bluuebunny/crossref_metadata_2025 huggingface dataset:
345 LOSS OF Brm AND Baf170, COMPONENTS OF THE ATP-DEPENDENT CHROMATIN REMODELING COMPLEX, FACILITATES REPROGRAMMING OF SOMATIC CELL
Authors: Jiang Z. , Tang Y. , Tian X.
Published: December 2014

The SWI/SNF [SWItch/Sucrose NonFermentable; also known as BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodelling complexes are epigenetic modifiers that change the structure of chromatin, and thereby modulate gene transcription. The BAF chromatin remodeling complexes undergo progressive changes in subunit composition during cellular differentiation. In embryonic stem cells (ESC), BAF complex, esBAF, contains Brg1 and Baf155, which are crucial for ESC self-renewal and also facilitate induced pluripotent stem cell (iPSC) reprogramming from somatic cells. Here we sought to determine the roles of somatic BAF components (Brm and Baf170, homologues of Brg1 and Baf155, respectively) in mouse iPSC reprogramming through shRNA-mediated knockdown studies. We found that the expression of Brm and Baf170 were inhibited in reprogramming by Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) activity, which is essential for pluripotency establishment. We further found that knockdown of Brm and Baf170 in mouse somatic cells promotes reprogramming efficiency. Specifically, loss of Brm and Baf170 during early (Days 3 and 6 after initial iPSC induction) and later-stage (Day 9) reprogramming, respectively, improves the numbers of iPSC colonies formed. These also led to significant upregulation of pluripotent genes, including Sox2, Nanog, Fgf5, and Tbx3. Although the somatic Brm and Baf170 are believed to be absent in ESC, the expression levels of Nanog and Tbx3 were increased significantly by knockdown of either Brm or Baf170 in ESC. Finally, we showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed cells (pre-iPSC). These data suggest that inhibiting somatic BAF complex improved complete reprogramming by facilitating the activation of the pluripotency circuitry. A reduced activity of the somatic BAF complex constitutes part of the Stat3 regulated epigenetic changes during pluripotency establishment.
35 EFFECT OF 6-DIMETHYLAMINOPURINE TREATMENT DURATION ON PRONUCLEAR FORMATION AND IN VIVO DEVELOPMENT OF CANINE CLONED EMBRYO
Authors: Oh H. J. , Kim G. A. , Kim M. J. , Jo Y. K. , Choi Y. B. , Setyawan E. M. N. , Lee S. H. , Kim H. J. , Lee B. C.
Published: December 2014

Artificial activation is an important step for successful somatic cell nuclear transfer (SCNT). In order to clone animals, diverse methods of activation have been studied to increase the developmental efficiency of cloned embryos. Here, we investigated the pronucleus formation and in vivo development of canine cloned embryos produced by different durations of 1.9 mM 6-dimethylaminopurine (DMAP) treatment. For canine SCNT, in vivo-matured oocytes were enucleated, microinjected into the perivitelline space with donor cells, and fused by electrical stimulation. For activation, the fused couplets were cultured for 4 min in 10 μM calcium ionophore, and then they were divided into 2 groups: (1) the 2DMAP group was cultured for 2 h in DMAP; (2) the 4DMAP group was cultured for 4 h in DMAP. Activated cloned embryos were subjected to 2 analyses: (1) observing the pronuclear formation by bromodeoxyuridine (BrdU) incorporation at 2 h, 4 h and 8 h post-activation (hpa), and (2) following fetus formation and pregnancy efficiency after embryo transfer into naturally synchronous recipients. Pregnancy diagnosis was performed by ultrasonography on Day 26 of embryo transfer. Data were analysed using Graph Prism software (GraphPad Software Inc., San Diego, CA, USA). All cloned embryos of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP embryos showed 77.7% BrdU incorporation at 2 hpa (P < 0.05). Incorporation of BrdU was detected in all cloned embryos of both experimental groups after 4 hpa and 8 hpa. A total of 370 cloned embryos were transferred to 24 surrogate mothers (182 cloned embryos into 12 recipients in 2DMAP group and 188 cloned embryos into 12 recipients in 4DMAP group). There was no significant difference in pregnancy rate (2DMAP; 41.6% v. 4DMAP; 33.3%) or implantation rates (2DMAP; 4.9% v. 4DMAP; 3.7%) between the 2 groups. In conclusion, DMAP exposure for 2 h in activation completed pronucleus development of canine reconstructed embryos. However, none of the applied tested treatments resulted in increased implantation rates.
This study was supported by RDA (#PJ008975022014), IPET (#311062–04–3SB010), Research Institute for Veterinary Science, Nestlé Purina PetCare and the BK21 plus program.
36 EFFECT OF SUBEROYLANILIDE HYDROXAMIC ACID TREATED DONOR CELLS ON DOG CLONING
Authors: Kim M. J. , Oh H. J. , Kim G. A. , Suh H. N. , Jo Y. K. , Choi Y. B. , Kim D. H. , Han H. J. , Lee B. C.
Published: December 2014

