bluuebunny/crossref_metadata_2025 huggingface dataset:
134 Generation of NANOS2 knockout goats using CRISPR/Cas9 and somatic cell nuclear transfer techniques
Authors: Fan Z. , Regouski M. , Liu Y. , Keim J. , Perisse I. , Oatley J. , Polejaeva I.
Published: December 2019

The NANOS2 gene, encoding an RNA binding protein, is known to play a critical role in the development of germline for all organisms studied to date. The male mice with biallelic NANOS2 knockouts (KOs) are sterile due to apoptosis of prospermatogonia shortly after birth but with morphologically intact seminiferous tubules. Thus, the choice of NANOS2 for targeting could be a viable strategy to develop germline ablated males that would serve as recipients for exogenous spermatogonial stem cell transplantation. The goat is a potential model of human physiology and an agriculturally important species. Here, we report successful generation of NANOS2 KO goats using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. We first designed 4 single-guide RNAs (sgRNAs) specific for the single exon of goat NANOS2 (GenBank: NC_030825.1). The targeting vectors were constructed by using the pX330 plasmid (Addgene: 42230) and transfected into sheep fetal fibroblasts. Mutation efficiency analysis showed that 3 of them (out of 4, 75.0%) were efficient in directing Cas9 to generate targeted cleavages, with mutation efficiencies of 10-30%. We established single cell-derived fetal fibroblast colonies by limiting dilution of the cells transfected with one of targeting vectors (sgRNA: GCTGGAGACCCAAGGGACTG). Colony screening with PCR/restriction fragment length polymorphism (RFLP) assays confirmed that we achieved biallelic mutations in the targeting site in 6 of 89 (6.7%) male and 6 of 172 (3.5%) female colonies. Sanger sequencing analysis of genomic DNA isolated from cell colonies with biallelic mutations showed that typical nucleotide deletions and insertions (indels), caused by repairing double-strand DNA breaks during the error-prone non-homologous end joining (NHEJ) process, were generated at the targeting site of NANOS2. Three male and two female colonies with NANOS2 null mutations were identified and used as cell donors for SCNT. In total, 202 cloned 1-cell stage embryos (130 male or 72 female) were generated and surgically transferred into 12 synchronized recipients. Six of them (6 of 12, 50.0%) were confirmed pregnant by ultrasonography on Day 40-45 of gestation. Four pregnancies developed to term, resulting in six offspring (five males and one female). Sequence analysis and PCR/RFLP assays showed that both male and female offspring carried the mutations in NANOS2, which were identical to the donor colonies from which they originated. Our results indicated that CRISPR/Cas9 combined with SCNT is an efficient system for generating NANOS2 KO goats. The phenotypic analysis to assess the effects of NANOS2 KO on the development of germline in male cloned goats is in progress.
184 Effects of cyanidin supplementation on invitro maturation of pig oocytes
Authors: Hicks E. , Mentler M. , Whitaker B. D.
Published: December 2019

Oxidative stress can have a negative effect on oocyte maturation during invitro production of pig embryos. Imbalance of reactive oxygen species and antioxidant levels can affect the progression of oocyte maturation up to the point of fertilization. Antioxidants are effective in maintaining more ideal reactive oxygen species levels, which help to protect oocytes from potential harmful effects of oxidative stress. Berries from the elder plant (Sambucus sp.) contain high levels of a broad spectrum of antioxidants. One of these antioxidants, cyanidin, when supplemented to maturation medium at 100μM concentrations, reduces reactive oxygen species formation and improves IVF and early embryonic development in pigs. However, changes in the enzyme mechanisms of action during oocyte maturation due to cyanidin supplementation are unknown. Therefore, the objective of this study was to characterise the intracellular oocyte enzyme mechanisms between oocytes supplemented with 100μM cyanidin during 40 to 44h of maturation (n=600) and oocytes without supplementation of cyanidin during maturation (n=558). At the end of maturation, oocytes were evaluated for either glutathione peroxidase (n=300), catalase (n=564), or superoxide dismutase (n=294) activities. Glutathione peroxidase activity was determined by following the rate of NADPH oxidation, catalase activity was determined by following the rate of hydrogen peroxide decomposition, and superoxide dismutase activity was determined by following the reduction rate of cytochrome c, utilising the xanthine-xanthine oxidase system. Data were analysed using ANOVA and Tukey's test. There were no significant differences between oocytes matured with 100μM cyanidin and those that were not when comparing glutathione peroxidase and superoxide dismutase activities. Supplementation of 100μM cyanidin to maturation medium increased (P<0.05) catalase activity in oocytes (0.78±0.15 units/oocyte) compared with no cyanidin supplementation (0.14±0.11 units/oocyte). These results indicate that supplementing 100μM cyanidin to the maturation medium of pig oocytes could reduce the negative effects of oxidative stress by increasing intracellular catalase activity during oocyte maturation.
192 The effect of invitro maturation on the PI3K-Akt pathway in bovine cumulus cells
Authors: Andrade G. , Del Collado M. , Nociti R. , Da Silva W. J. , Meirelles F. , Da Silveira J. , Perecin F.
Published: December 2019

