto flat plates.

  7. A cathepsin B inhibitor, E-64, improves the preimplantation development of bovine somatic cell nuclear transfer embryos.

    PubMed

    Min, Sung-Hun; Song, Bong-Seok; Yeon, Ji-Yeong; Kim, Jin-Woo; Bae, Jung-Ho; Park, Soo-Yong; Lee, Yong-Hee; Chang, Kyu-Tae; Koo, Deog-Bon

    2014-03-01

    Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos. PMID:24240170

  8. Transfer-printing of single DNA molecule arrays on graphene for high resolution electron imaging and analysis

    PubMed Central

    Cerf, Aline; Alava, Thomas; Barton, Robert A.; Craighead, Harold G.

    2011-01-01

    Graphene represents the ultimate substrate for high-resolution transmission electron microscopy, but the deposition of biological samples on this highly hydrophobic material has until now been a challenge. We present a reliable method for depositing ordered arrays of individual elongated DNA molecules on single-layer graphene substrates for high resolution electron beam imaging and electron energy loss spectroscopy analysis. This method is a necessary step towards the observation of single elongated DNA molecules with single base spatial resolution to directly read genetic and epigenetic information. PMID:21919532

  9. 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. PMID:23692072

  10. Extending light-harvesting of poly(3-hexylthiophene) through efficient energy transfer from infra-red absorbing nanocrystals: Single nanoparticle study

    NASA Astrophysics Data System (ADS)

    Grzelak, Justyna; Ciszak, Kamil; Nyk, Marcin; Mackowski, Sebastian; Piatkowski, Dawid

    2014-10-01

    We report on single nanocrystal fluorescence microscopy of blends composed of colloidal up-converting NaYF4 nanocrystals doped with rare-earth ions embedded in poly(3-hexylthiophene) (P3HT) polymer. By probing both steady-state and time-resolved fluorescence properties of individual nanocrystals excited with infra-red 980 nm laser, we demonstrate that upon up-conversion to the visible spectral range, the energy is efficiently transferred from the nanocrystals to P3HT. From the analysis of fluorescence lifetimes, the energy transfer efficiency for 550 nm emission of the nanocrystals was estimated to be 60%. This observation renders the up-converting nanocrystals as potential structures for improving light-harvesting efficiency of polymers in the near-infrared spectral region.

  11. 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 ?C and ?C-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 (?carbon: -4.0 0.2; ?nitrogen: -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. PMID:24851834

  12. Sensitivity function analysis of gravitational wave detection with single-laser and large-momentum-transfer atomic sensors

    NASA Astrophysics Data System (ADS)

    Tang, Biao; Zhang, Bao-Cheng; Zhou, Lin; Wang, Jin; Zhan, Ming-Sheng

    2015-03-01

    Recently, a configuration using atomic interferometers (AIs) had been suggested for the detection of gravitational waves. A new AI with some additional laser pulses for implementing large momentum transfer was also put forward, in order to reduce the effect of shot noise and laser frequency noise. We use a sensitivity function to analyze all possible configurations of the new AI and to distinguish how many momenta are transferred in a specific configuration. By analyzing the new configuration, we further explore a detection scheme for gravitational waves, in particular, that ameliorates laser frequency noise. We find that the amelioration occurs in such a scheme, but novelly, in some cases, the frequency noise can be canceled completely by using a proper data processing method. Supported by the National Natural Science Foundation of China.

  13. Reduced dielectric screening and enhanced energy transfer in single- and few-layer MoS2.

    PubMed

    Prins, Ferry; Goodman, Aaron J; Tisdale, William A

    2014-11-12

    We report highly efficient nonradiative energy transfer from cadmium selenide (CdSe) quantum dots to monolayer and few-layer molybdenum disulfide (MoS2). The quenching of the donor quantum dot photoluminescence increases as the MoS2 flake thickness decreases with the highest efficiency (>95%) observed for monolayer MoS2. This counterintuitive result arises from reduced dielectric screening in thin layer semiconductors having unusually large permittivity and a strong in-plane transition dipole moment, as found in MoS2. Excitonic energy transfer between a zero-dimensional emitter and a two-dimensional absorber is fundamentally interesting and enables a wide range of applications including broadband optical down-conversion, optical detection, photovoltaic sensitization, and color shifting in light-emitting devices. PMID:25289461

  14. Mass transfer of single- and double-charged anions through an MA-41L anion-exchange membrane

    SciTech Connect

    Kulikova, O.M.; Sharkova, O.V.; Kulikov, S.M.

