bluuebunny/crossref_metadata_2025 huggingface dataset:
352 THE USE OF PLASMA ANTI-MÜLLERIAN HORMONE IN SHEEP AS AN ENDOCRINE MARKER OF THE OVARIAN RESPONSE TO FOLLICLE-STIMULATING HORMONE IN MULTIPLE-OVULATION EMBRYO TRANSFER PROGRAMS
Authors: Lahoz B. , Alabart J. L. , Cocero M. J. , Monniaux D. , Fabre S. , Folch J.
Published: December 2014

The performance of MOET (multiple-ovulation embryo transfer) programs in sheep is limited, mainly due to variable ovarian responses to FSH superovulation treatments. In several mammalian species, anti-Müllerian hormone (AMH) has been demonstrated to be a good predictor of the ovarian follicle population able to respond to gonadotropins. Therefore, we aimed to evaluate its usefulness in ovine MOET programs. With this goal, two MOET trials involving 24 adult ewes in total were performed. Each ewe received a fluorogestone acetate sponge (Sincropart 30 mg, CEVA Animal Health SA, Barcelona, Spain) which was replaced by a new one after 6 days (T–4). Four days later (T0), the first FSH injection (Folltropin-V, Minitub Ibérica SL, Tarragona, Spain) of a superovulation treatment consisting in 280 IU of FSH administered in 8 decreasing doses was applied. Blood samples were taken at T–4 and T0 using lithium heparin tubes for AMH measurement. Ewes were inseminated 51 h after sponge removal. Eight days after sponge removal, ovulation rate was recorded and embryo recovery was carried out under general anaesthesia. After morphological evaluation, 2 embryos were transferred to each recipient previously synchronized. The plasma concentrations of AMH were determined using the AMH equine ELISA kit (AnshLab, Webster, TX, USA). The sensibility of the assay was 27.8 pg mL–1, and the intra-assay coefficient of variation was 4.8%. Relationships between the AMH concentration of each animal and the number of corpora lutea (CL), embryo recovered and lambs born per donor ewe were tested using the Pearson correlation coefficient. Normality of the variables was assessed by Kolmogorov–Smirnov test. The plasma AMH concentrations at T–4 were highly correlated with those at T0 (r = 0.95; P < 0.01), so both sampling times could be used indistinctly. The plasma AMH concentration at T0 was highly variable between animals, ranging from 0 to 309.1 pg mL–1 (mean ± s.e.m.: 98.4 ± 18.4 pg mL–1). Similarly, the number of CL ranged from 2 to 29 (12.2 ± 1.5), recovered embryos from 0 to 17 (7.6 ± 1.2), and lambs born per donor and session from 0 to 13 (4.5 ± 0.9). The AMH concentration at the beginning of the FSH treatment (T0) was highly correlated with the total number of CL (r = 0.70; P < 0.01), but significance was not attained for AMH with the other variables. The number of CL was also correlated with the number of recovered embryos (r = 0.69; P < 0.01) and lambs born (r = 0.58; P < 0.01). In conclusion, AMH concentrations measured in blood plasma before the FSH treatment could be used to predict the number of CL per donor ewe, and so to improve the efficiency of MOET programs. Further studies are necessary to assess the individual repeatability of a given ewe from session to session as well as the relationship of AMH with other embryo-related variables.
357 EARLY FETAL DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS GENERATED FROM FIBROBLASTS GENETICALLY MODIFIED BY piggyBac TRANSPOSITION
Authors: Alessio A. , Fili A. , Forcato D. , Olmos-Nicotra M. F. , Alustiza F. , Rodriguez N. , Sampaio R. V. , Sangalli J. , Bressan F. , Fantinato-Neto P. , Meirelles F. , Owens J. , Moisyadi S. , Kues W. A. , Bosch P.
Published: December 2014

Transposon-mediated transgenesis is a well-established tool for genome manipulation in small animal models. However, translation of this active transgenesis method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac (PB) transposon system can efficiently transpose transgenes into the bovine genome [Alessio et al. 2014 Reprod. Domest. Anim. 49 (Suppl. 1), 8]. The aims of the current study were a) to investigate the effectiveness of a hyperactive version of the PB transposase, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFF) were chemically transfected with either pmGENIE-3 (a helper-independent PB transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. 2010 PNAS USA 107, 8117–8122), pmhyGENIE-3 (carrying an hyperactive version of the PB transposase; Marh et al. 2012 PNAS USA 109, 19 184–19 189), or pmGENIE-3/Δ PB (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic-resistant and EGFP+ colonies were counted and data analysed by ANOVA and Tukey's test. For SCNT, pmhyGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were nonsurgically transferred to synchronized recipients. Conceptuses were recovered by Day 35 of gestation, observed under fluorescence excitation, and genotyped. The mean number of colonies in pmhyGENIE-3 group was significantly higher than those in pmGENIE-3 and the control group (324.0 ± 17.8 v. 100.0 ± 16.1 and 2.8 ± 0.8 respectively, n = 4–7; P < 0.05). The hyperactive transposase increased transgene integration efficiency 3.24 times compared with the conventional PB transposase. The SCNT and early fetal development data are summarised in Table 1. Phenotypic analysis revealed that both transgenic fetuses and the extraembryonic membranes expressed EGFP with no macroscopic evidence of variegated transgene expression. Molecular analysis by PCR confirmed that both fetuses carried the transposon DNA. Here, we demonstrate that a hyperactive version of the PB transposase is more active in bovine cells than the conventional PB transposase. In addition, SCNT embryos generated from genetically modified cells by the pGENIE transposon system can progress to early stages of fetal development.