Oocyte quality is influenced by invitro oocyte maturation (IVM) because the culture conditions can alter the metabolism and gene expression of cumulus cells. Proper oocyte development requires fine regulation of signalling pathways involved with cell proliferation and survival, such as the PI3K-Akt (phosphatidylinositol-3-kinase/protein kinase B) signalling pathway. The aim of this study was to determine the effect of IVM on the expression of PI3K-Akt-related genes in bovine cumulus cells. To this aim, cumulus cells associated with immature oocytes, associated with oocytes invitro-matured for 24h, or associated with oocytes invivo-matured were compared in terms of gene expression. Pools (n=4) of cumulus cells from 20 cumulus-oocyte complexes (COCs) per group were submitted to total RNA extraction using the TRizol protocol, libraries were prepared with TruSeq stranded mRNA sample prep kit (Illumina Inc.), and the sequencing was performed in the HisEqn 2500 V4 (Illumina Inc.). After quality check with FastQC and filtering with Trim Galore, read alignment was performed with STAR and analysis of differential gene expression was done using DESEqn 2 in R considering the Benjamini-Hochberg method for adjusted P-values<0.10, and absolute value of log2-fold change >0.5. Principal component analysis was able to separate, with 94% cumulative variance (81% and 13% for PC1 and PC2, respectively), the cumulus cells groups, especially the immature from the matured counterparts. Gene ontology and enrichment analysis showed that the PI3K-Akt signalling pathway was affected in immature cumulus cells compared with cumulus cells from invitro- or invivo-matured oocytes, with 77 and 88 genes from PI3K-Akt pathway being differentially expressed, respectively. A total of 51 genes were common in invivo- and invitro-matured oocytes cumulus cells groups compared with immature group. Regarding cumulus cells after the maturation process, 48 genes from the PI3K-Akt pathway were differentially expressed; of those, 26 genes were upregulated in cumulus cells from invitro-matured oocytes and 22 genes were upregulated in cumulus cells from invivo-matured oocytes. Comparing the invitro and invivo cumulus cells, the main genes of the pathway (AKT, PI3K, and PTEN) were not differentially expressed. The differences in expression between invitro and invivo cumulus cells were in genes responsible for different cellular functions controlled by the PI3K-Akt pathway, such as apoptosis, protein synthesis, and DNA repair, among others, which, in general, were increased in cumulus cells from invitro-matured oocytes. These results demonstrated the effect of culture conditions on cumulus cell gene expression modulating important pathways involved in oocyte competence acquisition, such as PI3K-Akt signalling.
Financial support was provided by FAPESP grants 2014/22887-0, 2018/01431-9, and 2018/13155-6.
212 Increased expression of YAP/TAZ encourages outgrowth establishment and three-dimensional colony formation and boosts plasticity of parthenogenetic stem cells
Authors: Arcuri S. , Pennarossa G. , Gandolfi F. , Brevini T.
Published: December 2019

Cell proliferation, apoptosis, and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs), which are able to proliferate maintaining pluripotency and, at the same time, give rise to all populations belonging to the three germ layers in response to specific stimuli. ESCs are, therefore, considered a well-established invitro model to study the complexity of these processes. We previously generated porcine and human diploid parthenogenetic embryonic stem cells (ParthESC) that showed many features and regulatory pathways common to biparental ESCs. However, we observed that monoparental cells demonstrated a greater ability to form outgrowths and generate stable three-dimensional (3D) spheroid colonies, which are distinctive signs of high plasticity (Brevini et al. 2010 STCR 6; https://doi.org/10.1007/s12015-010-9153-2). In the present study, we compared porcine and human biparental ESCs and ParthESC. Pluripotency gene expression was analysed and ELISA tests for global DNA methylation and total YAP levels were performed. Our results showed that ParthESCs showed significantly higher expression levels of YAP and TAZ than biparental ESCs. In contrast, the analysis of the upstream genes involved in the Hippo signalling pathway revealed no differences. We demonstrated that YAP remains in a dephosphorylated form, encouraging its nuclear compartmentalization and direct interaction with the nuclear transcription factors TEADs and SMADs, that are also upregulated and maintain pluripotency, repressing differentiation processes (Beyer et al. 2013 Cell Rep. 5, 1611-1624; https://doi.org/10.1016/j.celrep.2013.11.021). These complex regulatory interactions accompanied the overexpression of the pluripotency-related genes OCT4, NANOG, REX1, SOX2, UTF1, and TERT. In parallel, we observed a decrease of DNMT3 and DNMT4 activity that resulted in a global DNA hypomethylation and a chromatin high-permissive state. Altogether, our results demonstrate a significant upregulation of YAP/TAZ activity in monoparental cell lines. This may account for their greater ability to form outgrowths and generate 3D spheroid colonies and increased plasticity compared with biparental counterparts. It is interesting to note that YAP mRNA is supplied and expressed by the oocyte and is maternally inherited (Yu et al. 2016 Cell Res. 26, 275-287; https://doi.org/10.1038/cr.2016.20). Although further clarifications are needed, we hypothesise that its higher expression in ParthESC may be related to the strictly maternal origin of these cells.
This study was supported by the Carraresi Foundation. Authors are members of the COST Actions CA16119.
213 WNT inhibition by dickkopf WNT signalling pathway inhibitor 1 (DKK1) cannot replace IWR-1 during derivation of bovine embryonic stem cells
Authors: Xiao Y. , Amaral T. , Tribulo P. , Diffenderfer K. , Ross P. , Hansen P.
Published: December 2019