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACi), on dog cloning efficiency. Relative mRNA expression of the bax1, bcl2, and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 μM) of SAHA and durations (0, 20, 44 h) were assessed using real-time polymerase chain reaction. After determining an optimum concentration and duration, histone acetylation levels (H3K9, H4K5/K8/K12/K16) of SAHA-treated cells were analysed using immunostaining. The SAHA-treated cells were used as donor cells for somatic cell nuclear transfer, and activated reconstructed embryos were transferred to recipients. Pregnancy diagnosis was performed by ultrasonography at least 29 days after the embryo transfer. All experiments were repeated more than 3 times and the data were analysed using Graph Prism software (GraphPad Software Inc., San Diego, CA, USA). An unpaired t-test was used to compare transcripts levels and fluorescence intensities. A chi-squared test was used to compare the implantation rates. The bax1/bcl2 ratio of the 1 μM SAHA group was similar to that of control but significantly increased in the 10 μM and 50 μM groups. Expression of Dnmt1 was decreased in the 1 μM SAHA group, and the 10 μM and 50 μM groups showed the lowest expression compared with the control group. Although the bax1/blc2 ratio was not affected by the SAHA treatment duration, 20-h treatment group showed significantly decreased Dnmt1 levels compared with control group. As a pan-HDAC inhibitor, 1 μM for 20 h of SAHA treatment significantly increased acetylation of H3K9, H4K5, H4K8, and H4K16. For control and SAHA groups, a total of 76 and 64 cloned embryos were produced and transferred to 7 and 5 recipients, respectively. Three fetuses were diagnosed in both groups but there was no significant difference in the pregnancy rate. In conclusion, although SAHA treatment as used in this study significantly decreased bax/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 and H4 acetylation, it would not enough to increase in vivo developmental competence of cloned dog embryos.This study was supported by RDA (#PJ008975022014), IPET (#311062–04–3SB010), Research Institute for Veterinary Science and the BK21 plus program.
361 GROWTH HORMONE RECEPTOR MUTANT PIGS PRODUCED BY USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR) AND CRISPR-ASSOCIATED SYSTEMS IN IN VITRO-PRODUCED ZYGOTES
Authors: Kurome M. , Dahlhoff M. , Bultmann S. , Krebs S. , Blum H. , Kessler B. , Leonhardt H. , Wolf E.
Published: December 2014

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology is considered as an efficient strategy for generating gene edited large animals, such as pigs. Compared to somatic cell nuclear transfer, this new technology offers a relatively simple way to generate mutant pigs by direct injection of RNA into the cytoplasm of zygotes. Moreover, the use of in vitro produced zygotes would provide a highly effective and practical method for the production of porcine disease models for biomedical research. Here we examined the production efficiency of growth hormone receptor (GHR) mutant pigs by the combination of the CRISPR/Cas system and in vitro produced zygotes. In vitro maturation (IVM) of oocytes was performed as described previously (Kurome et al., Meth. Mol. Biol., in press). In all experiments, the same batch of frozen sperm was used. After IVM, around 20 oocytes with expanded cumulus cells were incubated with 5 × 104 spermatozoa in a 100-μL drop of porcine fertilization medium for 7 h. In vitro-produced embryos were assessed by the ratio of normal fertilization (eggs with 2 pronuclei) and blastocyst formation at Day 7. The Cas9 mRNA and a single guide RNA, recognising a short sequence of 20 base pairs in exon 3 of the GHR gene, were injected directly into the cytoplasm of the embryos 8.5 to 9.5 h after IVF. Injected embryos were transferred laparoscopically to recipient pigs, and 86.4% (57/66) of sperm-penetrated oocytes (66/96) exhibited normal fertilization. Incidence of polyspermy was relatively low (9/66, 13.6%). Developmental ability of in vitro-produced embryos to the blastocyst stage was 17.4% (24/138). In total, 426 RNA-injected embryos were transferred into 2 recipients, one of which became pregnant and gave birth to 8 piglets. All piglets were clinically healthy and developed normally. In 3 out of 8 piglets (37.5%), mutations were introduced. Next-generation sequencing revealed that all of them were mosaics: one with a single mutation (22% wild-type/78% mutant) and 2 piglets with 2 different mutations (80% wild-type/2% mutant_1/18% mutant_2 and 94% wild-type/4% mutant_1/2% mutant_2). Four out of 5 mutations caused a frameshift in the GHR gene. Our study reports for the first time generation of GHR mutant pigs by the use of the CRISPR/Cas system in in vitro-produced zygotes. Because all GHR mutant offspring were mosaic, Cas9 activation probably occurred after the 1-cell stage under our experimental conditions. The founder animal with the highest proportion of mutant GHR alleles will be used for breeding to establish a large animal model for Laron syndrome.This work is supported by the German Research Council (TR-CRC 127).
40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES
Authors: Do L. T. K. , Sato Y. , Taniguchi M. , Otoi T.
Published: December 2014