    1995-02-20

    Selective anion transfer through an MA-41L anion-exchange membrane in the Cl{sup -}-F{sup -}, Cl{sup -}-SO{sub 4}{sup 2-}, F{sup -}-SO{sub 4}{sup 2-}, and F{sup -}-CO{sub 3}{sup 2-} systems has been studied. The feasibility of partial anion separation in the chloride-sulfate system has been demonstrated. The separation of fluoride ions from accompanying anions was found to be practically impossible.

  15. [A role of trophoblastic cells in regulation of mouse blastocyst survival in vitro after microinjection and osmotic stress].

    PubMed

    Khramtsova, E A; Mezhevikina, L M; Fesenko, E E

    2014-01-01

    We have evaluated the morphology of the mouse preimplantation embryos at developmental stages from morula to late-blastocyst after two different impacts: microinjection of modified Witten's medium and osmotic stress in physiological osmolarity (310 mOsM), in 5% glucose (560 mOsM) at high concentration of NaCl (614 mOsM). Results of our research showed that these stresses caused similar changes in embryo morphology: volume was reduced followed by its recovery in culture medium (osmolality was less than a physiological value, 260 MOsM). The ability of embryos to recover the volume and morphology up to the initial level depends on a stage of embryo development and consequently competence of TB cells. In this study it was revealed that a key role in regulation of volume homeostasis after microinjection and after short-time (30-60 min) osmotic stress belongs to TB cells. Both physical effects induce the further embryo development in vitro up to the formation of primary colonies of embryonic and trophoblastic cells. These data could be used to develop the morphological criteria for a prediction of blastocyst-stage embryonic implantation potential. PMID:25702484

  16. Highly Efficient Simplified Single-Emitting-Layer Hybrid WOLEDs with Low Roll-off and Good Color Stability through Enhanced Förster Energy Transfer.

    PubMed

    Zhang, Dongdong; Cai, Minghan; Zhang, Yunge; Zhang, Deqiang; Duan, Lian

    2015-12-30

    Single-emitting layer hybrid white organic light-emitting diodes (SEL-hybrid-WOLEDs) usually suffer from low efficiency, significant roll-off, and poor color stability, attributed to the incomplete energy transfer from the triplet states of the blue fluorophores to the phosphors. Here, we demonstrate highly efficient SEL-hybrid-WOLEDs with low roll-off and good color-stability utilizing blue thermally activated delayed fluorescence (TADF) materials as the host emitters. The triplet states of the blue TADF host emitter can be up-converted into its singlet states, and then the energy is transferred to the complementary phosphors through the long-range Förster energy transfer, enhancing the energy transfer from the host to the dopant. Simplified SEL-hybrid-WOLEDs achieve the highest forward-viewing external quantum efficiency (EQE) of 20.8% and power efficiency of 51.2 lm/W with CIE coordinates of (0.398, 0.456) at a luminance of 500 cd/m(2). The device EQE only slightly drops to 19.6% at a practical luminance of 1000 cd/m(2) with a power efficiency of 38.7 lm/W. Furthermore, the spectra of the device are rather stable with the raising voltage. The reason can be assigned to the enhanced Förster energy transfer, wide charge recombination zone, as well as the bipolar charge transporting ability of the host emitter. We believe that our work may shed light on the future development of highly efficient SEL-hybrid-WOLEDs with simultaneous low roll-off and good color stability. PMID:26642836

  17. Catechol estrogen formation by pig blastocysts during the preimplantation period: biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity.

    PubMed

    Mondschein, J S; Hersey, R M; Dey, S K; Davis, D L; Weisz, J

    1985-12-01

    Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blastocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up to 30 min and with blastocyst protein concentrations of up to 100 micrograms in a reaction volume of 150 microliters. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein . 30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 microM, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein . 30 min), increased to a maximum on days 12 and 13 (628 +/- 153 and 516 +/- 227 pmol 2-OHE2/mg protein . 30 min, respectively), and declined by day 14 (63.2 +/- 32.9 pmol 2-OHE2/mg protein . 30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig. PMID:2998735

  18. 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.