Understanding the signalling pathways involved with derivation of embryonic stem cells could enhance our understanding of pluripotency in pre-implantation embryos. Recently, the small molecule IWR-1 has been shown to promote derivation of mouse epiblast stem cells and pluripotent bovine and porcine embryonic stem cells (ESC). IWR-1 blocks WNT signalling mediated by β-catenin-targeted gene expression through stabilisation of Axin2, a member of the destruction complex that induces β-catenin degradation. Here, we evaluated whether dickkopf WNT signalling pathway inhibitor 1 (DKK1) can replace IWR-1 for establishment of bovine pluripotent ESC. If so, it is likely that the actions of IWR-1 to promote pluripotency involve inhibition of WNT signalling. Treatment of bovine embryos with 100ngmL−1 recombinant human DKK1 beginning at Day 5 of development decreased (P=0.02) immunofluorescent labelling of β-catenin in the resulting blastocysts (n=41-45/group), indicating that bovine embryos are responsive to DKK1 treatment. For ESC derivation, blastocysts were plated on top of feeder cells and cultured in ESC medium supplemented with 2.5 µM IWR-1 (n=21), 100ngmL−1 DKK1 (n=34), or vehicle (n=23). Cells were passaged every 5 to 7 days in their respective treatment medium. Seven days after plating, 57.9±14.7% of blastocysts in IWR-1 ESC medium developed outgrowth, which was lower (P=0.02) than the proportion of blastocysts with outgrowth in DKK1 medium (92.4±5.2%) or vehicle (81.9±10.0%). Outgrowth size did not differ among treatments. Labelling with CDX2 indicated that the majority of cells in outgrowths were trophectoderm cells. Thus, IWR-1 inhibits competence of blastocysts to form trophectoderm outgrowths during derivation of ESC. The percent of blastocysts from which cell lines were derived after 4 passages were 48% (10/21) for IWR-1, 41% (14/34) for DKK1, and 48% (11/23) for vehicle. Immunolabelling for the pluripotency marker SOX2 showed that only cells grown in IWR-1 medium were positive, whereas most of the cells derived in the other two media were not. Thus, IWR-1 could not be replaced by DKK1 for maintaining pluripotency. Immunoreactive β-catenin was abundantly distributed on the membrane of cells cultured with IWR-1 but not with DKK1 or vehicle-treated cells. Thus, β-catenin distribution to the cell membrane is linked with bovine pluripotency. Overall, results indicate that maintenance of pluripotency by IWR-1 may involve mechanisms other than WNT inhibition, and may be related to the localization of β-catenin to the plasma membrane.
220 Exploring the use of mesenchymal stem cells for treatment of mastitis and metritis in cattle
Authors: Singh R. , Saini S. , Ansari S. , Jamwal S. , Malakar D.
Published: December 2019

The present study was carried out to isolate mesenchymal stem cells (MSCs) from adipose tissue of cattle (Bos indicus), characterise them, and apply them for the treatment of mastitis and metritis in the cow. Cattle MSCs were isolated from adipose tissue near the loin region of cow. Isolated adipose tissue was subjected to enzymatic digestion using 2% collagenase with agitation at regular intervals. The cells obtained after digestion were resuspended in cell culture flasks containing growth enriched medium and cultured under standard culture conditions. Alkaline phosphatase staining was used as one of the parameters to confirm cultured putative MSCs. Bovine Ad-MSCs were further characterised using real time-PCR by amplification of MSC-specific markers: CD73, CD90, and CD105 as positive markers and CD34, CD45, and CD79a as negative markers. Immunocytochemistry showed the presence of CD73, CD90, and CD105 on the cell surface. Three groups-control (C), local (L), and intravenous (IV)-with 6 cows suffering from mastitis were taken in each group and subjected to MSC transplantation through local and intravenous routes. Control group animals were subjected to antibiotic treatment only. Similarly, another three groups were taken with 6 cows in each group suffering from metritis. Post-transplantation wound healing, tissue repair, and reduction in inflammation were monitored for 26 days, at different time intervals; that is, after Days 1, 3, 7, and 15. Blood samples were also collected from animals at the same time intervals for real time-PCR. A similar examination was also done in metritis groups along with the analysis of the reduction in turbidity of cervical fluid at the abovementioned time intervals. Real time-PCR was performed to determine relative expression of genes for proliferative factors, anti-inflammatory cytokines, and antimicrobial peptides on cells isolated from blood collected at different time intervals. Gene expression in the local group of mastitis subjected to MSC injection was significantly higher than that of the IV and control group. The somatic cell count declined in both local and IV groups compared with the control group. Whereas the expression of the same genes in the IV group of metritis was significantly higher than that of the local and control groups of cows. The turbidity of cervical fluid and mucus was reduced in the IV group compared with the local group. In conclusion, we demonstrated the healing potential of MSCs in a cow model via MSC injection. Promising results were obtained in curing mastitis in both local and IV groups, whereas healing in the case of metritis was significantly higher in the IV group compared with both the control and local groups of cows. The study indicates the potential use of MSc for treatment of mastitis and metritis in cattle through wound healing and decreasing microbial infection.
26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters
Authors: Raphalalani Z. , Ramukhithi F. , Ndhlala R. , Nephawe K. , Nedambale T.
Published: December 2019