Table 1.SCNT and early fetal development of bovine fibroblasts transposed with piggyBac1

The financial support of UNRC, CONICET and ANPCyT from Argentina is gratefully acknowledged.
42 CONVERSION OF THE CHROMATIN OF SOMATIC CELLS INTO SPERMATID-LIKE STRUCTURES
Authors: Iuso D. , Czernik M. , Toschi P. , Zacchini F. , Shiota H. , Barral S. , Curtet S. , Buchou T. , Ptak G. , Khochbin S. , Loi P.
Published: December 2014

The post-meiotic phase of spermatogenesis is characterised by a radical reorganization of the chromatin, leading to its nucleosomal to toroid transition. The replacement of histones with protamine is a gradual process regulated to the hierarchical translation of repressed mRNAs leading to the following events: incorporation of testis-specific histone variants and general histone hyperacetylation, bromodomain proteins, transition proteins, concluded by protamine incorporation on DNA. In this work, we tested whether the induced expression of human protamine 1 (PR1) in sheep somatic cells could induce a protamine/toroid conformation of interphase nuclei. Sheep adult fibroblasts (SAF) were cultured in DMEM with 10% fetal bovine serum from second to eighth passage. Then, SAF at 80% confluence were transfected with 3 μg of pPR1-red fluorescent protein (RFP) and pRFP (CTR) with lipofectamine. At 4 h post-transfection, cells were treated with 5 nM trichostatin A (TSA; histone deacethylase inhibitor) for an additional 16 h. Transfected cells (visualised through the RFP tag) were analysed for nuclear morphology (transmission electron microscopy), PR1-RFP expression (confocal microscope, RT-PCR, Western blot), cytofluorimeter, DNA damage (comet assay, pH2A.X immune-detection), and chromatin immune-precipitation assays (ChIP). Moreover, to visualise the histone/protamine exchange, we transfected with PR1 mouse GFP-H2B fibroblasts. Protaminized cells were used as donors for nuclear transfer (NT) and TH2B (testis/oocyte-specific histone normally present in male pronucleus, a marker of reprogramming) was detected in pronuclear stage of NT zygotes. The χ2 test was used for statistical analyses. We demonstrated that PR1 translocates into the nuclei and gradually compacts them into elongating spermatid-like structures in 48 h; TSA treatment facilitates the process [TSA 83.3% (50/60); without TSA 55.2%; 58/105; P < 0.0003]. A complete histone-to-protamine exchange was also visualised in GFP-H2B nuclei (mouse fibroblasts) 40h after PR1 transfection. Cytofluorimetric analysis demonstrated that protamine incorporation occurs in any cell cycle stage and without DNA breaks. Next, protamine-protamine binding was excluded by ChIP analysis, which confirmed protamine-DNA binding. Finally, protaminized nuclei transplanted into enucleated oocytes incorporated maternal histone TH2B (3/8), whereas no signal was detected in control cells (0/9), suggesting that protaminized nuclei are better remodelled. We conclude that the induced expression of PR1 forces the somatic chromatin to acquire a structure overlapping elongated spermatid/spermatozoa, a conformation that perfectly matches the nuclear reprogramming machinery of the oocyte. Further work will determine whether protaminized cells are better reprogrammed upon nuclear transfer.
46 SPINDLE CONFIGURATION OF IN VITRO-MATURED BOVINE OOCYTES EXPOSED TO SODIUM CHLORIDE OR SUCROSE PRIOR TO CRYOTOP VITRIFICATION
Authors: Arcarons N. , Morató R. , Spícigo J. F. W. , Ferraz M. A. M. M. , Mogas T.
Published: December 2014