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.
56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY
Authors: Toishibekov Y. M. , Tursunova R. K. , Yermekova M. Sh.
Published: December 2014

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.
58 EFFECT OF MELATONIN IMPLANT ON BLANCA de RASQUERA BUCKS DURING SPRING ON SPERM MORPHOMETRY BEFORE AND AFTER CRYOPRESERVATION
Authors: Tabarez A. , García W. , Palomo M. J.
Published: December 2014

In order to improve sperm cryopreservation throughout the year and accelerate the process of preservation of this Catalonian goat breed in extinction danger, we proposed to assess the effect of melatonin implant application in Blanca de Rasquera males during spring on sperm head morphometry of fresh and thawed sperm. Therefore 8 bucks of 30 months old approximately were divided into 2 groups. In one of the groups, 2 melatonin implants (Melovine®, CEVA) were inserted into bucks 60 days before starting the collection of semen, and the other group was kept untreated. Briefly, fresh ejaculates from each group of 4 bucks were collected in spring, immediately mixed in equal quantities, and centrifuged twice (600 g for 10 min). Then the pellet was resuspended in a Tris-based medium containing 15% (v/v) of powdered egg yolk supplemented with 5% glycerol. Afterward, sperm samples were refrigerated at 5°C for 4 h before being frozen in LN vapour. Buck sperm head morphometry was analysed by computer-assisted sperm analysis (ISAS®) on fresh and thawed sperm previously stained with Diff Quick®. Data were analysed by GLM multivariate procedure (IBM SPSS, 2011; mean ± s.e., n = 6), showing significant differences among treatments in all the morphometric parameters except head perimeter and rugosity (Table 1). Our results suggest that melatonin application in bucks increases the ellipticity and elongation of fresh and thawed sperm, meanwhile the cryopreservation process reduces both parameters. Likewise melatonin implants increase significantly the head length only on thawed sperms as cryopreservation process increases the head width, area in sperms from implanted males and regularity only in sperms from nonimplanted bucks. These head changes on fresh and thawed sperm morphometry should be deeply investigated in order to know how they could affect sperm cryosurvival and fertility.

Table 1.Effect of melatonin implant on Blanca de Rasquera bucks during spring on morphometry of fresh and thawed sperm

This research was supported by INIA (RZ2009–00008–00–00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundación Carolina.
60 PREDICTING FROZEN BOAR SPERM FERTILITY USING NOVEL AND CONVENTIONAL ANALYSES
Authors: Daigneault B. W. , McNamara K. A. , Purdy P. H. , Rodriguez-Zas S. L. , Krisher R. L. , Knox R. V. , Miller D. J.
Published: December 2014

Cryopreserved boar sperm is seldom used for AI because fertility is reduced. Despite many potential advantages of frozen-thawed sperm for AI, lack of reliable fertility estimation of frozen ejaculates before AI limits the application of frozen sperm. Conventional post-thaw evaluation of sperm does not accurately estimate fertility. Identifying sperm traits that predict fertility would help select ejaculates that produce adequate litter sizes. Our objective was to identify traits of cryopreserved sperm that are related to boar fertility for AI through the use of novel and traditional laboratory analyses. Semen from 14 boars of several breeds was cooled to 15°C for shipping before freezing. Post-thaw motility was evaluated using a microscope and confirmed with computer-automated sperm analysis. Sperm viability and acrosome integrity were measured at 0, 30, and 60 min post-thaw. In addition to traditional analyses, each sperm sample was tested by IVF to record fertilization, cleavage, and blastocyst development. A sperm-oviduct binding assay was used to compare the number of sperm bound to epithelial aggregates harvested from the isthmus. Additionally, a competitive zona binding assay using 2 distinct fluorophores for boar identification was used to count the number of sperm from each boar bound to the zona. Frozen sperm from the same ejaculates subjected to laboratory analyses were used to determine actual boar fertility. Fertility was measured by AI of mature gilts using 4.0 × 109 total sperm from one boar at 24 h and a second boar at 36 h after the onset of oestrus, and AI order was reversed in consecutive replicates. Fertility was expressed as the percentage of the litter sired by each boar. Reproductive tracts were harvested at 32 days after AI, and fetal paternity was identified using microsatellite markers. The actual boar fertility was regressed against the mean of each laboratory evaluation by boar, and the assays that best predicted fertility were identified using stepwise logistic regression. The model generated was highly predictive of fertility (P < 0.001, r2 = 0.87) and included 5 traits: acrosome compromised sperm (0 and 30 min), percent live sperm (0 min), percent total motility (30 min), and the number of zona bound sperm. An additional model in which fertility was assessed by the number of piglets sired by boar also predicted fertility (P < 0.05, r2 = 0.57) and shared many of the same traits. These models were highly accurate when used to predict actual fertility of cryopreserved boar sperm. This approach may be used to screen ejaculates before AI and advance the use of frozen boar sperm by the swine industry.Research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2010-85112-20620 from the USDA National Institute of Food and Agriculture.
69 REACTIVE OXYGEN SPECIES EVALUATION OF DONKEY FROZEN SEMEN ADDED TO HOMOLOGOUS SEMINAL PLASMA ON POST-THAW
Authors: Oliveira P. V. L. , Oliveira J. V. , Ramires Neto C. , Sancler-Silva Y. F. R. , Freitas-Dell'aqua C. P. , Papa F. O.
Published: December 2014

