bluuebunny/crossref_metadata_2025 huggingface dataset:
173 EFFECTS OF GLUCOSE METABOLISM DURING IN VITRO MATURATION ON CYTOPLASMIC MATURATION OF GOAT OOCYTES
Authors: Tan J.-H. , Wang Y.-B. , Xie H.-L. , Li Q. , Liu X.-Y. , Zhou P. , Luo M.-J.
Published: December 2013

It is well known that oocyte maturation consists of 2 processes: nuclear maturation and cytoplasmic maturation. Nuclear maturation refers to resumption of the first meiosis and extrusion of the first polar body (PB1), and cytoplasmic maturation is manifested as acquisition of the ability to complete pre-implantation development. Although it is recognised that energy supply is essential for oocyte maturation and there have been many reports on the effect of glucose metabolism on oocyte nuclear maturation, studies on the effect of glucose metabolism on ooplasmic maturation are limited. In the present study, goat oocytes recovered from slaughterhouse ovaries were cultured for 24 h in a simplified CR1 (sCR1) medium (NaCl, KCl, NaHCO3, CaCl2, BSA, and eCG) supplemented with glucose (10 mM) and/or lactate (3.5 mM) in the presence or absence of pentose phosphate pathway (PPP) inhibitor dehydroepiandrosterone (DHEA, 100 μM) or glycolysis inhibitor iodoacetate (1 μM). At the end of maturation culture, oocytes with PB1 were either activated by treatment with ionomycin plus 6-DMAP to observe embryo development, or assayed for total glutathione concentrations (GSX) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios. Embryos were cultured for 9 days in CR1aa medium (NaCl, KCl, NaHCO3, calcium lactate, sodium pyruvate, glutamine, EAA, NEAA, and FCS) at 38.5°C under 5% CO2 in humidified air. In the absence of inhibitors, oocyte maturation rates of 82, 65, and 76%, and blastocyst rates of 7, 0, and 7%, were obtained, respectively, after oocytes were matured in sCR1 supplemented with glucose, lactate, or both. When oocytes were matured in sCR1 containing glucose and lactate in the presence of DHEA or iodoacetate, oocyte maturation rates were 69 and 67%, respectively, with no blastocyst produced in either case. However, whereas the presence of DHEA produced 12% morulae, no morulae were observed in the presence of iodoacetate. Furthermore, GSX concentrations (pmol/oocyte) were 8.5, 6.5, and 7.2, whereas GSH/GSSG ratios were 1.8, 0.3, and 0.5, respectively, after oocyte maturation without inhibitors or with 300 μM DHEA or 3 μM iodoacetate. The difference in GSX concentration was statistically significant (P < 0.05; one-way ANOVA) between DHEA and iodoacetate. In conclusion, using a culture system (sCR1 containing 3.5 mM lactate) that sustained oocyte nuclear maturation but did not support blastocyst development, we have studied the effect of PPP and glycolysis of glucose metabolism on the cytoplasmic maturation of goat oocytes. The results suggest that both PPP and glycolysis are essential for ooplasmic maturation of goat oocytes, and that both promote oocyte cytoplasmic maturation by increasing glutathione synthesis and reduction.
This study was supported by grants from the National Basic Research Program of China (Nos. 2012CB944403 and 2014CB138503) and the China National Natural Science Foundation (Nos. 31272444 and 30972096).
200 TELOMERASE ACTIVITY MODIFICATION WITH RESVERATROL IN CANINE ADIPOSE-DERIVED MESENCHYMAL STEM CELLS
Authors: Kim G. A. , Oh H. J. , Kim S. Y. , Shin Y. R. , Lee D. K. , Kang S. K. , Lee B. C.
Published: December 2013