  19. 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.

  20. 2.0-?m 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 ?m of Ho3+ ions are investigated. The maximum emission cross section of the above sample at 2.0 ?m is calculated to be 1.0810-20 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).

  1. 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%.

  2. The measurement of U(VI) and Np(IV) mass transfer in a single stage centrifugal contactor

    NASA Astrophysics Data System (ADS)

    May, I.; Birkett, E. J.; Denniss, I. S.; Gaubert, E. T.; Jobson, M.

    2000-07-01

    BNFL currently operates two reprocessing plants for the conversion of spent nuclear fuel into uranium and plutonium products for fabrication into uranium oxide and mixed uranium and plutonium oxide (MOX) fuels. To safeguard the future commercial viability of this process, BNFL is developing novel single cycle flowsheets that can be operated in conjunction with intensified centrifugal contactors.

  3. Single longitudinal dorsal incision approach to transfer the flexor digitorum longus tendon between the flexor digitorum brevis hemitendons: a cadaveric study.

    PubMed

    Becerro de Bengoa Vallejo, Ricardo; Losa Iglesias, Marta Elena; Rodriguez, Miguel Fuentes; Tirado, Fermn Viejo

    2013-01-01

    Transposition of the flexor digitorum longus tendon has been widely reported for the correction of flexible claw and hammertoe deformities. The most common technique uses two cutaneous incisions, one plantar and another dorsal. We performed a cadaveric study to determine whether the flexor digitorum longus tendon could be transferred to the dorsum of the proximal phalanx of the toe from its lateral or medial aspect through a unique single longitudinal central dorsal incision. The rationale for this novel approach was to minimize the risk of vascular compromise to the digit associated with the two-incision approach. Transposition of the flexor digitorum longus tendon was attempted in 120 toes of cadaveric feet (60 each second and third digits) through a central longitudinal dorsal incision. The flexor digitorum longus tendon segment was long enough to be successfully transposed between the flexor digitorum brevis hemitendons of the second and third toes in 100% of the cases using the central longitudinal dorsal incision approach, with a resection arthroplasty at the proximal interphalangeal joint. Transfer of the flexor digitorum longus tendon to the dorsum of the proximal phalanx can be performed for the correction of claw and hammertoe deformities in the second and third digits. The meticulous longitudinal incision of the flexor tendon sheath to expose the flexor digitorum brevis tendon and its longitudinal incision are essential to the successful transfer of the flexor digitorum longus tendon between the flexor digitorum brevis hemitendons. PMID:24072373

  4. 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.

  5. An inter-subspecies cloned buffalo (Bubalus bubalis) obtained by transferring of cryopreserved embryos via somatic cell nuclear transfer.

    PubMed

    Yang, B Z; Yang, C Y; Li, R C; Qin, G S; Zhang, X F; Pang, C Y; Chen, M T; Huang, F X; Li, Z; Zheng, H Y; Huang, Y J; Liang, X W

    2010-10-01

    The aim of this study was to explore the feasibility of cryopreservation of inter-subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum-starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross-bred buffalo (Murrah Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13-month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter-subspecies cloned embryos is feasible to produce buffalo offspring. PMID:19788521

  6. WNT/?-catenin signaling affects cell lineage and pluripotency-specific gene expression in bovine blastocysts: prospects for bovine embryonic stem cell derivation.

    PubMed

    Madeja, Zofia Eliza; Hryniewicz, Kamila; Orsztynowicz, Maciej; Pawlak, Piotr; Perkowska, Anna