It has been previously described that a simple treatment with medium containing elevated NaCl or sucrose concentrations increases the cryotolerance and developmental competence of in vitro-matured porcine oocytes after vitrification and parthenogenetic activation (Lin et al. 2009 Reprod. Fertil. Dev. 21, 338–344). This work was designed to study whether the exposure to increased concentrations of NaCl or sucrose before vitrification improves cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, in vitro-matured oocytes were exposed to different NaCl and sucrose concentrations (from 375 to 808 mOsm) for 1 h. In Experiment 2, and according to the results obtained in the first experiment, oocytes were exposed to 375 mOsm NaCl or sucrose solution, vitrified, and warmed. Nontreated oocytes were used as controls. In both experiments, oocytes were fixed after treatment and microtubule, and chromosome distribution was analysed by immunocitochemistry. All statistical analyses were conducted with the IBM SPSS 19 for Windows (IBM corp., Chicago, IL). ANOVA was performed to analyse differences in meiotic spindle. Statistical significance was set at P < 0.05. After exposure to 375 mOsm of NaCl or sucrose, similar percentages of oocytes showing normal chromosome distribution were obtained compared to the control group (83.4, 71.8, and 85.0%, respectively). Groups treated with higher concentrations (443 to 808 mOsm) triggered significantly lower proportions of normal spindles. After vitrification/warming, no significant differences were observed between nonvitrified oocytes (71.3%) and those treated with NaCl before vitrification/warming procedure (41.9%) when normal chromosome organisation was analysed. Significantly higher percentages of normal chromosome configuration were observed when oocytes were exposed to sucrose before vitrification (34.2%) compared with control-vitrified oocytes (23.3%). However, pretreatment with NaCl or sucrose before vitrification did not trigger significant differences in terms of percentages of normal microtubule configuration (41.9 and 32.9%, respectively) compared with control-vitrified oocytes (40.2 and 24.4%, respectively), although both treatments differed significantly from control (79.1 and 81.7%, respectively). In conclusion, this study showed that a 375-mOsm NaCl or sucrose pretreatment of bovine oocytes before vitrification did not have a deleterious effect on the organisation of the meiotic spindle of vitrified/warmed bovine oocytes. Further experiments are required to investigate whether in vitro-matured oocytes subjected to this osmotic treatment could improve their development competence after being vitrified/warmed.
5 PROPYLENE GLYCOL FEEDING SUPPLEMENTATION MODIFIES INSULIN-LIKE GROWTH FACTOR SYSTEM GENE EXPRESSION IN CUMULUS–OOCYTE COMPLEXES AND THE EXPRESSION OF SELECTED CANDIDATE GENES IN EMBRYOS PRODUCED IN VITRO IN FEED-RESTRICTED HEIFERS
Authors: Gamarra G. , Ponsart C. , Lacaze S. , Nuttinck F. , Mermillod P. , Le Guienne B. , Monniaux D. , Humblot P. , Ponter A. A.
Published: December 2014

Dietary supplementation with propylene glycol (PG) increases the rate of grade 1 embryos produced from feed restricted females (Gamarra et al. 2014 Reprod. Fertil. Dev.). The aim of this study was to evaluate if a PG feeding supplement could modify the expression profile of selected candidate genes that are important for in vitro embryo development and the gene expression patterns of the insulin-like growth factor (IGF) system in oocytes and cumulus cells in feed-restricted heifers. Feed-restricted heifers (n = 16, growth rate of 600 g day–1) received a single daily drench of 400 mL of water (group restricted, R) from Day 1 to Day 9 of a first synchronized oestrous cycle followed by 400 mL of PG from Day 1 to Day 9 of the second synchronized oestrous cycle (group restricted + PG, RPG). Ovum pick-up (OPU) was performed following superovulation, on Day 5 of the oestrous cycle to produce embryos in vitro and on Day 9 without superovulation to obtain oocytes and cumulus cells. The same protocol was used in control animals (n = 6, growth rate of 800 g day–1). Real-time PCR was used to determine the relative abundance of genes involved in lipid metabolism and storage (PLIN2, SCD), energy metabolism (ATP5A1, GLUT1), membrane permeability (AQP3), epigenetic marks (DNMT3a), apoptosis (BAX, TP53), and protein processing (HSPA9B) in grade 1 blastocysts, IGF1, IGF1R, IGFBP2, IGFBP4 in cumulus cells, and IGF1R and IGFBP2 in oocytes. Mann-Whitney nonparametric tests were performed to analyse gene expression results. The expression of PLIN2, ATP5A1, GLUT1, AQP3, DNMT3a, BAX, and HSPA9B were decreased in embryos collected from restricted compared with control animals. The expression levels of these genes were restored when females were supplemented with PG. The expression of TP53 and SCD were not affected. In cumulus cells, the expression levels of IGF1, IGF1R, and IGFBP4 were decreased in restricted compared with control animals. The expression levels of IGF1 and IGF1R were restored with PG supplementation. No differences were observed for the IGFBP2 gene. In the oocytes, no differences were observed for the expression levels of IGF1R and IGFBP2 genes. In conclusion, this work shows for the first time that feed restriction and dietary supplementation by PG in heifers produced changes in gene expression in blastocysts and modified the pattern of the IGF system in cumulus cells. These results suggest the existence of an epigenetic regulation induced by PG during follicular growth, which can regulate the level of gene expression up to the blastocyst stage. In general, PG supplementation of feed-restricted donors restored gene expression at the levels observed after normal feeding.
73 APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD TO THE CRYOPRESERVATION OF HIGHLY CRYOSENSITIVE EMBRYOS
Authors: Uchikura A. , Matsunari H. , Nakano K. , Hatae S. , Matsumura Y. , Asano Y. , Takeishi T. , Nakauchi H. , Nagashima H.
Published: December 2014