It has been established that the telomerase activity of donor cells is causally linked to the reprogramming efficiency, which includes somatic cell nuclear transfer (SCNT). Among the many cell source as donor cells of SCNT, canine adipose-derived stem cells (Ad-MSCs), a form of adult stem cells, are donor cells that have been recently used. Resveratrol (trans-3,5,4′-trihydroxystilbene), a polyphenolphytoalexin, possesses diverse biochemical and physiological action, including antiplatelet and reducing cellular senescence via telomerase activity modification. However, appropriate dose and time of resveratrol for telomerase activity modification in Ad-MSCs have not been investigated. In addition, unlike most normal somatic cells which have negative telomerase activities; low to moderate levels of the enzyme in mesenchymal stem cells have been described. In the present study, we compared the cellular telomerase activity of Ad-MSCs according to the dose of resveratrol and passages of Ad-MSCs. Cells were isolated from collected adipose derived tissues of beagle at age 7 and cultured in RCME-P provided from RNL Bio incorporation. For identification of telomerase activity in ad-MSCs, adult fibroblasts derived from same dog were used as negative control. After 2 days of cultivation, Ad-MSCs were treated with 2 μM, 10 μM, or 25 μM resveratrol or without resveratrol at 39°C for 24 h in a humidified atmosphere of 5% CO2 in air. Ad-MSCs with passage at 1, 4, and 7 were used for analysis. Telomerase activity was measured by the telomeric repeat amplification protocol assay. Statistical analysis was performed by the two-way ANOVA. P < 0.05 was considered as showing a statistically significant difference between means. It revealed that Ad-MSCs have telomerase activity significantly higher than those shown in fibroblasts (negative control). Resveratrol increased telomerase activity, with maximal increase at 10, 25 μM at passage of 1, 4 (P < 0.05). However, increase of telomerase activity in Ad-MSCs treated with 10, 25 μM at passage 7 was not shown and the telomerase activities of Ad-MSCs at passage 7 were lower than those of Ad-MSCs at passage 1 and 4 (P < 0.05). In conclusion, the telomerase activity was detectable in canine Ad-MSCs, which may suggest that canine Ad-MSCs have similar telomere biology compared to that of other adult stem cells. Furthermore, resveratrol can enhance activation of telomerase activity with dose dependent increase. Further studies are warranted on efficiency of establishing a stable donor cells for SCNT using Ad-MSCs treated with resveratrol.
This study was supported by R&E (#550–20130027), IPET (#311062–04–2-SB010), RNL Bio (#550–20130013), RDA (PJ008975022013), the Research Institute for Veterinary Science, and TS Corporation.
208 SUPEROVULATORY RESPONSE AND EMBRYO PRODUCTION IN HOLSTEIN COWS FED DIETS ENRICHED IN OLEIC, LINOLEIC, OR α-LINOLENIC ACID
Authors: Salehi R. , Colazo M. G. , Behrouzi A. , Ruiz-Sanchez A. , Oba M. , Dyck M. K. , Ambrose D. J.
Published: December 2013

Early embryonic development is enhanced in Holstein cows fed diets enriched in linoleic and α-linolenic acids compared to those fed a diet enriched in stearic fatty acid (Thangavelu et al. 2007; Theriogenology 68, 949–957). A positive effect of oleic acid on oocyte competence in vitro has been reported (Aardema et al. 2011; Biol. Reprod. 85, 62–69), but the influence of oleic acid on early embryonic development has not been investigated. Therefore, our objective was to evaluate the effects of diets enriched in oleic, linoleic, or α-linolenic acid on superovulatory response and embryo production. Non-lactating Holstein cows were blocked by parity and bodyweight, and assigned to one of three diets supplemented with canola (CAN, n = 8; 62% oleic acid), sunflower (SUN, n = 7; 73% linoleic acid), or flax (FLX, n = 8; 57% α-linolenic acid). Cows were individually offered hay (9.4 kg) and concentrate mixture (4.1 kg) supplemented with one of the three rolled oilseeds (~8% of dietary dry matter). After a minimum of 21 days of diet adaptation, cows were given FSH (300 mg total dose, eight 12-hourly injections), artificially inseminated twice, 12 h apart, with semen of the same sire and ova/embryos recovered non-surgically 7.5 days post-insemination. Once started on a diet, cows continued to receive the same diet until the end of the experiment. Thirty-five collections (flushing) were performed (CAN: 12, SUN: 11 and FLX: 12) and data were analysed using the GENMOD procedure of SAS (SAS Institute Inc., Cary, NC, USA); results are presented as mean ± s.e. Number of corpora lutea (12.8 ± 2.6, 14.5 ± 1.2 and 13.1 ± 1.5), anovulated follicles (3.0 ± 1.4, 2.0 ± 1.1 and 2.6 ± 0.9), and total ova/embryos (7.5 ± 2.0, 8.6 ± 1.7 and 7.3 ± 1.2) did not differ (P > 0.05) for CAN, SUN, and FLX treatment, respectively. Cows fed SUN tended (P = 0.06) to produce more transferable embryos (6.1 ± 1.7) than those fed CAN (4.3 ± 1.5), but did not differ from those fed FLX (4.8 ± 1.3). There were fewer (P < 0.05) unfertilized ova in cows fed SUN (0.6 ± 0.5) than in those fed either CAN (1.3 ± 0.6) or FLX (1.8 ± 0.9). Conversely, cows fed FLX had fewer (P < 0.05) degenerated embryos (0.7 ± 0.4) compared to those fed either CAN (1.9 ± 0.6) or SUN (1.9 ± 0.5). The number of expanded blastocysts was greater (P < 0.05) in cows fed CAN (1.08 ± 0.57) than in those fed FLX (0.25 ± 0.25), but it was not different from those fed SUN (0.90 ± 0.36). In summary, the type of dietary fatty acid did not affect superovulatory response, but a diet enriched in oleic acid resulted in more expanded blastocysts, whereas a diet enriched in linoleic acid significantly reduced the number of unfertilized ova and numerically increased transferable embryos. The diet enriched in α-linolenic acid resulted in fewer degenerated embryos.
This study was supported by Alberta Meat and Livestock Agency, Alberta Milk, and Alberta Innovates-BioSolutions.
29 GENERATION OF TRANSGENIC FIBROBLASTS EXPRESSING PANCREAS-SPECIFIC AND DOX-INDUCIBLE ICER Iγ IN A MINIATURE PIG MODEL OF HUMAN DIABETES MELLITUS
Authors: Kang H. Y. , Jung E. M. , Jeung E. B.
Published: December 2013