    2015-10-15

    Despite many attempts, true bovine embryonic stem cells (bESC) still remain elusive. The WNT pathway has been associated with stem cell control in vertebrates and its role in pluripotency maintenance has been proven for several mammalian species, including rodents and primates. Thus, we have aimed to investigate the effect of WNT activation on pluripotency marker gene expression in the inner cell mass (ICM) and the trophectoderm (TE) and to study the derivation potential of primary bESC lines from blastocysts obtained in the presence of the glycogen synthase kinase 3 inhibitor (GSK3i). WNT activity clearly exerted a positive effect on pluripotency gene expression in developing bovine embryos, manifested by upregulation of OCT4, NANOG, REX1, SOX2, c-MYC, and KLF4 in the ICM and downregulation of CDX2 in the TE. However, the prolonged exposition of preimplantation embryos to the GSK3i resulted in reduced potential to form primary ESC-like colonies. The results of bESC derivation experiments allowed us to speculate that the derived cell lines may share features of both nave and primed ESCs. Similar to mouse epiblast stem cells and human ESCs, the derived lines grew as flat monolayer colonies intolerant to passaging as single cells. JAK/STAT signaling was indispensable for proper colony formation and proliferation, yet LIF alone was inefficient to support self-renewal. Concomitant with the nave state of mouse ESCs, WNT activity supported by LIF had beneficial effects on cell culture propagation, survival after passage, morphology, and pluripotency-related marker gene expression. Moreover, colonies derived in the presence of LIF and GSK3i maintained KLF4 transcription over several passages, whereas EpiSCs virtually do not express KLF4. PMID:26119137

  7. Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid-Protein-Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

    PubMed Central

    Liu, Hsiao-Wei; Cosa, Gonzalo; Landes, Christy F.; Zeng, Yining; Kovaleski, Brandie J.; Mullen, Daniel G.; Barany, George; Musier-Forsyth, Karin; Barbara, Paul F.

    2005-01-01

    The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3?/5? termini, termed the zipper complex, or nucleation through the hairpin loops in a kissing complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways. PMID:16100256

  8. 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 Frster 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

  9. Probing Driving Force and Electron Accepting State Density Dependent Interfacial Electron Transfer Dynamics: Suppressed Fluorescence Blinking of Single Molecules on Indium Tin Oxide Semiconductor.

    PubMed

    Govind Rao, Vishal; Dhital, Bharat; Lu, H Peter

    2016-03-01

    Photoinduced, interfacial electron transfer (ET) dynamics between m-ZnTCPP and Sn-doped In2O3 (ITO) film has been studied using single-molecule photon-stamping spectroscopy. The observed ET dynamics of single m-ZnTCPP adsorbed on ITO was compared with that of m-ZnTCPP adsorbed on TiO2 NPs with and without applied electric potential. Compared to m-ZnTCPP on the TiO2 NP surface, m-ZnTCPP on the ITO surface shows a reduced lifetime as well as suppressed blinking and a quasi-continuous distribution of fluorescence intensities, presumably due to higher electron density in ITO. The higher electron density leads to the occupancy of CB acceptor states/trap states, which supports a higher backward electron transfer (BET) rate that results in a quasi-continuous distribution of fluorescence intensities. The dependence of BET rate on electron density and charge trapping is consistent with our previous observations of quasi-continuous distribution of fluorescence intensities of m-ZnTCPP on TiO2 NPs with applied negative potential across the dye-TiO2 interface. The quasi-continuous distribution of fluorescence intensities in both cases of m-ZnTCPP on the ITO surface and m-ZnTCPP on TiO2 NPs with applied negative potential indicates that the electron density in the semiconductor plays a dominant role in dictating the changes in rates of charge transfer, rather than the relative energetics between electrons in the semiconductor and the oxidized sensitizer. PMID:26468609

  10. Single oocyte bisulfite mutagenesis.

    PubMed

    Denomme, Michelle M; Zhang, Liyue; Mann, Mellissa R W

    2012-01-01

    Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting. One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level. Development of the bisulfite mutagenesis assay has advanced from those originally reported towards ones that are more sensitive and reproducible. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos. Taking clues from agarose embedding of minute DNA amounts including oocytes, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos. PMID:22782232

  11. Slow Proton Transfer Coupled to Unfolding Explains the Puzzling Results of Single-Molecule Experiments on BBL, a Paradigmatic Downhill Folding Protein

    PubMed Central

    Cerminara, Michele; Campos, Luis A.; Ramanathan, Ravishankar; Muñoz, Victor

    2013-01-01

    A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6–11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ∼7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ∼15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2–0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7–8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form. PMID:24205082

  12. 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.