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P<0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.
46 Presence of porcine TET3L isoform in oocytes: Potential involvement in the DNA demethylation process
Authors: Uh K. , Wax N. , Farrell K. , Lee K.
Published: December 2019

Fertilized oocytes undergo genome-wide DNA demethylation with the exception of imprinted genes and certain repetitive elements. Ten-eleven translocation 3 (TET3) protein has been known to be responsible for the DNA demethylation process by catalysing oxidation of 5-methylcytosine. Recent studies in the mouse indicated that multiple Tet3 isoforms exist in oocytes, implying differential actions of the isoforms. Previously, we reported the sequence of TET3 in porcine oocytes (GenBank: KC137685). Here, we investigated the presence of TET3 isoforms in porcine oocytes and cumulus cells, and followed changes in the TET3 expression during oocyte maturation to further understand the mechanism of TET3 action in the DNA demethylation process. To identify porcine TET3 isoforms, 5′ RACE (rapid amplification of cDNA ends) was conducted using mRNAs isolated from oocytes and cumulus cells at both germinal vesicle (GV) and MII stages. Gene-specific primers for 5′RACE were designed to recognise conserved regions of TET3 that are present in all isoforms, based on EST databases and the cloned sequence in our previous study. The PCR-amplified 5′RACE products were cloned into a vector and subsequently sequenced. The 5′RACE revealed 3 different TET3 isoforms from GV and MII cumulus cells; no amplification was detected using oocytes, potentially due to a low amount of mRNA. Among the isoforms, the longest variant (TET3L) contained sequences for the CXXC domain, known to carry DNA binding properties. Then, RT-PCR was used to detect the presence of the isoforms in porcine oocytes. Interestingly, the expression of TET3 isoform containing the CXXC domain (TET3L) was only verified from the RT-PCR, suggesting that the isoform may be the predominant isoform in porcine oocytes. To characterise transcript abundance of porcine TET3L, RNAs were isolated from different cells and/or tissues including cumulus cells, oocytes, brain, spleen, and lung. The RT-qPCR was performed using the RNAs and ΔΔCT method was used to analyse the data; GAPDH was used as an internal control. Three biological replicates were used for analysis of RT-qPCR data and P-values of less than 0.05 from one-way analysis of variance were considered significant. The expression level of TET3L was much higher in MII oocytes compared with that in somatic tissues; MII oocytes expressed the TET3 isoform over 350-fold higher than MII cumulus cells and 18-fold higher than lung cells. Interestingly, the expression level of TET3L increased over 3-fold during oocyte maturation (i.e. from GV to MII stage oocytes), indicating that TET3L may have a significant role in DNA demethylation after fertilisation. In conclusion, the TET3 isoform containing CXXC domain (TET3L) is predominantly expressed in matured porcine oocytes, suggesting an important role of the TET3 CXXC domain in DNA demethylation in zygotes.
47 Inhibition of endoplasmic reticulum stress during invitro maturation improves the developmental competence of bovine cumulus-oocyte complexes
Authors: Khatun H. , Ihara Y. , Takakura K. , Wada Y. , Yamanaka K.-I.
Published: December 2019

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many physiological responses including embryonic development. Evidence shows that the ER stress-induced unfolded protein response signaling pathway is associated with impairment of oocyte maturation and pre-implantation embryonic development; supplementation of culture medium with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, improved the developmental process of oocytes and embryos by attenuating ER stress. However, no reports are available on the role of TUDCA in reducing ER stress during IVM of bovine oocytes. Therefore, the aim of this study was to examine the mechanism of TUDCA on reducing ER stress in maturation of bovine cumulus-oocyte complexes (COCs) and whether inhibition of ER stress during maturation can promote subsequent embryonic development. Bovine ovaries were collected from a local slaughterhouse, and after aspiration COCs were matured with/without TUDCA (50, 100, and 200 µM) for 22h at 38.5°C in a humidified atmosphere of 5% CO2. After IVM, we examined the maturation rate, reactive oxygen species, apoptosis, protein/mRNA expression levels, and subsequent embryonic development after IVF. The data were analysed using analysis of variance followed by the Tukey-Kramer multiple comparison test. As a result, the dose-dependent experiment shows that a 100μM concentration of TUDCA significantly increased the maturation rate and decreased the percentage of apoptotic cells in COCs and reactive oxygen species levels in denuded oocytes. Subsequently, the expression of ER stress inducible protein GRP78/BIP significantly decreased in COCs treated with 100 µM TUDCA compared with the control COCs. In addition, the mRNA expression of ER stress and pro-apoptotic markers (GRP78/BIP, PERK, IER1, ATF4, XBP1, CHOP, and BAX) in COCs were significantly decreased by TUDCA (100 µM) treatment, whereas it increased anti-apoptotic BCL2 expression. Moreover, we show that TUDCA (100 µM) supplementation enhances embryonic development by significantly increasing the blastocyst formation rate (43.6±1.8% vs. 49.7±1.3%) and decreasing the number of apoptotic cells (7.7±1.1% vs. 5.03±0.6%) in blastocysts. These findings suggest that existence of ER stress during maturation alters the developmental competence of bovine COCs. Therefore, for the first time, we demonstrate that application of TUDCA during IVM plays a crucial role in reducing ER stress and improves the meiotic maturation, oocyte quality, and subsequent embryonic development invitro.
54 Choline alters the pattern of DNA methylation and lipid content of pre-implantation bovine embryos
Authors: Estrada-Cortés E. , Hansen P. J.
Published: December 2019