Inducible cyclic AMP (cAMP) early repressor (ICER) Iγ, consisting of a DNA binding domain, plays a role as a competitor to the cAMP responsive element-binding protein (CREB), and disrupts the transcriptional regulation of CREB responsive genes. Overexpression of the ICER Iγ gene in a rodent model has been reported to cause impairment of insulin production, reduction of pancreatic β cell number, and induction of glucagon synthesis in the same way in human diabetes mellitus. As mentioned above, overexpression of ICER Iγ causes severe diabetes. Therefore, we focused our attention on an ICER Iγ overexpression vector containing a tissue-specific and an adjustable promoter. In our previous study, ICER Iγ-overexpression controlled by the pancreas-specific human insulin promoter region induced significant decline of glucose-mediated insulin transcription in the mouse pancreatic β cell line, MIN6. Using this promoter region, we established a doxycycline-inducible ICER Iγ expression system via the tet-controlled transactivator (TA) with TA response element (TRE). We developed the unitary tet-on system, which combined an activator cassette under the control of the human insulin promoter with a responder cassette containing ICER Iγ and the green fluorescence protein, which are controlled by the TRE promoter. To determine whether enhanced ICER Iγ by doxycycline influences insulin expression, we first introduced the tet-on ICER Iγ-vector into MIN6 cells, and then treated them with 0.1 to 1 mg mL–1 doxycyline. We observed a robust increase in ICER Iγ expression and a decrease in insulin expression. This in vitro system could be useful for study of human diabetes mellitus and pre-diabetes using tissue-specific promoter and tet-on inducible transgene. In addition, we generated porcine transgenic fibroblasts containing doxycycline-inducible ICER Iγ cDNA.
30 A TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE (TALEN)-MEDIATED UNIVERSAL GENE KNOCK-IN STRATEGY FOR MAMMARY GLANDS-SPECIFIC EXPRESSION OF RECOMBINANT PROTEINS IN DAIRY CATTLE
Authors: Lee S. , Park H. , Kong I. , Wang Z.
Published: December 2013

To harness the great capability of producing biologically active recombinant proteins with animal mammary glands, active research has been carried out in the past several decades to develop transgenic animals as bioreactors. However, when a transgene is introduced in the animal genome by random integration, the transgene tends to be subjected to epigenetic silencing, due to the so-called position effect from the chromatin environments surrounding the transgene integration sites, thereby resulting in low-level expression or total suppression. We report a universal transgenic strategy to knock in (KI) transgenes into the bovine β-casein gene locus allowing the expression of a transgene to be totally under the control of the endogenous regulatory sequences of the bovine β-casein gene. This universal KI strategy comprises two key components: one is the design of transcription activator-like effector nuclease (TALEN) constructs targeting the start codon region of bovine β-casein gene, and the other is the design of KI vectors in which a transgene of choice is flanked with homologous arms isolated from the ~500-bp bovine genomic DNAs immediately 5′ and 3′, respectively, of the translation start codon of the bovine β-casein gene. By using the human erythropoietin (hEPO) as the model transgene, we demonstrated that a transgene can be highly efficiently integrated immediately after the translation start codon of the bovine β-casein gene. In brief, the TALEN constructs were assembled by using the Golden Gate protocol. To KI the hEPO transgene, early passage (<5) of fibroblasts established from Holstein dairy cattle were cultured into full confluence in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), harvested with 0.25% trypsin-EDTA, and co-transfected with KI vector and the TALEN constructs by the Amaxa Nucleofector system. For each experiment, 106 cells were transfected with 5 μg of KI vector and 5 μg of TALEN constructs. After 72 h post-transfection, cells were harvested and subjected to limiting dilution to obtain single-cell derived colonies. To screen for single-cell derived colonies carrying the correctly KI of hEPO in the β-casein locus, we performed genomic PCR amplifying the genomic junctions created by the KI of hEPO gene into the bovine genome. We identified and established 2 hEPO transgenic bovine fibroblast cell lines after screening 10 single-cell derived colonies from the transfected cells (20%). The genotype of these 2 colonies was also confirmed by sequencing the PCR products. We have initiated the effort to produce hEPO transgenic cattle by somatic cell nuclear transfer (SCNT), and the animal cloning results will be reported at the conference.
37 RESTORING REPROGRAMMING ABNORMALITIES IN A CLONED DOG HAVING ECTOPIC LIVER AND GALL BLADDER BY RECLONING
Authors: Kim M. J. , Oh H. J. , Kim G. A. , Jo Y. K. , Choi J. , Lee B. C.
Published: December 2013