  13. 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 scheme as due to both Fe2+-Fe3+ (16500 cm?1) and Fe2+-Ti4+ (20000 cm?1) CT, rather than to Fe2+-Ti4+ CT only with two extreme types of Fe-Ti dimers [- v - Fe2+ - Ti4+ - v -] and [- M - Fe2+ - Ti4+ - v -], where v stands for vacancy. We discuss the implications in terms of energy and of short-range ordering of vacancies in the octahedral single chains of the ellenbergerite and dumortierite groups. Optical spectroscopy appears as a very sensitive structural probe of minor or trace elements. [1] Chopin C. & Langer, K. (1988): Fe2+-Ti4+ charge transfer between face-sharing octahedra: polarized absorption spectra and crystal chemistry of ellenbergerite. Bull. Minral., 111, 17-27. [2] Platonov, A.N., Langer, K., Chopin, C., Andrut, M., Taran, M.N. (2000): Fe2+-Ti4+ charge-transfer in dumortierite. Eur. J. Mineral., 12, 521-528.

  14. In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

    PubMed

    Verma, P J; Du, Z T; Crocker, L; Faast, R; Grupen, C G; McIlfatrick, S M; Ashman, R J; Lyons, I G; Nottle, M B

    2000-11-01

    The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred. PMID:11013434

  15. Establishment of bovine trophoblast stem-like cells from in vitro-produced blastocyst-stage embryos using two inhibitors.

    PubMed

    Huang, Xianghua; Han, Xuejie; Uyunbilig, Borjigin; Zhang, Manling; Duo, Shuguang; Zuo, Yongchun; Zhao, Yuhang; Yun, Ting; Tai, Dapeng; Wang, Chen; Li, Jinhua; Li, Xueling; Li, Rongfeng

    2014-07-01

    The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-?, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90%, NANOG 74.40%, and SOX2 8.50%) compared with blastocysts (OCT4 8.90%, NANOG 34.4%, and SOX2 3.80%). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80% and 3.90%) compared with blastocysts (18.80% and 9.10%). The TSC lines that were established in this study may be used either for basic research that is focused on peri-implantation and placenta development or as donor cells for transgenic animal production. PMID:24605918

  16. 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 (0.190.01 pmoles L-citrulline mg prot?1 h?1) compared to days 4 (0.340.03) and 5 (0.350.02) of pregnancy and to day 6 implantation sites (0.330.01). 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 (0.700.02 vs 0.400.04) and URB-597 (1.080.09 vs 0.830.06) 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 (0.250.04 vs 0.400.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.170.02 vs 0.270.02), a CB2 antagonist decreased it (0.170.02 vs 0.120.01). 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 establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies. PMID:21559512

  17. 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

  18. 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.

  19. No evidence of association between blastocyst aneuploidy and morphokinetic assessment in a selected population of poor-prognosis patients: a longitudinal cohort study.

    PubMed

    Rienzi, L; Capalbo, A; Stoppa, M; Romano, S; Maggiulli, R; Albricci, L; Scarica, C; Farcomeni, A; Vajta, G; Ubaldi, F M

    2015-01-01

    Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening. PMID:25458852

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

    PubMed Central

    Shankar, Madhira Bhawani; Munuswamy, Deecaraman

    2010-01-01

    Purpose To determine the effects of ?-tocopherol supplementation to oocyte maturation media and embryo culture media on the yield of ovine embryos. Methods ?-tocopherol, at concentrations of 0, 50, 100, 200, 400 and 500M was supplemented to ovine oocyte or embryo culture media and cultured at 5% or 20% O2 levels. Percentages of cleavage, morula and blastocyst, total cell count and comet assay were taken as indicators of developmental competence of embryos. Results 200M ?-tocopherol in embryo culture medium at 20% O2 level significantly increased the rates of cleavage (P?blastocyst (P?blastocyst total cell number (P?blastocyst formation were also significantly higher in 100M (P?

  1. Deletion of Mylk1 in oocytes causes delayed morula-to-blastocyst transition and reduced fertility without affecting folliculogenesis and oocyte maturation in mice.