For many years the pregnancy rate of donkey frozen semen presented lower results in donkey jennies; however, a recent study showed an increase in pregnancy rates using frozen semen added to seminal plasma on post-thaw. A hypothesis for this result is the higher uterine inflammation response after breeding when using seminal plasma. The same studies demonstrated higher uterine inflammation in the presence of higher reactive oxygen species concentration. The aim of the present study was to evaluate the content of reactive oxygen species in donkey frozen semen added to homologous seminal plasma on post-thaw. Five ejaculates from each 3 donkeys were used. Semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The semen was frozen with Botu-CryoTM extender (Botupharma, Brazil) in an isothermal box in straws containing 100 × 106 of total sperm. The samples were thawed at 46°C for 20 s. After this, the straws of each donkey were divided in 2 group: control group (CG), in which the semen was incubated at 37°C for 5 min, and plasma seminal group (PG), in which the semen was incubated at 37°C for 5 min with 70% of homologous seminal plasma. Sperm kinetic parameters were evaluated by computer-assisted semen analysis, and the plasma membrane integrity (propidium iodide and fluorescein isothiocyanate -PSA) and reactive oxygen species (5–6-carboxi-2,7-diclorodihidrofluoresceindiacetate) were evaluated by flow cytometer. Comparison of sperm parameters was performed by t-test. Total motility (%, CG = 75.4 ± 8.2a v. PG = 57.5 ± 16.4b), progressive motility (%, CG = 42.0 ± 8.7a v. PG = 33.3 ± 13.2b), progressive angular velocity (μm/s, CG = 95.8 ± 10.8a v. PG = 88.9 ± 10.9b), and percentage of rapid sperm (%, CG = 58.4 ± 12.5a v. PG = 41.0 ± 17.3b) were higher in CG compare with PG. No difference (P < 0.05) was observed in membrane integrity (%, CG = 20.7 ± 7.4 v. PG = 20.6 ± 7.8); however, reactive oxygen species (%, CG = 12.3 ± 10.6a v. PG = 81.8 ± 32.5b) were higher in PG. The results of this study showed that the addition of homologous seminal plasma on post-thaw decreases the sperm kinetic parameters and viability of donkey frozen semen but increases reactive oxygen species, and this may cause higher uterine inflammation response in donkey jennies and increase their fertility.
80 DNA METHYLATION OF INSULIN-LIKE GROWTH FACTOR 2 (IGF2) GENE IN DAY 14 IN VITRO-PRODUCED BOVINE EMBRYOS OF DIFFERENT SIZES
Authors: Carvalho J. O. , Franco M. M. , Machado G. M. , Dode M. A. N.
Published: December 2014