The risk of reprogramming abnormalities such as placental hyperdevelopment, excessive fetal growth, or abnormalities of the immune system in cloned neonates is one of the major concerns in cloning research. However, until now, relatively few studies about birth defects have been reported in dog cloning, which might be due to the short in vitro manipulated procedure in this species. Here, we report a cloned dog having abnormal liver and investigated whether the abnormal liver was due to genetic modification. A cloned beagle was produced from a fibroblast derived from a 10-year-old donor, but accidentally died due to cannibalism of a nanny dog on the day of birth. During autopsy, an abnormal liver structure was found; 7 lobes were presented at the normal liver position inside the abdomen, but there was no gall bladder. Interestingly, 3 additional lobes with a gall bladder were found in between the rib and the skin. There were no other macroscopic anomalies observed in this puppy. To evaluate the heredity of this liver abnormality, the liver structure of the donor dog was diagnosed by computed tomography (CT). Also, to assess the possibility of restoring the liver abnormality, recloning was performed using a fibroblast cell line established from the dead pup, and liver positions in the recloned dogs were diagnosed by CT after puberty. In results, 2 recipients delivered 5 recloned dogs with birthweights of 510, 250, 460, 400, and 410 g. The smallest one showed severe bow-legged phenotype in its hind leg, and a unique coat pattern that showed the largest white coat surface. The pup died 10 days after birth, and no other abnormal phenotype was found during autopsy. The other 4 pups showed normal morphology at birth. The CT results showed normal positioning of the liver and gall bladder in all experimental dogs, including the original cell donor dog and recloned dogs. To our knowledge, there have been no reported cases of an ectopic liver and gall bladder present between rib and skin in both cloned and noncloned animals, and we consider that these abnormalities are not a due to genetic cause. Further studies regarding aberrant epigenetic reprogramming in the abnormal liver formation are needed.
50 DEVELOPMENT OF A MODIFIED STRAW LOADING METHOD FOR VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS
Authors: Ha A.-N. , Park H.-S. , Lee K.-L. , Kim Y.-G. , Song S.-H. , Park P.-R. , Kim T. , White K. L. , Kong I.-K.
Published: December 2013

This study evaluated a modified plastic straw method for vitrification of in vitro-produced (IVP) bovine blastocysts. A modified straw was used that has a depressed area on its inner surface to which embryos attach. The IVP blastocysts were randomly assigned into 3 groups: (1) attachment of embryos to the inner surface of a plastic straw (aV), (2) attachment of embryos to the inner surface of a modified plastic straw (maV), and (3) non-vitrified (control). The recovery rates of blastocysts were not significantly different between the aV and maV groups (95.8 v. 94.3%; P > 0.05). The survival of post-thaw blastocysts did not significantly differ between the aV and maV groups (86.4 v. 88.2%; P > 0.05). The total cell number of blastocysts was significantly higher in the control group than in the aV and maV groups (142 ± 21.8 v. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not different between the aV and maV groups. The mRNA levels of the pro-apoptosis-related genes Bax and caspase-3 were significantly higher in the aV and maV groups than in the control group. By contrast, the mRNA levels of Bcl-2 and Mcl-1, which are anti-apoptotic genes, and of MnSOD and Prdx5, which are antioxidant-related genes, were significantly lower in the aV and maV groups than in the control group (P < 0.05). In conclusion, the aV and maV methods can be used to vitrify IVP bovine blastocysts, and embryos are more easily loaded using the maV method than using the aV method.
This work was partly supported by the Next-Generation BioGreen 21 Program (grant no. PJ009587022013), IPET (grant no. 110020-5 and 112020-3), and a scholarship from the BK21 Program, Republic of Korea.
60 EFFECT OF CAKE COLLAPSE ON THE INTEGRITY OF FREEZE-DRIED BULL SPERMATOZOA
Authors: Hara H. , Tagiri M. , Hirabayashi M. , Hochi S.
Published: December 2013