    PubMed

    Liang, Qiu-Xia; Zhang, Qing-Hua; Qi, Shu-Tao; Wang, Zhong-Wei; Hu, Meng-Wen; Ma, Xue-Shan; Zhu, Min-Sheng; Schatten, Heide; Wang, Zhen-Bo; Sun, Qing-Yuan

    2015-04-01

    The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation. PMID:25761595

  2. Tracer Tests in a Fractured Dolomite: 3. Analysis of Mass Transfer in Single-Well Injection-Withdrawal Tests

    SciTech Connect

    Haggerty, R.; Fleming, S.W.; Meigs, L.C.; McKenna, S.A.

    1999-03-04

    We investigated multiple-rate diffusion as a possible explanation for observed behavior in a suite of single-well injection-withdrawal (SWIW) tests conducted in a fractured dolomite. We first investigated the ability of a conventional double-porosity model and a multirate diffusion model to explain the data. This revealed that the multirate diffusion hypothesis/model is most consistent with all available data, and is the only model to date that is capable of matching each of the recovery curves entirely. Second, we studied the sensitivity of the SWIW recovery curves to the distribution of diffusion rate coefficients and other parameters. We concluded that the SWIW test is very sensitive to the distribution of rate coefficients, but is relatively insensitive to other flow and transport parameters such as advective porosity and dispersivity. Third, we examined the significance of the constant double-log late-time slopes ({minus}2. 1 to {minus}2.8), which are present in several data sets. The observed late-time slopes are significantly different than would be predicted by either conventional double-porosity or single-porosity media, and are found to be a distinctive feature of multirate diffusion under SWIW test conditions. Fourth, we found that the estimated distributions of diffusion rate coefficients are very broad, with the distributions spanning a range of at least 3.6 to 5.7 orders of magnitude.

  3. Transfer of melamine from feed to milk and from milk to cheese and whey in lactating dairy cows fed single oral doses.

    PubMed

    Battaglia, M; Cruywagen, C W; Bertuzzi, T; Gallo, A; Moschini, M; Piva, G; Masoero, F

    2010-11-01

    A study was conducted to evaluate the excretion pattern, after a single oral dose, of melamine from feed into milk, and the subsequent transfer to cheese and whey. The transfer of cyanuric acid was also investigated. Twenty-four lactating Holstein cows were randomly allocated to 4 treatments and received single doses of melamine as follows: 0.05, 0.50, 5.00, and 50.00 g/cow for groups D1, D2, D3, and D4, respectively. Individual milk samples were collected for melamine and cyanuric acid analyses on d 1, 2, 3, 4, 5, and 7. Milk collected individually from the second milking after melamine ingestion was used to make cheese on a laboratory scale. Melamine and cyanuric acid were extracted using a solid-phase extraction cartridge, and analyses were carried out by liquid chromatography-mass spectrometry/mass spectrometry. Maximal melamine concentrations occurred between 6 and 18 h after treatment and increased with log dose (linear and quadratic), ranging from 0.019 to 35.105 mg/kg. More than 60% of the melamine that was transferred to the milk was observed within 30 h after melamine ingestion. Melamine was not detected (limit of detection was 0.002 mg/kg) in milk 5 d after treatment in group D1, and 7 d after treatment in groups D2, D3, and D4. Blood urea nitrogen was not influenced by melamine ingestion. During cheese making, melamine was transferred mainly to the whey fraction. Cyanuric acid was not detected in any of the samples (milk, cheese, or whey). The excretion pattern of melamine in milk and whey may represent a health concern when cows ingest more than 0.50 g of melamine/d. However, only at intake levels of 5 and 50 g/d did cheese exceed the limits as set forth by the European Union. The results confirmed that melamine contamination of milk and milk products may be related not only to direct contamination, but also to adulteration of animal feeds. PMID:20965350

  4. Direct excitation energy transfer as a technique for in situ measurements of ion-exchange processes in single polymer particles.