Choline is a methyl donor for DNA methylation and a precursor for phosphatidylcholine, which is the major phospholipid of cell membranes. Early embryonic development involves processes of DNA demethylation and remethylation as well as synthesis of new cell membranes. Addition of choline chloride (ChCl) to culture medium of embryos produced invitro increased birthweight of Brahman calves after embryo transfer. The objective was to determine whether the addition of ChCl to culture medium alters the pattern of DNA methylation and lipid content of pre-implantation embryos produced invitro. Embryos were incubated after fertilisation in BBH7 culture medium containing 0.0, 0.004, 1.3, or 1.8mM ChCl (adjusted with NaCl to maintain isotonicity). Concentrations were chosen to approximate free choline (0.004mM) and total choline (1.30mM) in plasma of cows at week 1 postpartum and concentration of total choline in plasma of cows fed rumen-protected choline (1.8mM). Cleavage and blastocyst rate (n=8 replicates) were evaluated at Days 3 and 7.5 post-insemination, respectively. Embryos ≥8 cells (range 8 to 24 cells; stages near embryonic genome activation) and expanded blastocysts were harvested at Day 3.75 (n=232) and 7.5 (n=204) to estimate global DNA methylation by immunostaining for 5-methyl-cytosine. Methylation of DNA was estimated by calculating the ratio of fluorescence for 5-methyl-cytosine to that of propidium iodide (DNA). Another group of expanded blastocysts was harvested (n=99) to estimate lipid content using Nile Red. Embryo development was analysed by GLIMMIX procedure and fluorescence data by GLM procedure of SAS (SAS Institute Inc.). The proportion of zygotes that cleaved after fertilisation (77.5±2.3, 78.1±2.3, 74.5±2.4, and 80.1±2.2% for 0.0, 0.004, 1.3, and 1.8mM ChCl; P=0.2736) and cleaved embryos that became blastocysts (37.8±4.4, 41.5±4.6, 42.8±4.6, and 39.6±4.4%; P=0.5764) was similar between treatments. The DNA methylation at both days was affected by treatment (P<0.001). At Day 3.75, 1.3mM choline reduced methylation and there were no effects of other concentrations (1.13a±0.03, 1.04a±0.03, 0.92b±0.03, and 1.13a±0.04; means with different superscripts differ at P<0.05). For blastocysts, in contrast, DNA methylation was increased for embryos treated with 1.3 and 1.8mM choline (0.98a±0.04, 1.04ab±0.03, 1.25c±0.03, and 1.11b±0.03). Lipid content in blastocysts was also affected by treatment (P=0.0139). In particular, lipid content was higher for embryos treated with 1.3 and 1.8mM choline (409.1a±54.3, 542.3ab±62.3, 651.3b±54.3, and 583.9b±55.0). In conclusion, addition of ChCl to culture medium altered DNA methylation in bovine pre-implantation embryos produced invitro in a manner dependent on developmental stage and choline concentration. Likewise, ChCl increased lipid content in the resultant blastocysts.
Support was provided by the Red Larson Endowment.
Reference point and case
Authors: Park Chongwon
Published: December 2019

This monograph answers the rarely discussed questions of why complicated grammatical case phenomena exist in Korean and what the connection is between the case forms and their functions. The author argues that the case forms in Korean reflect patterns of the human cognitive process. While this approach may seem rather obvious to non-linguists, it is indeed a novel claim in contemporary linguistic theory. In order to provide technical analyses of Korean case phenomena such as multiple nominative/accusative, non-nominative subject, and adverbial case constructions, this book adopts an independently established descriptive construct known as reference point in the framework of Cognitive Grammar. The author demonstrates that the notion of reference point not only explains a substantially wider set of data, but also leads to a more reasonable generalization. The intended readership of this book are researchers who are interested in case phenomena, irrespective of their theoretical orientation.
Voltage-controlled ON switching and manipulation of magnetization via the redox transformation of β-FeOOH nanoplatelets
Authors: Nichterwitz Martin , Neitsch Sabine , Röher Stefan , Wolf Daniel , Nielsch Kornelius , Leistner Karin
Published: December 2019