During freezing, a solution changes into an amorphous phase at the glass transition temperature of the maximally freeze-concentrated phase (T′g). The solution exhibits a cake-like porous structure under the optimal freeze-drying process. However, if the product temperature is higher than theT′g during the drying phase, the glassy material will undergo viscous flow, resulting in loss of the porous structure. This is defined as the collapse phenomenon and may be related to instability of the freeze-dried products. The purpose of the present study was to investigate the effect of cake collapse on freeze-dried bull spermatozoa. One-way ANOVA was used for comparison of T′g, DNA damage, and blastocyst yield. When the ANOVA was significant, differences among means were analysed by a Tukey test. In Experiment 1, factors affecting the T′g were investigated. Using differential scanning calorimetry, theT′g of an EGTA buffer (10 mM TRIS-HCl, 50 mM EGTA, and 50 mM NaCl, pH8.0) that has been conventionally used for sperm freeze-drying was determined to be –45.0°C. Modification of the EGTA buffer composition by complete removal of NaCl and addition of 0.5 M trehalose (referred to hereafter as mEGTA buffer) resulted in an increase in theT′g up to –27.7°C. The T′g of the mEGTA buffer cooled by direct immersing into liquid nitrogen (–29.4°C) was slightly lower (P < 0.05) than that cooled slowly at 20 and 1°C min–1 (–27.6 and –27.2°C, respectively). In Experiment 2, the integrity of freeze-dried and rehydrated bull spermatozoa was investigated. Spermatozoa from a Japanese Black bull were suspended into mEGTA buffer (3 × 107 cells mL–1), cooled at 20°C min–1, and then processed for drying for 6 h at 0, –15, and –30°C (ALPHA2-4; Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Cakes were collapsed when the sperm suspension was dehydrated either at 0 or –15°C. In vitro-matured bovine oocytes were injected with rehydrated sperm, chemically activated (5 μM ionomycin, 7% ethanol, and 2 mM 6-DMAP), and then cultured for 8 days. Blastocyst yields after injection of sperm dried at 0 and –15°C, calculated from cleaved oocytes, were significantly lower than that of sperm dried at –30°C (0.7–3.7% v. 14.2%; P < 0.05). The level of DNA damage, assessed by the alkaline comet assay, was not different between the sperm populations dried at 0 and –30°C. Transmission electron microscopic observation revealed that the sperm membrane dried at 0°C was more damaged compared with that dried at –30°C (P < 0.05; chi-squared test with Bonferroni correction). In conclusion, incidence of collapse in freeze-dried cake may be a detrimental factor for maintenance of sperm integrity after freeze-drying, and can be inhibited by controlling the T′g of the buffer and drying phase temperature.
H. Hara is Research Fellow of the Japan Society for the Promotion of Science (JSPS). This work was supported by a grant-in-aid for basic research from JSPS (no. 24580407) to S. Hochi.
76 RELATIONSHIP BETWEEN BLASTOCYST CELL NUMBER AT EMBRYO TRANSFER AND CONCEPTUS ELONGATION ON DAY 14: CAN SUPPLEMENTARY PROGESTERONE RESCUE SMALL EMBRYOS?
Authors: O'Hara L. , Forde N. , Kelly A. K. , Lonergan P.
Published: December 2013

Conceptus size on Day 14 following multiple embryo transfer of Day 7 in vitro-produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development following the transfer of Day 7 blastocysts containing a low total cell number. The oestrous cycles of crossbred beef heifers were synchronised using an 8-day CIDR (1.38 g of P4) treatment with administration of a prostaglandin F2α analogue (Estrumate™, Merck Animal Health, Summit, NJ, USA) on the day before CIDR removal. Only those heifers recorded in standing oestrus (Day 0) were used. Heifers were randomly assigned to 1 of 4 treatment groups: (1) Control: large blastocysts (high total cell number), (2) Control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU of hCG (Chorulon®, Merck Animal Health) on Day 2 after oestrus, or (4) small blastocysts plus insertion of a vaginal progesterone insert (PRID, 1.55 g of P4) between Days 3 to 5 after oestrus. In vitro-produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer) and conceptuses were recovered at slaughter on Day 14. A sample of blastocysts was fixed and stained using Hoechst 33342 to determine the mean total cell number per group. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute Inc. Cary, NC, USA). Total cell number on Day 7 was significantly lower in small compared with large blastocysts (72.4 ± 3.93 v. 144.8 ± 3.90; P < 0.05). Conceptus recovery rate was 53.8% overall (140/260) and was highest in the large blastocyst group (68.3%, 41/60) compared with the other groups (45.7–55.0%). Concentrations of serum P4 were similar in the 2 unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm), whereas insertion of a PRID from Day 3 to 5 increased conceptus length ~5-fold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.
Supported by Science Foundation Ireland (07/SRC/B1156).
87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS
Authors: Demyda-Peyrás S. , Hidalgo M. , Dorado J. , Moreno-Millan M.
Published: December 2013

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.
The early Cold War and the Western welfare state
Authors: Petersen Klaus
Published: December 2013

This paper analyses possible links between the early Cold War and the Western welfare state. These have so far attracted far too little interest among both welfare state and Cold War scholars. The paper argues that the nature of the relationship between the Cold War and the welfare state is too complex to be formulated into a general theory and proposes a research strategy focussing on different mechanisms of interactions. It identifies and discusses four such mechanisms: The possible trade-offs between military and social expenses, the use of the Cold War in political and ideological debates on social reforms, social reforms as an anti-communist strategy, and finally how the Cold War influenced transnational social policy learning.
Vespucci: a system for building annotated databases of nascent transcripts
Authors: Allison Karmel A. , Kaikkonen Minna U. , Gaasterland Terry , Glass Christopher K.
Published: December 2013