    PubMed

    Kim, H B; Habuchi, S; Hayashi, M; Kitamura, N

    1998-01-01

    Ion-exchange processes of a cationic dye (Malachite Green, MG) are studied for individual polymer particles (diameter of 20 or 13 ?m) by laser trapping microspectroscopy. When a cation-exchange resin, preadsorbed homogeneously with Rhodamine B (RhB), is soaked in an aqueous MG solution, MG is adsorbed in the surface layer of the particle in the initial stage and then diffuses into the inner volume with time. In the MG-diffused layer, RhB fluorescence is quenched by excitation energy transfer from the excited state of RhB to the ground-state MG, as revealed by fluorescence microspectroscopy of individual resin particles in the dye solution. On the basis of the time dependence of the quenching efficiency, the thickness of the MG-diffused layer at a given soaking time is estimated, and the diffusion coefficient of the dye in the particle is determined to be 8 10(-)(11) cm(2) s(-)(1). This method is shown to be very useful for determining the diffusion coefficient of a nonfluorescent ion in individual ion-exchange resin microparticles. PMID:21644605

  5. Transferring the state of a quantum register to a single oscillator: a simple circuit verses numerical optimization

    NASA Astrophysics Data System (ADS)

    Jacobs, Kurt; Wang, Xiaoting; Strauch, Frederick

    2012-02-01

    We consider the problem of swapping a quantum state between a register of qubits and a single quantum oscillator. We design a mesoscopic quantum circuit to do this, using an off-resonant interaction, based on the concept of coherent feedback control. We consider an explicit realization of this circuit, and perform simulations of its performance. We then take a different approach, in which we couple the register directly to the resonator, including inter-qubit couplings and local controls, and use numerical optimization to search for a control protocol that will achieve the swap with very high fidelity. Our results show that the protocols found using numerical searches are superior in speed and fidelity to the manually-designed circuit. We also explore how the time and complexity of the protocols increases with the problem size.

  6. 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

  7. Inhibition of MAP2K and GSK3 signaling promotes bovine blastocyst development and epiblast-associated expression of pluripotency factors.

    PubMed

    Harris, Daina; Huang, Ben; Oback, Bjrn

    2013-03-01

    Cells in the mammalian blastocyst segregate into three distinct lineages, namely, trophoblast, hypoblast, and epiblast. During development, these will form extraembryonic and embryonic tissues, respectively. In mouse, only epiblast cells can be directly converted into cultured pluripotent embryonic stem cells, capable of forming all adult cell types. This conversion is promoted by the double inhibition (i.e., 2i) of mitogen-activated protein kinase kinase (Map2k), antagonizing Fgf signaling, and of glycogen synthase kinase 3 (Gsk3), stimulating the Wnt pathway. We investigated the effect of 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro fertilized (IVF) embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAP2K (0.4 ?M PD0325901) and GSK3 (3 ?M CHIR99021) from the zygote (Day 1) stage. Compared to vehicle controls, 2i conditions increased the abundance of cumulus cells in bovine IVF cultures, which compromised blastocyst formation. Following cumulus removal, 2i accelerated blastocyst development and increased inner cell mass (ICM) and trophoblast cell numbers by 30% and 27%, respectively. These developmental and morphological changes were accompanied by alterations in gene expression. Signal inhibition increased transcription of putative epiblast markers NANOG and SOX2 while repressing putative hypoblast marker GATA4. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the ICM and not due to ectopic expression in the trophoblast. Expression of other pluripotency-related (POU5F1, KLF4, DPPA3) or trophoblast-enriched (CDX2) genes was not affected. In summary, 2i conditions reprogrammed the transcriptional profile of bovine ICM but not trophoblast cells. By shifting the balance from hypoblast- to epiblast-associated gene expression, 2i culture may prime bovine epiblast for subsequent derivation of pluripotent stem cell cultures. PMID:23390165

  8. Granulocyte-macrophage colony-stimulating factor stimulates mouse blastocyst inner cell mass development only when media lack human serum albumin.