In mammals, a correct DNA methylation reprogramming and the maintenance of genomic imprinting after fertilization are essential for embryo development and pregnancy. One important imprinted gene, related to embryo development and placentation, is the insulin-like growth factor 2 (IGF2) gene. Therefore, embryos with different sizes could show differences in the methylation pattern of IGF2 gene. The aim of this study was to evaluate the methylation pattern of the differentially methylated region (DMR) located within exon 10 of the IGF2 gene, of in vitro-produced Nellore bovine embryos that were different in size on day D14 of development. The embryos were produced from oocytes obtained by follicular aspiration of slaughter house ovaries. On D7 after in vitro fertilization only grade I blastocysts were selected and, in groups of 10 embryos, were transferred non-surgically to the uteri of previously synchronized recipients with similar conditions. Seven days after being transferred, embryos were collected (Day 14 of development) and measured using Motic Images Plus 2.0 program (Motic, Richmond, BC, Canada). Embryos >45 mm were considered large (L) and those <25 mm were considered small (S). After being measured, a portion of each trophoblast layer was biopsied and used to determine the methylation status of the IGF2 gene by bisulfite sequencing. The methylation pattern was evaluated on individual embryos considered as separate replicates. At least 5 to 8 clones were evaluated per embryo and the sequences were analysed with the BiQAnalyser software (Max-Planck-Institut für Informatik, Saarbrücken, Germany), using the GenBank sequence NM_174087.3 as reference. The methylation pattern of the different groups was compared using Kruskal-Wallis test (P < 0.05). No differences in DNA methylation were found between S (26.7 ± 8.3%, n = 37 clones, 5 embryos) and L (34.8 ± 2.9%, n = 20 clones, 4 embryos) embryos. It is already known that the region studied is hypermethylated in sperm and hypomethylated in oocytes and, in some somatic cell types, it is expected to be around 50% methylated, being an imprinted region. Although we found a lower percentage of methylation than that expected for an imprinted region, this pattern may be the physiological pattern for trophoblast cells. This is the first report describing the methylation pattern of this region of the IGF2 gene in Day 14 bovine embryos of different sizes. It can be concluded that the methylation pattern of the intragenic DMR on exon 10 of IGF2 gene of in vitro-produced embryos on Day 14 of development is not affected by embryo size.This work was supported by CNPq, FAP-DF.
Intrinsically organized resting state networks in the human spinal cord
Authors: Kong Yazhuo , Eippert Falk , Beckmann Christian F. , Andersson Jesper , Finsterbusch Jürgen , Büchel Christian , Tracey Irene , Brooks Jonathan C. W.
Published: December 2014

Significance

The human brain displays an enormous amount of intrinsic activity in the absence of any task or external stimulation. Here we demonstrate that the human spinal cord, the brain’s principal interface with the body, also shows such resting-state activity. We observed biologically plausible and spatially distinct networks that reflect the functional organisation of the spinal cord: networks in the anterior part likely relating to motor function and distinct networks in the posterior part likely reflecting sensory function. These networks were grouped along the spinal cord, consistent with motor output to, and sensory input from, the body. Together with previous brain imaging studies, our data suggest that resting-state activity constitutes a major functional signature of the entire central nervous system.
Effect of distracting faces on visual selective attention in the monkey
Authors: Landman Rogier , Sharma Jitendra , Sur Mriganka , Desimone Robert
Published: December 2014

Significance

Primates express a natural interest in faces. The viewing of faces with an emotional expression affects emotion circuits in the brain, even when they are not directly attended. This has led to a debate about whether faces attract attention automatically. We tested the influence of emotional faces as irrelevant distracters in an attention task in monkeys. Task performance was most affected when facial expression was threatening, especially when presented for durations longer than 200 ms. We conclude that, in monkeys, emotional distractors do attract attention away from other tasks but not instantly. Administration of the hormone oxytocin reduced the effect. Among the brain systems likely involved are areas where oxytocin receptors are abundant.
Well-posedness for the fifth-order KdV equation in the energy space
Authors: Kenig Carlos , Pilod Didier
Published: December 2014

We prove that the initial value problem (IVP) associated to the fifth-order KdV equation
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Food Insecurity and Food Access in U.S. Metropolitan Areas
Authors: Bonanno Alessandro , Li Jing
Published: December 2014

Household food insecurity in the United States has reached its highest levels to date. As public and private initiatives have emerged to help improve diets by fostering access to food, the availability of more food stores may result in lower levels of food insecurity. In this article, we assess the relationship between adult food insecurity and food store density in metropolitan areas of the United States. We find that while small grocery/convenience stores show a mitigating effect on adult food insecurity across different samples of households, the effects of large supermarkets/grocery stores and supercenters vary. We also find that Supplemental Nutrition Assistance Program participation and food access can have a simultaneously beneficial effect in reducing adult food insecurity. Implications for policies aiming to improve food security by fostering access to food stores are discussed.
The chemical component dictionary: complete descriptions of constituent molecules in experimentally determined 3D macromolecules in the Protein Data Bank
Authors: Westbrook John D. , Shao Chenghua , Feng Zukang , Zhuravleva Marina , Velankar Sameer , Young Jasmine
Published: December 2014

Summary: The Chemical Component Dictionary (CCD) is a chemical reference data resource that describes all residue and small molecule components found in Protein Data Bank (PDB) entries. The CCD contains detailed chemical descriptions for standard and modified amino acids/nucleotides, small molecule ligands and solvent molecules. Each chemical definition includes descriptions of chemical properties such as stereochemical assignments, chemical descriptors, systematic chemical names and idealized coordinates. The content, preparation, validation and distribution of this CCD chemical reference dataset are described.