Global run-on sequencing (GRO-seq) is a recent addition to the series of high-throughput sequencing methods that enables new insights into transcriptional dynamics within a cell. However, GRO-sequencing presents new algorithmic challenges, as existing analysis platforms for ChIP-seq and RNA-seq do not address the unique problem of identifying transcriptional units de novo from short reads located all across the genome. Here, we present a novel algorithm for de novo transcript identification from GRO-sequencing data, along with a system that determines transcript regions, stores them in a relational database and associates them with known reference annotations. We use this method to analyze GRO-sequencing data from primary mouse macrophages and derive novel quantitative insights into the extent and characteristics of non-coding transcription in mammalian cells. In doing so, we demonstrate that Vespucci expands existing annotations for mRNAs and lincRNAs by defining the primary transcript beyond the polyadenylation site. In addition, Vespucci generates assemblies for un-annotated non-coding RNAs such as those transcribed from enhancer-like elements. Vespucci thereby provides a robust system for defining, storing and analyzing diverse classes of primary RNA transcripts that are of increasing biological interest.
Detection and Quantification ofXanthomonas albilineansby qPCR and Potential Characterization of Sugarcane Resistance to Leaf Scald
Authors: Garces F. F. , Gutierrez A. , Hoy J. W.
Published: December 2013

Leaf scald is an important disease of sugarcane with erratic symptom expression. Latency represents a threat to germplasm exchange, and erratic symptom development makes accurate evaluation of disease resistance during breeding and selection problematic. Real-time quantitative polymerase chain reaction (qPCR) assays for Xanthomonas albilineans, the causal agent of leaf scald, were developed and evaluated for the sensitive, specific detection and quantification of the pathogen. Assays with SYBR Green primers and TaqMan probe and primers derived from the albicidin toxin biosynthesis gene cluster efficiently and reproducibly amplified X. albilineans. Detection was more sensitive with qPCR compared with conventional PCR. Assays were specific for X. albilineans and sap extracts did not inhibit the qPCR reaction. Leaf-scald-resistant and -susceptible cultivars were distinguished by infection incidence, disease severity, and X. albilineans population determined by SYBR Green qPCR in both greenhouse and field experiments. Populations of X. albilineans varied in different tissues. Differences were the greatest within tissues in resistant cultivars, and bacterial populations in systemically infected, young, not yet fully emerged leaves exhibited the greatest differences between resistant and susceptible cultivars. The results demonstrate that qPCR is a highly sensitive method for the detection of X. albilineans that could provide a reliable method for leaf scald resistance screening.
First Report of Podosphaera xanthii Race 1W Causing Powdery Mildew of Watermelon in California
Authors: Mercier J. , Muscara M. J. , Davis A. R.
Published: December 2013

In September and October 2012, powdery mildew was detected on watermelon (Citrullus lanatus var. lanatus) plants of various breeding lines growing in field plots in Davis, California. Plants had partially necrotic leaves, yellowing to brown in color, with white surface mycelium and faint sporulation. No teleomorph was observed. Infected leaves were collected for examination and a spore suspension of the field isolate was made in water with 0.01% Tween 20 to spray inoculate watermelon seedlings of cultivar Dixie Lee with two true leaves. Plants were incubated in a growth chamber (22 to 26°C, 12-h photoperiod) for approximately 10 days, until sporulation was apparent. Microscopic observation of conidial chains showed that they had clearly crenate edges indicative of Podosphaera xanthii (4). To confirm the identity of the pathogen, we used Podosphaera-specific primers PFITS-F (5′-CCAACTCGTGCTGAGTGT-3′) and PF5.8-R (5′-TGTTGGTTTCTTTTCCTCCG-3′) to amplify and sequence the internal transcribed spacer regions of the nuclear rDNA. The 326-bp sequence had 98% homology to the GenBank sequence (accessions JQ340082.1 and AB774158.1) for P. xanthii. Infected ‘Dixie Lee’ leaves were used to make a spore suspension (approximately 5 × 104 conidia/ml) as described above to inoculate watermelon, melon, and squash seedlings (2 to 3 plants per cultivar) in a greenhouse. It caused severe symptoms on all watermelon plants cv. Charleston 76, P8, and Sugar Baby in the form of a powdery mildew with surface mycelium and chains of conidia, with leaves becoming gradually more necrotic and eventually dying, with the appearance of a melting down. Non-inoculated plants did not develop symptoms. The isolate also infected all squash plants ‘Zucchini Elite’ and melon powdery mildew differentials Iran H and ‘Védrantais.’ On these plants, the pathogen produced a powdery mildew (white surface mycelium with sporulation) but did not cause extensive necrosis. All other melon powdery mildew differentials (‘PMR5,’ ‘PMR45,’ WMR29, MR1, PI 124112, and PI 313970) did not develop any powdery mildew. A follow-up test in a growth chamber (22 to 26°C, 12-h photoperiod) with the same set of species and cultivars gave the same results. Based on these results, we conclude that this isolate belongs to race 1W (1,2). The presence of race 1W could have implications in disease management for this crop in the Central Valley of California as most cultivars are not resistant to it and the disease has been shown to cause severe damage in other states (1,3).