Redox-based metal/metal oxide transformations achieved via electrolytic gating recently emerged as a novel, magneto-ionic route for voltage control of magnetism. So far, mainly metal or oxide thin films and nanoporous metal alloy structures are used as starting materials. The present study demonstrates a magneto-ionic transformation starting from a stable electrodeposited FeOOH nanoplatelet structure. The application of a low voltage in a Li-based electrolyte results in the reduction of the virtually non-magnetic FeOOH into ferromagnetic Fe, yielding an ON switching of magnetization. The magnetization can be tuned in a large range by the time of voltage application and remains stable after voltage-switch off. A reversible magneto-ionic change of magnetization of up to 15% is achieved in the resulting iron films with a thickness of about 30 nm. This large magneto-ionic effect is attributed to the enhanced roughness of the iron films obtained from the nanoplatelet structure. The robust, voltage-controlled, and non-volatile ON switching of magnetism starting from a stable oxide structure is promising for the development of energy-efficient magnetic switches, magnetic actuation and may offer new avenues in magnetoelectronic devices.
Q-switched Thulium-doped fibre laser using Bismuth (III) Telluride-based saturable absorber
Authors: Rusdi M F M , Cheng X S , Latiff A A , Rosol A H A , Ahmad M T , Rahman M F A , Harun SW
Published: December 2019

We demonstrate a passive Q-switched thulium-doped fibre laser (TDFL) pulse generation using bismuth (III) telluride (Bi2Te3) thin film as saturable absorber. The Bi2Te3 was fabricated by a simple processing technique; simply by embedding the Bi2Te3 into the polyvinyl alcohol (PVA) film. By sandwiching 1 mm x 1 mm Bi2Te3 thin film between two fibre ferrules in a TDFL ring cavity, a stable Q-switching pulses train was generated. The repetition rate and pulse width were tuneable from 10.72 kHz to 39.9 kHz and 13.08 µs to 5.98 µs, respectively, as the pump power increased from 309 mW to 454 mW. The slope efficiency was 4.51%, while, the maximum pulse energy was calculated as 0.17 µJ.
Development and preliminary data of integrated temperature-Insensitive lateral force sensor based on linear chirp Fiber Bragg Grating
Authors: Abushagur Abdulfatah A G , Bakar Ahmad Ashrif A , Arsad Norhana
Published: December 2019

Lateral force measurement using a cantilever ruler-beam (CRB) sensor based on linear chirp fiber Bragg grating (LCFBG) is proposed and experimentally demonstrated. The lateral force is determined by altering the reflection optical power of LCFBG of which an axial-strain gradient along its sensing is induced. The reflection spectrum intensity responds linearly and monotonically with lateral force increasing from 0 to ≍ 0.6 N. Experimental results demonstrate that the CRB force sensor can successfully achieve lateral forces prediction with less than 0.0015 N resolution using simply Thorlabs PM100USB optical power meter. And can accomplish an adequate accuracy with RMS error < 0.04N of full scale (0-0.749N). The CRB is chosen in one way or another to resemble steerable catheter flexibility as our intention is to developing catheter force sensor based on temperature-insensitive contact force measurement.
Structural and magnetic properties of RE-Al substituted nanocrystalline hexaferrites (Sr1−xRExAl2Fe10O19)
Authors: Al-Garalleh Ghada A , Mahmood Sami H , Bsoul Ibrahim , Loloee Riza
Published: December 2019

We describe the synthesis and characterization of magnetic materials with improved magnetic properties for permanent magnet applications. Sr1−x
RE
x
Al2Fe10O19 (RE = La, Ce, Tb, and Dy; x = 0.0 and 0.1) were prepared by ball milling and sintering at 1200 °C for 6 h. The effects of (Al3+–RE3+) substitution on the structural and magnetic properties of SrM hexaferrites were investigated by x-ray diffraction, scanning electron microscopy, and magnetic measurements at low (5 K), and high temperatures (from room temperature to above Curie temperature). The sample with x = 0, and that with La substitution, consisted of a single SrM phase. The rest of the samples contained traces of secondary oxide phases, and exhibited a small reduction in lattice parameters compared with the unsubstituted SrFe12O19. The coercivity of the samples (8800 ≤ H

cM
≤ 9750 Oe) was more than double the standard value of the unsubstituted compound, and the Curie temperatures was >300 °C for all samples. Also, the saturation magnetization (32.5–46.4 emu g−1) was high enough to make the overall magnetic properties of these compounds potentially important for the permanent magnet industry.
Dry sliding wear performance of an annealed TiNi alloy with different nickel contents
Authors: Meddah Soumaya , Chadli Hacene , Bourebia Mounira , Montagne Alex , Iost Alain , Labaiz Mohamed , Taleb Ahlem
Published: December 2019