Availability and implementation: The CCD is updated regularly in conjunction with the scheduled weekly release of new PDB structure data. The CCD and amino acid variant reference datasets are hosted in the public PDB ftp repository at ftp://ftp.wwpdb.org/pub/pdb/data/monomers/components.cif.gz, ftp://ftp.wwpdb.org/pub/pdb/data/monomers/aa-variants-v1.cif.gz, and its mirror sites, and can be accessed from http://wwpdb.org.

Contact:  jwest@rcsb.rutgers.edu.

Supplementary information:  Supplementary data are available at Bioinformatics online.
A rationale for reduced-frequency dosing of anidulafungin for antifungal prophylaxis in immunocompromised patients
Authors: Brüggemann R. J. M. , Van Der Velden W. J. F. M. , Knibbe C. A. J. , Colbers A. , Hol S. , Burger D. M. , Donnelly J. P. , Blijlevens N. M. A.
Published: December 2014

Objectives

Reduced-frequency dosing strategies of anidulafungin may offer a more convenient way of providing adequate antifungal prophylaxis to patients at high risk of invasive fungal diseases. We aimed to provide the pharmacological rationale for the applicability of reduced-frequency dosing regimens.

Methods

We defined two groups of 10 patients that were to receive anidulafungin at 200 mg every 48 h or 300 mg every 72 h. Blood samples were drawn daily and two pharmacokinetic curves were constructed after 1 and 2 weeks of treatment. A population pharmacokinetic model was developed using non-linear mixed-effects modelling. ClinicalTrials.gov identifier: NCT01249820.

Results

The AUC over a 6 day period (IQR) for a typical patient on 200 mg every 48 h or 300 mg every 72 h resulted in 348 mg · h/L (310.6–386.7) and 359 mg · h/L (319.1–400.9), respectively, comparable to the licensed regimen [397.0 mg · h/L (352.4–440.5)]. In the final model, the volume of distribution proved to be dependent on the lean body mass and CL of cyclosporine A. All three regimens resulted in comparable dose-normalized exposure over time.

Conclusions

We now have sufficient evidence to start using less frequent dosing regimens and demonstrate their value in clinical practice. These less frequently applied infusions enable more personalized care in an outpatient setting with reduced costs.
Vibration mode structure and simplified modelling of cyclically symmetric or rotationally periodic systems
Authors: Shi Chengzhi , Parker Robert G.
Published: December 2014

This paper proves the modal vibration properties of general rotating, cyclically symmetric (or rotationally periodic) systems, including those with central components to which cyclically symmetric substructures are attached. This cyclic symmetry results in structured modal properties with only two possible mode types referred to as substructure and coupled modes. For systems with uncoupled central component translations and rotations, which is the usual case, all eigenvectors fall into one of three categories: substructure, translational and rotational modes. The properties of the system equations of motion resulting from the cyclic symmetry are discussed first. These properties are then used to prove the modal decomposition of general rotating, cyclically symmetric systems. The development leads to modelling and computational efficiencies. The vibration modes and natural frequencies for each mode type are determined from reduced eigenvalue problems that are much smaller than the full system eigenvalue problem. The full system matrices, although not needed for the current purposes, can be generated from much smaller matrices derived from simple subsystem models.
Large-amplitude internal waves benefit corals during thermal stress
Authors: Wall M. , Putchim L. , Schmidt G. M. , Jantzen C. , Khokiattiwong S. , Richter C.
Published: December 2014

Tropical scleractinian corals are particularly vulnerable to global warming as elevated sea surface temperatures (SSTs) disrupt the delicate balance between the coral host and their algal endosymbionts, leading to symbiont expulsion, mass bleaching and mortality. While satellite sensing of SST has proved a reliable predictor of coral bleaching at the regional scale, there are large deviations in bleaching severity and mortality on the local scale that are poorly understood. Here, we show that internal waves play a major role in explaining local coral bleaching and mortality patterns in the Andaman Sea. Despite a severe region-wide SST anomaly in May 2010, frequent upslope intrusions of cold sub-pycnocline waters due to breaking large-amplitude internal waves (LAIW) mitigated coral bleaching and mortality in shallow waters. In LAIW-sheltered waters, by contrast, bleaching-susceptible species suffered severe bleaching and total mortality. These findings suggest that LAIW benefit coral reefs during thermal stress and provide local refugia for bleaching-susceptible corals. LAIW are ubiquitous in tropical stratified waters and their swash zones may thus be important conservation areas for the maintenance of coral diversity in a warming climate. Taking LAIW into account can significantly improve coral bleaching predictions and provide a valuable tool for coral reef conservation and management.
Cheaper is not always worse: strongly protective isolates of a defensive symbiont are less costly to the aphid host
Authors: Cayetano Luis , Rothacher Lukas , Simon Jean-Christophe , Vorburger Christoph
Published: December 2014