We recently demonstrated that the hollow fibre vitrification (HFV) method (Matsunari et al. 2012) could effectively be applied to the cryopreservation of embryos from diverse species. In this study, we applied the HFV method to the cryopreservation of highly cryosensitive specimens, such as in vitro matured (IVM)/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, as well as IVM/IVF-derived cattle embryos at early cleavage stages. Porcine parthenogenetic morulae (d-4) derived from IVM oocytes were treated with 0.25% pronase to remove zona pellucidae. The resulting blastomeres were isolated from the zona-free morulae by a decompaction treatment followed by gentle pipetting. Bovine IVM-IVF embryos at the 2 to 4 cell (d-1), 8 to 16 cell (d-3), and morula stages (d-5) were then subjected to vitrification. The HFV procedure was performed as described previously using 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M trehalose as cryoprotectants. Four to twenty embryos, or all of the blastomeres isolated from a single morula, were individually loaded into a cellulose acetate hollow fibre (25 mm long, 185 μm φ, 15 μm membrane thickness) and vitrified. Survival of the vitrified embryos was assessed by in vitro development to blastocysts. Blastomeres recovered after vitrification were aggregated in micro-wells to examine their ability to form blastocysts. The HFV method was demonstrated to be effective for cryopreserving zona-free in vitro-produced porcine morulae and the blastomeres isolated from them (Table 1), as well as bovine IVM-IVF embryos at early cleavage stages. These data demonstrate that the HFV method is effective for highly cryosensitive specimens, such as IVM/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, and IVM/IVF-derived cattle embryos at early cleavage stages. These achievements may expand the technological options in the production of cloned and genetically modified pigs that are useful for biomedical research.

Table 1.Survival of zona-free porcine morulae and isolated blastomeres after vitrification (top) and blastocyst formation rates in bovine early-stage in vitro matured-IVF embryos after vitrification (bottom)

This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, and MUIIBR.
76 EX VIVO MODEL TO STUDY THE EFFECT OF ACUTE EXPOSURE TO DI-(2-ETHYLHEXYL) PHTHALATE ON BOVINE OOCYTE MATURATION AND DEVELOPMENTAL COMPETENCE
Authors: Kalo D. , Hadas R. , Roth Z.
Published: December 2014

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolites are environmental toxicants that potentially affect mammalian health. However, their effects on ovarian function, and in particular oocyte developmental competence, are less known. We established a model of acute exposure to DEHP to examine its immediate and long-term effects on follicles and oocyte competence. Lactating Holstein cows were synchronized and gavaged with DEHP (OXPLAST®O, ZAK, Kędzierzyn-Koźle, Poland; 100 mg kg–1 per day, n = 4) or water (n = 5) for 3 days. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed high DEHP metabolite levels in both the urine and plasma during DEHP exposure (acute phase), followed by relatively low levels through the subsequent 34 days (chronic phase). In the chronic phase, cows were synchronized with prostaglandin (PG)2α-gonadotropin-releasing hormone (GnRH) (day 0) and ovaries were monitored by ultrasonography (Aloka SSD-900, 7.5 MHz; Aloka, Tokyo, Japan) through an entire oestrous cycle. On Day 18 of the cycle, cows were administered with PG2α and within 30 h, follicular fluid (FF) was aspirated from the preovulatory follicle by ultrasonic scanner connected to a vaginal sector transducer (7.5-MHz, PieMedical, Maastricht, the Netherlands). For each group, the FF were pooled and analysed for DEHP metabolites (MEHP, MEHOH, MEHHP) by LC-MS/MS. These FF further served as maturation medium for in vitro embryo production. For all cows, FF aspirated before DEHP administration did not contain any of the examined metabolites. However, during the chronic phase, the level of MEHP (but not MEHOH, MEHHP) was higher (22.31 v. 0.00 nM) in the FF aspirated from treated cows (FF-DEHP, n = 4) relative to controls (FF-control, n = 5). To examine the effect of MEHP on oocyte maturation and developmental competence, cumulus-oocyte complexes aspirated from abattoir ovaries (n = 250 × 5 replicates) were matured (22 h, 38.5°C, 5% CO2) in FF-control or FF-DEHP, then in vitro fertilized (18 h, 38.5°C, 5% CO2) and cultured for 8 days in KSOM (38.5°C, 5% CO2, 5% O2). The proportion of oocytes with expanded cumulus cells (84.5 v. 81.2%) and distribution of oocyte within cortical granule types (I–III) did not differ between groups at the end of maturation. The proportion of oocytes with metaphase II plate and first polar body (i.e. nuclear-matured) was lower in the DEHP-treated group relative to the control (34.7 v. 64.3%, n = 100/group; P < 0.0001). Moreover, a decreased proportion of 2- to 4-cell-stage embryos (53.12 v. 67.61%; P < 0.04) and 7-day blastocysts (4.2 v. 12%; P < 0.08) was noted for the FF-DEHP group. In summary, the findings reveal long-lasting effects of DEHP exposure, expressed by MEHP incorporation into the FF, suggesting potential deleterious effects on developmental competence of the follicle-enclosed oocyte.
85 LOW-MOLECULAR-WEIGHT METABOLITES IN BOVINE IN VITRO PRODUCTION CULTURE MEDIA AS EMBRYO QUALITY MARKERS
Authors: Nõmm M. , Mark E. , Kilk K. , Kõks S. , Jaakma Ü.
Published: December 2014