References: (1) A. R. Davis et al. J. Am. Soc. Hort. Sci. 132:790, 2007. (2) J. D. McCreight. Amer. Soc. Hort. Sci. 131:59, 2006. (3) A. Y. Tetteh et al. Crop Sci. 50:933, 2010. (4) T. A. Zitter. Page 28 in: Compendium of Cucurbit Diseases, The American Phytopathological Society, St. Paul, MN, 1996.
Bee pollination improves crop quality, shelf life and commercial value
Authors: Klatt Björn K. , Holzschuh Andrea , Westphal Catrin , Clough Yann , Smit Inga , Pawelzik Elke , Tscharntke Teja
Published: December 2013

Pollination improves the yield of most crop species and contributes to one-third of global crop production, but comprehensive benefits including crop quality are still unknown. Hence, pollination is underestimated by international policies, which is particularly alarming in times of agricultural intensification and diminishing pollination services. In this study, exclusion experiments with strawberries showed bee pollination to improve fruit quality, quantity and market value compared with wind and self-pollination. Bee-pollinated fruits were heavier, had less malformations and reached higher commercial grades. They had increased redness and reduced sugar–acid–ratios and were firmer, thus improving the commercially important shelf life. Longer shelf life reduced fruit loss by at least 11%. This is accounting for 0.32 billion US$ of the 1.44 billion US$ provided by bee pollination to the total value of 2.90 billion US$ made with strawberry selling in the European Union 2009. The fruit quality and yield effects are driven by the pollination-mediated production of hormonal growth regulators, which occur in several pollination-dependent crops. Thus, our comprehensive findings should be transferable to a wide range of crops and demonstrate bee pollination to be a hitherto underestimated but vital and economically important determinant of fruit quality.
Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin
Authors: Mallinjoud Pierre , Villemin Jean-Philippe , Mortada Hussein , Polay Espinoza Micaela , Desmet François-Olivier , Samaan Samaan , Chautard Emilie , Tranchevent Léon-Charles , Auboeuf Didier
Published: December 2013

Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type–specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution.
Regulatory modules controlling maize inflorescence architecture
Authors: Eveland Andrea L. , Goldshmidt Alexander , Pautler Michael , Morohashi Kengo , Liseron-Monfils Christophe , Lewis Michael W. , Kumari Sunita , Hiraga Susumu , Yang Fang , Unger-Wallace Erica , Olson Andrew , Hake Sarah , Vollbrecht Erik , Grotewold Erich , Ware Doreen , Jackson David
Published: December 2013

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.
Morphological changes of the hair roots in alopecia areata: A scanning electron microscopic study
Authors: Karashima Tadashi , Tsuruta Daisuke , Hamada Takahiro , Ishii Norito , Ono Fumitake , Ueda Akihiro , Abe Toshifumi , Nakama Takekuni , Dainichi Teruki , Hashimoto Takashi
Published: December 2013

Alopecia areata is a chronic inflammatory condition causing non‐scarring patchy hair loss. Diagnosis of alopecia areata is made by clinical observations, hair pluck test and dermoscopic signs. However, because differentiation from other alopecia diseases is occasionally difficult, an invasive diagnostic method using a punch biopsy is performed. In this study, to develop a reliable, less invasive diagnostic method for alopecia areata, we performed scanning electron microscopy of the hair roots of alopecia areata patients. This study identified four patterns of hair morphology specific to alopecia areata: (I) long tapering structure with no accumulation of scales; (II) club‐shaped hair root with fine scales; (III) proximal accumulation of scales; and (IV) sharp tapering of the proximal end of hair. On the basis of these results, we can distinguish alopecia areata by scanning electron microscopic observation of the proximal end of the hair shafts.
Sociologies of Moderation
Authors: Smith Alexander Thomas T. , Holmwood John
Published: December 2013

What does moderation mean in the twenty-first century? And what might a reasoned project of moderation look like, intellectually, politically and in practice? This paper argues for a sociological reappraisal of the historical origins, intellectual foundations and contemporary salience of moderation. Too often wrongly defined by a sense of what it is not – by its apparent absence of ideology – moderation has come, in recent years, to be associated with ‘bland’ and incoherent notions of centrism. However, moderation presents sociologists and other scholars with particular analytical and theoretical challenges and opportunities. In a post-secular liberal democracy like Britain, an exploration of what moderation might mean demands an interrogation of the often taken-for-granted assumptions of the social sciences and the ideological extremes more generally. Moderation is about the relations among publics and the possibilities of a deep pluralism that is respectful of difference. However, publics are under threat by markets and the future of sociology, tied to the future of publics, without revivified social and political practices of moderation, may be bleak.
Functional magnetic resonance imaging (fMRI) response to alcohol pictures predicts subsequent transition to heavy drinking in college students
Authors: Dager Alecia D. , Anderson Beth M. , Rosen Rivkah , Khadka Sabin , Sawyer Broderick , Jiantonio‐Kelly Rachel E. , Austad Carol S. , Raskin Sarah A. , Tennen Howard , Wood Rebecca M. , Fallahi Carolyn R. , Pearlson Godfrey D.
Published: December 2013

Background and Aims

Young adults show the highest rates of escalating drinking, yet the neural risk mechanisms remain unclear. Heavy drinkers show variant functional magnetic resonance imaging (fMRI) blood oxygen level‐dependent (BOLD) response to alcohol cues, which may presage increasing drinking. In this longitudinal study, we ascertained whether BOLD response to alcohol pictures predicted subsequent heavy drinking among college students.