Defences against parasites are typically associated with costs to the host that contribute to the maintenance of variation in resistance. This also applies to the defence provided by the facultative bacterial endosymbiont
Hamiltonella defensa,
which protects its aphid hosts against parasitoid wasps while imposing life-history costs. To investigate the cost–benefit relationship within protected hosts, we introduced multiple isolates of
H. defensa
to the same genetic backgrounds of black bean aphids,
Aphis fabae
, and we quantified the protection against their parasitoid
Lysiphlebus fabarum
as well as the costs to the host (reduced lifespan and reproduction) in the absence of parasitoids. Surprisingly, we observed the opposite of a trade-off. Strongly protective isolates of
H. defensa
reduced lifespan and lifetime reproduction of unparasitized aphids to a lesser extent than weakly protective isolates. This finding has important implications for the evolution of defensive symbiosis and highlights the need for a better understanding of how strain variation in protective symbionts is maintained.
Does coping style predict optimization? An experimental test in a wild passerine bird
Authors: Nicolaus Marion , Mathot Kimberley J. , Araya-Ajoy Yimen G. , Mutzel Ariane , Wijmenga Jan J. , Kempenaers Bart , Dingemanse Niels J.
Published: December 2014

A number of studies have suggested that avian brood size is individually optimized. Yet, optimal reproductive decisions likely vary owing to among-individual differences in environmental sensitivity. Specifically, ‘proactive’ individuals who do not track environmental changes may be less able to produce optimal brood sizes than ‘reactive’ individuals who have more precise local environmental knowledge. To test this, we quantified exploratory behaviour (a proxy for proactivity) in a great tit (
Parus major
) population, manipulated brood sizes (reduced, control, enlarged) and evaluated whether individuals of dissimilar coping style differed in their level of optimization. If reactive females behaved optimally, any deviation from their original brood size should lower fitness, whereas this should not be the case for proactive females. Reactive females indeed performed best at their natural brood size, whereas proactive females performed best when raising an enlarged brood. These findings imply that proactive females produced sub-optimal brood sizes. We speculate that proactive females might (i) take decisions based on biased perception of their environment, (ii) face energetic constraints in offspring production and/or (iii) be more willing to invest into current reproduction when given the option. Our findings provide experimental evidence for coping style-related differences in optimal reproductive decisions and life-history strategies.
A mechanistic model of the S-shaped population growth
Authors: Kalmykov Lev V.
Published: December 2014

The main idea of this note is to show the most basic and purely mechanistic model of population growth, which has been used by us to create models of interspecific competition for verification of the competitive exclusion principle (1,2). Our logical deterministic individual-based cellular automata model demonstrates a spatio-temporal mechanism of the S-shaped population growth.
Extra‐linguistic influences on sentence comprehension in Italian‐speaking children with and without specific language impairment
Authors: Pettenati P. , Benassi E. , Deevy P. , Leonard L. B. , Caselli M. C.
Published: December 2014

Background

Many children with specific language impairment (SLI) in sentence comprehension. These deficits are usually attributed to limitations in the children's understanding of syntax or the lexical items contained in the sentences. This study examines the role that extra‐linguistic factors can play in these children's sentence comprehension.

Aims

Extra‐linguistic demands on sentence comprehension are manipulated directly by varying the nature of the materials used.

Methods & Procedures

Forty‐five Italian‐speaking children participated: 15 with SLI (mean age = 4;5), 15 typically developing children matched for age (TD‐A, mean age = 4;5), and 15 younger typically developing children matched according to language comprehension test scores (TD‐Y, mean age = 3;9). The children responded to sentence comprehension items that varied in their length and/or the number and type of foils that competed with the target picture.

Outcomes & Results

The TD‐A children were more accurate than the TD‐Y children and the children with SLI, but, for all groups, accuracy declined when task demands increased. In particular, sentences containing superfluous adjectives (e.g., Il topo bello copre l'uccello allegro, ‘The nice mouse covers the happy bird’ where all depicted mice were nice and all birds were happy) yielded higher scores than similar sentences in which each adjective had to be associated with the proper character (e.g., Il cane giallo lava il maiale bianco, ‘The yellow dog washes the white pig’, where foils included a yellow dog washing a pink pig, and a brown dog washing a white pig). Many errors reflected recency effects, probably influenced by the fact that adjectives modifying the object appear at the end of the sentence in Italian.