The need for noninvasive embryo quality assessment techniques has increased as the in vitro production of cattle embryos has become more popular and necessary in the beef and milk production industries. In this study, we assessed the metabolomic profile of embryo culture media to determine whether it is possible to evaluate differences in low-molecular-weight metabolites in the culture media composition of morula stage embryos compared with embryos that develop to the blastocyst stage. Single bovine embryos were cultured in 60-µL SOF+0.4% BSA droplets under mineral oil. Twenty microliters of culture media was removed at Day 2, 5, and 8 post-fertilization. Cultured droplets without a zygote served as the control samples. A total of 42 samples were analysed using liquid chromatography-mass spectrometry (Q-Trap 3200, Ab Sciex, Framingham, MA, USA), followed by principal component analysis. Our preliminary results indicated significant differences (P < 0.00001) in 10 low-molecular-weight compounds between the groups. Three of those compounds (588, 589, and 702 Da) were represented in higher concentrations only in embryos that advanced into the blastocyst stage. These first results could allow the identification of embryos with improved viability and give better understanding of the development of pre-implantation embryo.This study was supported by CCRMB, Project ANIREP (3.2.0701.12–0036) and institutional grant IUT8–1.
86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER
Authors: Hoelker M. , Kassens A. , Held E. , Wrenzycki C. , Besenfelder U. , Havlicek V. , Sieme H. , Tesfaye D. , Schellander K.
Published: December 2014

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.
93 THE INVOLVEMENT OF E-CADHERIN IN THERMOPROTECTIVE FUNCTION OF INSULIN-LIKE GROWTH FACTOR-1 IN 4-CELL HAMSTER EMBRYOS
Authors: Trejo A. C. , Abad I. B. , Meza V. M. V. , Villa A. M. , Abad J. Z. , Navarro-Maldonado M. C. , Ambriz D. G.
Published: December 2014

Studies have demonstrated that the early pre-implantation embryo is very sensitive to effects of heat stress in vitro. Heat stress reduces the total cell number in blastocysts and increases apoptosis in blastomeres. Insulin-like growth factor-1 (IGF-1) has been widely studied as a thermoprotective agent for its anti-apoptotic actions. Addition of IGF-1 to the culture medium decreases the effects of heat stress on blastocysts but has no effects on 2-cell embryos. Molecular mechanisms by which IGF-1 decreases apoptosis involve activation of the PI3K/Akt pathway. It is also known that adherens junctions contribute to PI3K/AKT activation mediated by the transmembrane glycoprotein E-cadherin, which is involved in Ca2+-dependent cell-cell adhesion. Within 2- to 8-cell embryos, E-cadherin is mainly inactive and has cytoplasmic localization. 6-Dimethylaminopurine (6-DMAP) induces premature cell flattening and E-cadherin redistribution to adhesion sites in 4-cell embryos. The aim of this study was to induce E-cadherin redistribution in 4-cell hamster embryos and evaluate the thermoprotective function of IGF-1 in these embryos. Four-cell embryos were incubated in the presence of 6-DMAP to induce E-cadherin redistribution to adhesion sites and cultured for 24 h under conditions of heat stress and compared with controls without 6-DMAP. Culture medium was supplemented with IGF-1. At the end of culture, developmental stage and rate of apoptosis were determined and analysed by ANOVA using the General Linear Model (GLM) of SAS (SAS Institute Inc., Cary, NC, USA) procedure with statistical significance at P < 0.05. E-Cadherin redistribution induced by 6-DMAP increased development to the 6-cell stage after 24 h (63.57% v. 38.81%, respectively; P < 0.05) and reduced apoptosis (25% v. 33%, respectively; P < 0.05) under heat-stress conditions. In conclusion, we hypothesise a role for E-cadherin-mediated cell flattening in promoting IGF-1-mediated thermoprotection in pre-compact 4-cell hamster embryos. Further studies are required to confirm this link.
Kant on the Acquisition of Geometrical Concepts
Authors: Callanan John J.
Published: December 2014

It is often maintained that one insight of Kant’s Critical philosophy is its recognition of the need to distinguish accounts of knowledge acquisition from knowledge justification. In particular, it is claimed that Kant held that the detailing of a concept’s acquisition conditions is insufficient to determine its legitimacy. I argue that this is not the case at least with regard to geometrical concepts. Considered in the light of his pre-Critical writings on the mathematical method, construction in the Critique can be seen to be a form of concept acquisition, one that is related to the modal phenomenology of geometrical judgement.
A note on groups generated by involutions and sharply 2-transitive groups
Authors: Glauberman George , Mann Avinoam , Segev Yoav
Published: December 2014

LetGGbe a group generated by a setCCof involutions which is closed under conjugation. Letπ\pibe a set of odd primes. Assume that either (1)GGis solvable, or (2)GGis a linear group.