The wear behaviour of the TiNi alloy tested at different conditions (2 and 4 N as applied loads and 40, 50 an 60 wt % as nickel contents) was investigated. For this purpose, two main experimental techniques consisting of wear and indentation tests were used. Scanning electron microscopy (SEM) and laser source profilometry were employed to reveal the wear mechanisms and the affected worn surfaces. Furthermore, design of experiments planning introducing factorial design as well as response surface was adopted to attempt predicting the coefficient friction according to the planned test parameters. Nanoindentation results showed that all the TiNi alloys are harder than a TA6V4 alloy which is commonly used in dental implant. Particularly, the TiNi60 alloy exhibited superior superelasticity, characterized by a lower E/H ratio and a higher depth recovery ratio than the TA6V4 alloy. Besides, the wear rates underwent a substantial increase with the increase of the applied load but a decrease with increasing the Ni content. The worn surfaces analyses revealed an enhanced resistance to adhesive and abrasive wear with increasing nickel content.
Comparing perspectives of family caregivers and healthcare professionals regarding caregiver burden in dementia care: results of a mixed methods study in a rural setting
Authors: Krutter Simon , Schaffler-Schaden Dagmar , Essl-Maurer Roland , Wurm Laura , Seymer Alexander , Kriechmayr Celine , Mann Eva , Osterbrink Juergen , Flamm Maria
Published: December 2019

Background

Persons with dementia (PwD) need support to remain in their own homes as long as possible. Family caregivers, homecare nurses and general practitioners (GPs) play an important role in providing this support, particularly in rural settings. Assessing caregiver burden is important to prevent adverse health effects among this population. This study analysed perceived burden and needs of family caregivers of PwD in rural areas from the perspectives of healthcare professionals and family caregivers.

Methods

This was a sequential explanatory mixed methods study that used both questionnaires and semi-structured interviews. Questionnaires measuring caregiver burden, quality of life and nursing needs were distributed to the caregivers; health professionals received questionnaires with adjusted items for each group. Additionally, in-depth qualitative interviews were carried out with eight family caregivers.

Results

The cross-sectional survey population included GPs (n = 50), homecare nurses (n = 140) and family caregivers (n = 113). Healthcare professionals similarly assessed the psychosocial burden and stress caused by behavioural disturbances as most relevant. Psychological stress, social burden and disruptive behaviour (in that order) were regarded as the most important factors from the caregivers’ perspective. It was found that 31% of caregivers reported permanent or frequent caregiver overload. Eight themes related to caregiver burden emerged from the subsequent interviews with caregivers.

Conclusions

Professional support at home on an hourly basis was found to be highly relevant to prevent social isolation and compensate for lack of leisure among caregivers of PwD. Improvement of interprofessional dementia-related education is needed to ensure high-quality primary care.
Atrial fibrillation progression risk factors and associated cardiovascular outcome in well-phenotyped patients: data from the AF-RISK study
Authors: De With Ruben R , Marcos Ernaldo G , Dudink Elton A M P , Spronk Henri M , Crijns Harry J G M , Rienstra Michiel , Van Gelder Isabelle C
Published: December 2019

Aims

Atrial fibrillation (AF) is a progressive disease, but identifying patients at risk for AF progression is challenging. We aimed to identify factors associated with AF progression.

Methods and results

Atrial fibrillation progression was assessed in 392 patients with recent-onset paroxysmal or persistent AF included in the prospective, observational, multicentre identification of a risk profile to guide atrial fibrillation (AF-RISK) study. Progression of AF was assessed by Holter monitoring and 2-week event recorder at baseline and 1-year follow-up. AF progression was defined as: (i) doubling in AF burden at 1 year compared to baseline with a minimum AF burden of 10% in paroxysmal AF; or (ii) transition from paroxysmal to persistent or permanent AF; or (iii) persistent to permanent AF. Age was 60 ± 11 years, 62% were men, and 83% had paroxysmal AF. At 1 year, 52 (13%) had AF progression (11% in paroxysmal; 26% in persistent AF). Multivariable logistic regression showed that left atrial volume [odds ratio (OR) per 10 mL 1.251, 95% confidence interval (CI) 1.078–1.450; P < 0.001], N-terminal pro-B-type natriuretic peptide (NT-proBNP; OR per standard deviation increase 1.583, 95% CI 1.099–2.281; P = 0.014), and plasminogen activator inhibitor-1 (PAI-1; OR per standard deviation increase 0.660, 95% CI 0.472–0.921; P = 0.015) were associated with AF progression. In an additional follow-up of 1.9 (0.9–3.3) years patients with AF progression developed more cardiovascular events and all-cause mortality (12.4%/year vs. 2.3%/year, P < 0.001).

Conclusion

Atrial fibrillation progression occurred in 13% of patients with recent-onset AF during 1-year follow-up. Left atrial volume, NT-proBNP, and PAI-1 were associated with AF progression. Patients with AF progression had a higher event rate.