    PubMed

    Karagenc, Levent; Lane, Michelle; Gardner, David K

    2005-04-01

    The aim of the current study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and differentiation of preimplantation mouse embryos from different strains and under different culture conditions. Embryos from F1 hybrid mice were cultured in a modified G1 medium lacking amino acids and EDTA (simple G1), human tubal fluid medium (HTF) or in G1/G2 sequential media, supplemented with GM-CSF (0, 2, 4, 8, and 16 ng/ml). Embryos from CF1 mice were subsequently cultured in G1/G2 with (5 mg/ml) or without HSA, in the absence or presence of GM-CSF (2 ng/ml). GM-CSF had no effect at any concentration on F1 embryo development and blastocyst cell numbers, irrespective of the culture media used. Similarly, GM-CSF had no effect on CF1 blastocyst development. However, a stimulatory effect of GM-CSF was evident on total blastocyst cell number and ICM development when CF1 embryos were cultured in the absence of HSA. When HSA was present in the media the beneficial effect of GM-CSF was negated. There was no difference in the number of apoptotic cells in CF1 blastocysts when G1/G2 were supplemented with GM-CSF with or without HSA. These data indicate that there is no beneficial effect of supplementing either simple (simple G1 or HTF) or more complete (G1/G2) media with GM-CSF when protein is present in the medium. However, when culture conditions are suboptimal and non-physiological, i.e. the absence of protein, GM-CSF stimulates development of both total cell numbers and ICM development of CF1 blastocysts. PMID:15901460

  9. MT3 melatonin binding site, MT1 and MT2 melatonin receptors are present in oocyte, but only MT1 is present in bovine blastocyst produced in vitro

    PubMed Central

    2012-01-01

    Background Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP. Methods The presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [125I]-iodomelatonin (17 pM) in embryos. The kind of melatonin site was investigated in oocytes and embryos by immunocytochemistry. In vitro fertilized bovine embryos produced from in vitro maturated oocytes supplemented with melatonin (0.0001 to 1000 nM) were analysed to determine their cleavage and blastocyst formation rates. Results The [125I]-iodomelatonin (17 pM) binding in blastocysts was blocked by pre-incubation with melatonin (30000 nM), showing the presence of the high affinity melatonin receptors. MT1, MT2 and NQO2 immunoreactivity was observed in oocytes. MT1 immunoreactivity was observed in hatched blastocysts, however MT2 and NQO2 were not observed in this embryonic stage. Melatonin (pM) triggered significant difference in both cleavage and blastocysts formation rates. Conclusions The high affinity MT1 melatonin receptor must be taking part in IVM events; furthermore it is the first melatonin receptor to appear during bovine embryo development in vitro. PMID:23207065

  10. 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 endoergonicities greater than 30 kcal/mol with benzaldehyde, so these react by direct polar additions. A working hypothesis is that butyllithium reagents undergo polar additions, despite redox potentials which indicate less than 13 kcal/mol endoergonic electron transfer, because of the great exoergonicity associated with the two-electron addition, which is responsible for a low barrier for polar reactions.

  11. 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 continues; this work is supported by the new DOE BES grant.

  12. The effect of estrogen administration during early pregnancy upon the survival of single implanted pig embryos.

    PubMed

    Kawarasaki, T; Enya, S; Otsu, Y

    2012-12-01

    In the present study, we investigated the influence of exogenous estrogen on embryo survival after transfer into prepubertal gilts in which estrus had been induced. In the first experiment, estrus was induced in prepubertal gilts by the administration of 1,000 IU of eCG and 750 IU of hCG every 72 h. Several blastocysts were recovered on d 6 (d 0 is the day of hCG administration), and 1 embryo was transferred to the tip of 1 side of the uterine horn on d 6 (Control). In treated groups, after embryo transfer, 5 mg of estradiol benzoate (EB) was administered on d 11 (EB5mg-1) or d 11, d 13, and d 15 (EB5mg-3) or d 11, 12, 13, 14, and 15 (EB5mg-5) or 20 mg of estradiol dipropionate (EDP) was administered on d 11 (EDP20mg-1) or d 11 and d 14 (EDP20mg-2). Autopsy examinations were performed on d 53 to 60. Although nontreated gilts did not become pregnant, gilts in each of the estradiol-treated groups became pregnant. The greatest pregnancy rate (77.8%, 7/9) was obtained with EDP20mg-2 (EDP20mg-2 > control: P < 0.05). In a second experiment, 1 blastocyst was transferred to prepubertal gilts and treated with EDP20mg-2. Pregnancy in recipient pigs was confirmed by ultrasonography, and pigs were allowed to farrow. Embryo survival rate was high on d 30 of pregnancy (75%, 9/12) but had a tendency (P = 0.0995) to