We show that if the product of any two involutions inCCis aπ\pi-element, thenGGis solvable in both cases andG=Oπ(G)⟨t⟩G=O_{\pi }(G)\langle t\rangle, wheret∈Ct\in C.

If (2) holds and the product of any two involutions inCCis a unipotent element, thenGGis solvable.

Finally we deduce that ifG\mathcal {G}is a sharply22-transitive (infinite) group of odd (permutational) characteristic, such that every33involutions inG\mathcal {G}generate a solvable or a linear group; or ifG\mathcal {G}is linear of (permutational) characteristic0,0,thenG\mathcal {G}contains a regular normal abelian subgroup.
Sil1, a nucleotide exchange factor for BiP, is not required for antibody assembly or secretion
Authors: Ichhaporia Viraj P. , Sanford Tyler , Howes Jenny , Marion Tony N. , Hendershot Linda M.
Published: December 2014

Sil1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone BiP, and mutations in this gene lead to Marinesco–Sjögren syndrome (MSS), a debilitating autosomal recessive disease characterized by multisystem defects. A mouse model for MSS was previously produced by disrupting Sil1 using gene-trap methodology. The resulting Sil1Gtmouse phenocopies several pathologies associated with MSS, although its ability to assemble and secrete antibodies, the best-characterized substrate of BiP, has not been investigated. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1Gtmouse was indistinguishable from wild-type age-matched controls in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1Gtmice. ER chaperones were expressed at the same level in Sil1WTand Sil1Gtmice, indicating that there was no evident compensation for the disruption of Sil1. Finally, these results were confirmed and extended in three human EBV-transformed lymphoblastoid cell lines from individuals with MSS, leading us to conclude that the BiP cofactor Sil1 is dispensable for antibody production.
A novel role for WAVE1 in controlling actin network growth rate and architecture
Authors: Sweeney Meredith O. , Collins Agnieszka , Padrick Shae B. , Goode Bruce L.
Published: December 2014

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.
The Effect of Hepatitis C Virologic Clearance on Cardiovascular Disease Biomarkers in Human Immunodeficiency Virus/Hepatitis C Virus Coinfection
Authors: Chew Kara W. , Hua Lei , Bhattacharya Debika , Butt Adeel A. , Bornfleth Lorelei , Chung Raymond T. , Andersen Janet W. , Currier Judith S.
Published: December 2014

Background.  Successful hepatitis C virus (HCV) treatment may reduce cardiovascular disease (CVD) risk and improve levels of CVD biomarkers produced outside the liver (nonhepatic biomarkers).

Methods.  Stored serum or plasma from before and 24 weeks after end of HCV treatment (EOT) from human immunodeficiency virus (HIV)/HCV-coinfected subjects who received up to 72 weeks of peginterferon/ribavirin, 27 with and 27 without sustained virologic response (SVR) matched by race, ethnicity and sex, were tested for nonhepatic (soluble intercellular adhesion molecule-1 [sICAM-1], soluble P-selectin [sP-selectin], interleukin [IL]-6, d-dimer, and lipoprotein-associated phospholipase A2 [Lp-PLA2]) and hepatic (cholesterol and high-sensitivity C-reactive protein) CVD and macrophage activation markers (soluble CD163 [sCD163] and soluble CD14). Changes in biomarkers and their association with SVR were examined by t tests or Wilcoxon tests and regression models.

Results.  Of the 54 subjects, 30 were white, 24 were black, and 44 were male. Pretreatment levels of nonhepatic biomarkers were high: sICAM-1 overall median, 439.2 ng/mL (interquartile range [IQR], 365.6–592.8]; sP-selectin, 146.7 ng/mL (IQR, 94.1–209.9), and IL-6, 2.32 pg/mL (IQR, 1.61–3.49). Thirty-seven of 52 (71%) subjects had Lp-PLA2 >235 ng/mL. Sustained virologic response was associated with decrease in sICAM-1 (P = .033) and sCD163 (P = .042); this result was attenuated after controlling for changes in the alanine aminotransferase level. At 24 weeks after EOT, 17 (63%) SVRs had Lp-PLA2 >235 ng/mL vs 25 (93%) non-SVRs (P = .021).

Conclusions.  Hepatitis C virus clearance may reduce hepatic and, subsequently, systemic inflammation and CVD risk in HIV/HCV coinfection.
Evolutionary relatedness does not predict competition and co-occurrence in natural or experimental communities of green algae
Authors: Alexandrou Markos A. , Cardinale Bradley J. , Hall John D. , Delwiche Charles F. , Fritschie Keith , Narwani Anita , Venail Patrick A. , Bentlage Bastian , Pankey M. Sabrina , Oakley Todd H.
Published: December 2014