Conclusions & Implications

Differences between conditions were observed even when lexical content, syntactic structure and sentence length were controlled. This finding suggests the need for great care when assessing children's comprehension of sentences. The same syntactic structure and lexical content can vary in difficulty depending on the number and types of foils used in combination with the target picture.
Viscosity of dysphagia‐oriented cold‐thickened beverages: effect of setting time at refrigeration temperature
Authors: Kim Sung‐Gun , Yoo Byoungseung
Published: December 2014

Background

Although extensive literature is available on the viscosity of thickened beverages with food thickeners, no attempt has been made to study the effect of setting time on the viscosity of pudding‐like cold‐thickened beverages with xanthan gum (XG)‐based thickeners by using a rheometer. In particular, it is of considerable practical importance to investigate the effect of setting time on their viscosity at 5°C because some cold‐thickened beverages will be prepared in the kitchen in bulk and stored at 5°C before serving or consuming rather than serving immediately upon mixing with thickeners.

Aims

To examine the effect of different setting times (15–120 min) on the viscosity of cold‐thickened beverages prepared with various XG‐based food thickeners, and also to compare the viscosity differences among the various cold beverages and XG‐based food thickeners in beverage‐thickener mixture systems.

Methods & Procedures

Four commercially available XG‐based food thickeners (A–D) and three cold beverages (water, orange juice and milk) were used for the preparation of cold‐thickened beverages. The thickened sample was portioned into six samples for the designated setting times and then stored at 5°C over setting time. Their apparent viscosity (ηa,50) at 50 s−1 was measured using a rheometer.

Outcomes & Results

The largest increases in ηa,50 values for thickened beverages, except for water, were observed at 15 min (p < 0.05), showing a pudding‐like fluid, and at longer time periods their ηa,50 values gradually increased or were constant with an increase in setting time. The percentage increase in viscosity values at different setting times (15–120 min) as compared with the control (0 min) was less pronounced in the thickened orange juice and milk samples with thickener A over setting time, indicating that the thickened beverages with thickener A had more stable structure compared with those with other thickeners (B–D) over time. Statistical analysis showed that changes in the viscosity of cold‐thickened beverages over setting time are greatly influenced by the type of beverages and thickeners.

Conclusions & Implications

Cold‐thickened beverages should be carefully prepared with instant commercial XG‐based food thickeners because they produced different thickening patterns over setting time which clinicians must consider for a safe and easy swallowing. The information presented in this study will provide both clinicians and patients with additional knowledge to prepare cold‐thickened beverages with the corrected viscosity for safe swallowing.
DNA barcoding of Rhododendron (Ericaceae), the largest Chinese plant genus in biodiversity hotspots of the Himalaya–Hengduan Mountains
Authors: Yan Li‐Jun , Liu Jie , Möller Michael , Zhang Lin , Zhang Xue‐Mei , Li De‐Zhu , Gao Lian‐Ming
Published: December 2014

The Himalaya–Hengduan Mountains encompass two global biodiversity hotspots with high levels of biodiversity and endemism. This area is one of the diversification centres of the genus Rhododendron, which is recognized as one of the most taxonomically challenging plant taxa due to recent adaptive radiations and rampant hybridization. In this study, four DNA barcodes were evaluated on 531 samples representing 173 species of seven sections of four subgenera in Rhododendron, with a high sampling density from the Himalaya–Hengduan Mountains employing three analytical methods. The varied approaches (nj, pwg and blast) had different species identification powers with blast performing best. With the pwg analysis, the discrimination rates for single barcodes varied from 12.21% to 25.19% with ITS < rbcL < matK < psbA‐trnH. Combinations of ITS + psbA‐trnH + matK and the four barcodes showed the highest discrimination ability (both 41.98%) among all possible combinations. As a single barcode, psbA‐trnH performed best with a relatively high performance (25.19%). Overall, the three‐marker combination of ITS + psbA‐trnH + matK was found to be the best DNA barcode for identifying Rhododendron species. The relatively low discriminative efficiency of DNA barcoding in this genus (~42%) may possibly be attributable to too low sequence divergences as a result of a long generation time of Rhododendron and complex speciation patterns involving recent radiations and hybridizations. Taking the morphology, distribution range and habitat of the species into account, DNA barcoding provided additional information for species identification and delivered a preliminary assessment of biodiversity for the large genus Rhododendron in the biodiversity hotspots of the Himalaya–Hengduan Mountains.