Methods

Participants were 43 18–21‐year‐olds in the United States who underwent BOLD scanning and completed monthly substance use surveys over the following year. Participants were categorized according to baseline and follow‐up drinking into 13 continuously moderate drinkers, 16 continuously heavy drinkers and 14 transitioners who drank moderately at baseline but heavily by follow‐up. During fMRI scanning at baseline, participants viewed alcohol and matched non‐alcohol beverage images.

Results

We observed group differences in alcohol cue‐elicited BOLD response in bilateral caudate, orbitofrontal cortex, medial frontal cortex/anterior cingulate and left insula (clusters > 2619 ml, voxelwise F(2,40) > 3.23, P < 0.05, whole‐brain corrected P < 0.05), where transitioners hyperactivated compared with moderate and heavy drinkers (all Tukey P < 0.05). Exploratory factor analysis revealed a single brain network differentiating those who subsequently increased drinking. Exploratory regressions showed that, compared with other risk factors (e.g. alcoholism family history, impulsivity), BOLD response best predicted escalating drinking amount and alcohol‐related problems.

Conclusions

Neural response to pictures of alcohol is substantially enhanced among United States college students who subsequently escalate drinking. Greater cue‐reactivity is associated with larger increases in drinking and alcohol‐related problems, regardless of other baseline factors. Thus, neural cue‐reactivity could uniquely facilitate identifying individuals at greatest risk for future problematic drinking.
Hepatic transcriptome profiling identifies differences in expression of genes associated with changes in metabolism and postnatal growth between Hereford and Holstein‐Friesian bulls
Authors: Lisowski Pawel , Kościuczuk Ewa M. , Gościk Joanna , Pierzchała Mariusz , Rowińska Barbara , Zwierzchowski Lech
Published: December 2013

This study examined liver transcriptomic profiles of cattle distinctly different in meat and milk production capacity. It was performed on bulls of two different genetic backgrounds: Herefords (H), a meat breed, and Holstein‐Friesians (HF), a dairy breed. Using bovine long oligo‐microarrays and qPCR, we identified 128 genes that are differentially expressed between the two breeds. In H bulls, we observed up‐regulation of genes involved in fatty acid biosynthesis and lipid metabolism (CD36, CAT, HSD3B1, FABP1, ACAA1) and involved in insulin signaling (INSR, INSIG2, NR4A1) and down‐regulation of genes involved in somatotropic axis signaling (IGF1, GHR, IGFBP3) as compared to HF. Transcriptome profiling of these two breeds allowed us to pinpoint the transcriptional differences between Holstein and Hereford bulls at hepatic level associated with changes in metabolism and postnatal growth.
Ethical and legal aspects of anaesthesia for the elderly
Authors: White S. M.
Published: December 2013

The elderly have the ethical and legal equivalence of younger adults, yet are treated differently by society. Numerous recent reports have exposed poor inpatient care resulting in part from institutional ageism, which has moral and legal implications for healthcare providers. Morally, there is an argument for positive ‘exceptionalism’ in elderly peri‐operative care, pursuing quality improvement through use of a dignity agenda. Legally, numerous changes in human rights, equality, consent, capacity, and end‐of‐life laws and professional guidance have consistently re‐emphasised the need for greater communication between doctors, patients, their relatives and carers. This review describes current ethical thinking and legal precedent (in England and Wales), and directs readers to consider areas in which the law might change in the near future, particularly with regard to the end‐of‐life care of elderly surgical patients.
Academic Self‐Efficacy and Performance of Underrepresented STEM Majors: Gender, Ethnic, and Social Class Patterns
Authors: MacPhee David , Farro Samantha , Canetto Silvia Sara
Published: December 2013

This longitudinal study examined academic self‐efficacy and performance among Science/Technology/Engineering/Math (STEM) majors who are underrepresented in STEM education and occupations; i.e., women, specific ethnic minorities, and low‐socioeconomic status (SES) individuals. Performance on academic tests and self‐perceptions of academic skills were assessed at admission and graduation from a STEM mentoring program. At admission, women perceived themselves as academically weaker than men despite similar academic performance. However, by graduation, women's academic self‐efficacy was equivalent to men's. In addition, students with double STEM‐minority statuses, by ethnicity and SES, had lower academic self‐efficacy and performance than d id students with single STEM‐minority status. Exploratory analyses of change over time by ethnic/SES groups showed varying patterns of change that depended on the outcome variable. This study's finding of an increase in academic self‐efficacy for women and students with STEM‐minority status by both ethnicity and SES at graduation from a mentoring program is perhaps an indication of the positive impact of mentoring. The mixed findings at program completion for students with single versus double STEM‐minority status call for attention to the complex relationship between social disadvantage, academic self‐efficacy, and academic performance.