The competition-relatedness hypothesis (CRH) predicts that the strength of competition is the strongest among closely related species and decreases as species become less related. This hypothesis is based on the assumption that common ancestry causes close relatives to share biological traits that lead to greater ecological similarity. Although intuitively appealing, the extent to which phylogeny can predict competition and co-occurrence among species has only recently been rigorously tested, with mixed results. When studies have failed to support the CRH, critics have pointed out at least three limitations: (i) the use of data poor phylogenies that provide inaccurate estimates of species relatedness, (ii) the use of inappropriate statistical models that fail to detect relationships between relatedness and species interactions amidst nonlinearities and heteroskedastic variances, and (iii) overly simplified laboratory conditions that fail to allow eco-evolutionary relationships to emerge. Here, we address these limitations and find they do not explain why evolutionary relatedness fails to predict the strength of species interactions or probabilities of coexistence among freshwater green algae. First, we construct a new data-rich, transcriptome-based phylogeny of common freshwater green algae that are commonly cultured and used for laboratory experiments. Using this new phylogeny, we re-analyse ecological data from three previously published laboratory experiments. After accounting for the possibility of nonlinearities and heterogeneity of variances across levels of relatedness, we find no relationship between phylogenetic distance and ecological traits. In addition, we show that communities of North American green algae are randomly composed with respect to their evolutionary relationships in 99% of 1077 lakes spanning the continental United States. Together, these analyses result in one of the most comprehensive case studies of how evolutionary history influences species interactions and community assembly in both natural and experimental systems. Our results challenge the generality of the CRH and suggest it may be time to re-evaluate the validity and assumptions of this hypothesis.
Temperature dependence of predation depends on the relative performance of predators and prey
Authors: Öhlund Gunnar , Hedström Per , Norman Sven , Hein Catherine L. , Englund Göran
Published: December 2014

The temperature dependence of predation rates is a key issue for understanding and predicting the responses of ecosystems to climate change. Using a simple mechanistic model, we demonstrate that differences in the relative performances of predator and prey can cause strong threshold effects in the temperature dependence of attack rates. Empirical data on the attack rate of northern pike (Esox lucius) feeding on brown trout (Salmo trutta) confirm this result. Attack rates fell sharply below a threshold temperature of +11°C, which corresponded to a shift in relative performance of pike and brown trout with respect to maximum attack and escape swimming speeds. The average attack speed of pike was an order of magnitude lower than the escape speed of brown trout at 5°C, but approximately equal at temperatures above 11°C. Thresholds in the temperature dependence of ecological rates can create tipping points in the responses of ecosystems to increasing temperatures. Thus, identifying thresholds is crucial when predicting future effects of climate warming.
The forest or the trees: preference for global over local image processing is reversed by prior experience in honeybees
Authors: Avarguès-Weber Aurore , Dyer Adrian G. , Ferrah Noha , Giurfa Martin
Published: December 2014

Traditional models of insect vision have assumed that insects are only capable of low-level analysis of local cues and are incapable of global, holistic perception. However, recent studies on honeybee (Apis mellifera) vision have refuted this view by showing that this insect also processes complex visual information by using spatial configurations or relational rules. In the light of these findings, we asked whether bees prioritize global configurations or local cues by setting these two levels of image analysis in competition. We trained individual free-flying honeybees to discriminate hierarchical visual stimuli within a Y-maze and tested bees with novel stimuli in which local and/or global cues were manipulated. We demonstrate that even when local information is accessible, bees prefer global information, thus relying mainly on the object's spatial configuration rather than on elemental, local information. This preference can be reversed if bees are pre-trained to discriminate isolated local cues. In this case, bees prefer the hierarchical stimuli with the local elements previously primed even if they build an incorrect global configuration. Pre-training with local cues induces a generic attentional bias towards any local elements as local information is prioritized in the test, even if the local cues used in the test are different from the pre-trained ones. Our results thus underline the plasticity of visual processing in insects and provide new insights for the comparative analysis of visual recognition in humans and animals.
Stochasticity and negative feedback lead to pulsed dynamics and distinct gene activity patterns
Authors: Zambrano Samuel , Bianchi Marco E. , Agresti Alessandra , Molina Nacho
Published: December 2014

Gene expression is an inherently stochastic process that depends on the structure of the biochemical regulatory network in which the gene is embedded. Here we study the interplay between stochastic gene switching and the widespread negative feedback regulatory loop. Using a simple hybrid model, we find that stochasticity in gene switching by itself can induce pulses in the system. Furthermore, we find that our simple model is able to reproduce both exponential and peaked distributions of gene active and inactive times similar to those that have been observed experimentally. Our hybrid modelling approach also allows us to link these patterns to the dynamics of the system for each gene state.
Optimization of Supercritical Fluid Consecutive Extractions of Fatty Acids and Polyphenols from Vitis Vinifera Grape Wastes
Authors: Aizpurua‐Olaizola Oier , Ormazabal Markel , Vallejo Asier , Olivares Maitane , Navarro Patricia , Etxebarria Nestor , Usobiaga Aresatz
Published: December 2014

In this study, supercritical fluid extraction has been successfully applied to a sequential fractionation of fatty acids and polyphenols from wine wastes (2 different vitis vinifera grapes). To this aim, in a 1st step just fatty acids were extracted and in a 2nd one the polyphenols. The variables that affected to the extraction efficiency were separately optimized in both steps following an experimental design approach. The effect of extraction temperature flow, pressure, and time were thoroughly evaluated for the extraction of fatty acids, whereas the addition of methanol was also considered in the case of the polyphenols extraction. A quantitative extraction with high efficiency was achieved at a very short time and low temperatures. Concerning quantification, fatty acids were determined by means of gas chromatography coupled to mass spectrometry after a derivatization step, whereas the polyphenols were analyzed by means of high performance liquid chromatography coupled to tandem mass spectrometry and the Folin–Ciocalteu method.
Mechanical and Microstructural Properties of “Wet” Alginate and Composite Films Containing Various Carbohydrates
Authors: Harper B. Allison , Barbut Shai , Smith Alexandra , Marcone Massimo F.
Published: December 2014

Composite “wet” alginate films were manufactured from alginate–carbohydrate solutions containing 5% alginate and 0.25% pectin, carrageenan (kappa or iota), potato starch (modified or unmodified), gellan gum, or cellulose (extracted or commercial). The “wet” alginate films were used as a model to understand co‐extruded alginate sausage casings that are currently being used by several sausage manufacturers. The mechanical, optical, and microstructural properties of the calcium cross‐linked composite films were explored. In addition, the water holding capacity and textural profile analysis properties of the alginate–carbohydrate gels were studied. The results indicate that the mechanical properties of “wet” alginate films/casings can be modified by adding various carbohydrates to them. Alginate films with pectin, carrageenan, and modified potato starch had significantly (P < 0.05) greater elongation values than pure alginate films. The alginate–pectin films also had greater (P < 0.05) tensile strengths than the pure alginate films. Alginate films with extracted cellulose, commercial cellulose, and modified potato starch had lower (P < 0.05) puncture force, distance, and work values than the alginate control films. Transmission electron microscopy images showed a very uniform alginate network in the control films. Several large cellulose fibers were visible in the films with extracted cellulose, while the cellulose fibers in the films with commercial cellulose were difficult to distinguish. Despite these apparent differences in cellulose fiber length, the 2 cellulose films had similar puncture and tensile properties.
Use of the Screening Tool of Older Persons' Prescriptions (STOPP) and the Screening Tool to Alert doctors to the Right Treatment (START) in hospitalised older people
Authors: Manias Elizabeth , Kusljic Snezana , Lam Di‐Luu
Published: December 2014

Aims

To determine the prevalence and nature of potentially inappropriate medications (PIMs) and potential prescribing omissions (PPOs) in patients aged 65 years and over.

Method

A retrospective clinical audit was undertaken (N = 200) in an Australian metropolitan teaching hospital.

Results

The prevalence of at least one PIM was 51% (n = 101) whereas the prevalence of at least one PPO was 74% (n = 147). The most common PIM was prescribing aspirin to patients with no history of coronary, cerebral or peripheral arterial disease or occlusive arterial events. The most commonly detected PPO was the failure to prescribe statins to patients with a documented history of coronary, cerebral or peripheral vascular disease. Overall, 80 (24%) of the 335 PIMs identified were possibly associated with an adverse clinical outcome experienced by patients.

Conclusions

Inappropriate prescribing continues to be a problem as shown by complexities associated with the risk–benefit trade‐offs of managing medications in older patients.
Feedback in technology‐based instruction: Learner preferences
Authors: Lefevre David , Cox Benita
Published: December 2014

This research investigates learner preferences for the format of feedback when using technology‐based instruction (TBI).

The primary method of data collection was to provide subjects with a range of options for TBI feedback following responses to multiple‐choice questions and then observe their choices. A software tool both presented the feedback options and tracked subsequent interactions. Over a 3‐year period, data were collected relating to 2066 postgraduate students studying one or more of three items of TBI. These students responded to a total of 61 929 questions. In addition, a series of semi‐structured interviews were conducted which investigated why subjects made their choices and the factors that affect these choices.

Recommendations to educators regarding the content, timing and delivery of TBI feedback are suggested.
A social network analysis of teaching and research collaboration in a teachers' virtual learning community
Authors: Lin Xiaofan , Hu Xiaoyong , Hu Qintai , Liu Zhichun
Published: December 2014

Analysing the structure of a social network can help us understand the key factors influencing interaction and collaboration in a virtual learning community (VLC). Here, we describe the mechanisms used in social network analysis (SNA) to analyse the social network structure of a VLC for teachers and discuss the relationship between face‐to‐face and online collaborations. In contrast to previous research applying SNA to analyse measuring indexes alone, we emphasise the mechanisms combining SNA, questionnaires, content analysis and focus group interviews—the key methodology to analyse complex interaction in a VLC. On this basis, we present an analysis model for teachers' VLC and apply it to a teachers' VLC known as ‘IRIS’. The study participants comprised 172 K12 teachers aged between 25 and 55 years. This study collected collaboration data from 2006 to 2012 and analysed the social network structure using sociograms, centrality, cohesive subgroups, clique phenomenon, and matrix correlation of SNA. These findings suggest that face‐to‐face and online collaborations are both indispensable in teaching and in research and continuously supplement and remedy each other in professional development. Moreover, the model succeeded in accessing, describing and analysing the social network structure of a VLC.