Trial registration number

Clinicaltrials.gov NCT01510210.
Secondary stroke prophylaxis in atrial fibrillation patients with chronic kidney disease: a nationwide cohort study
Authors: Christensen Mathias Aagaard , Fosbøl Emil Loldrup , Bonde Anders Nissen , Olesen Jonas Bjerring , Gislason Gunnar H , Torp-Pedersen Christian , Gundlund Anna
Published: December 2019

Aims

Oral anticoagulation (OAC) therapy as secondary stroke prophylaxis in atrial fibrillation (AF) patients with chronic kidney disease (CKD) remains unexplored and poses a clinical treatment dilemma. We assessed the long-term risk of thromboembolic events according to post-stroke OAC therapy in AF patients with CKD after their first ischaemic stroke.

Methods and results

We identified Danish AF patients with CKD who presented with first-time ischaemic stroke from 2005 to 2014. Chronic kidney disease was defined as a diagnosis code for CKD before baseline, defined as 100 days after stroke discharge. Post-stroke antithrombotic therapy (OAC therapy and antiplatelet therapy) was identified from prescription claims from discharge to baseline. Cumulative incidences and adjusted hazard ratios (HRs) of thromboembolic events according to post-stroke OAC therapy were examined. Of 1252 AF patients with CKD presenting with ischaemic stroke, 631 (50.4%) patients were on OAC therapy and 621 (49.6%) were on antiplatelet therapy alone at baseline [median age 76 (interquartile range, IQR 71–83) and 80 (IQR 72–86), respectively]. The median follow-up period was 1.9 years (IQR 0.8–3.6). Cumulative incidence rates of thromboembolic events and bleeding showed no significant difference between those on OAC therapy and antiplatelet therapy. The results from the multivariable analysis revealed similar results: thromboembolic risk was not modified by OAC treatment [adjusted HR 0.89, 95% confidence interval (CI) 0.73–1.09] nor was the risk of bleeding (adjusted HR 0.88, 95% CI 0.67–1.17).

Conclusion

Oral anticoagulation in patients with CKD and prior stroke was not associated with a reduced risk of recurrent thromboembolic events compared with antiplatelet therapy.
Parental education associated with immune function in adolescence
Authors: Barroso Isaac , Cabral Maria , Ramos Elisabete , Guimarães João T
Published: December 2019

Background

The immune system is affected and shaped by several internal and external factors. Among the external variables, the socioeconomic status is known to influence the immune system since the early years of life and throughout life.

Methods

In this study, we assessed the relationship between parental education with the white blood cells and its subtypes in 1213 adolescents from the EPITeen cohort, assessed at the age of 13. Beta coefficients (β) and 95% confidence intervals (CI) were fitted using linear regression models to quantify the association and were adjusted for sex, body mass index and chronic disease.

Results

After adjustment, parental education presented a negative association with white blood cells, which was significant among those with higher high-sensitivity C-reactive protein (hs-CRP) median levels [−0.05 mg/l (95% CI −0.08, −0.01)]. On the contrary, a positive association with lymphocytes was observed, which was, significant among those with lower hs-CRP [0.17 mg/l (95% CI 0.02, 0.32)]. A neutrophil-to-lymphocyte ratio significant decrease was also observed with the increment of parental education (P<0.001).

Conclusion

We found that parental education was positively associated with a higher proportion of lymphocytes and a lower proportion of neutrophils, suggesting that parental education is associated with offsprinǵs innate immune system regulation. These results may contribute to clarify the relationships between childhood socioeconomic status and increased risk of adverse cardiovascular outcomes and other immune-related diseases.
Simulating the Effectiveness of Improvement Cuts and Commercial Thinning to Enhance Fire Resistance in West Coast Dry Mixed Conifer Forests
Authors: Jain Theresa B , Fried Jeremy S , Loreno Sara M
Published: December 2019

Nine multipurpose silvicultural treatments, formulated as a synthesis of recently implemented prescriptions offered by forest managers, were simulated to evaluate their effectiveness at enhancing fire resistance. The Forest Vegetation Simulator was applied, within the BioSum Framework, on over 3,000 Forest Inventory and Analysis plots representing 5 million hectares of dry mixed conifer forests in eastern Washington and Oregon and California’s Sierra Nevada Mountains. We developed a composite fire-resistance score based on four fuel modification principals and metrics: fuel strata gap, canopy bulk density, proportion of basal area in resistant species, and predicted tree survival. The trajectories of stands with and without treatment were compared to evaluate effectiveness immediately post-treatment, and over the three decades that followed. Seventy percent of these forests could be effectively treated in the short term by at least one prescription. Pretreatment forest condition, particularly fire-resistant species abundance, strongly influenced short-term treatment success, and the post-treatment stand dynamics that limit treatment longevity. Treatment effectiveness endured only 10 or 20 years, depending on fire-resistant species abundance, owing to growing space for crown expansion generated by treatment plus regeneration and release and growth of understory tree strata.
Finite-frequency imaging of the global 410- and 660-km discontinuities using SS precursors
Authors: Guo Zhen , Zhou Ying
Published: December 2019

We report finite-frequency imaging of the global 410- and 660-km discontinuities using boundary sensitivity kernels for traveltime measurements made on SS precursors. The application of finite-frequency sensitivity kernels overcomes resolution limits in previous studies associated with large Fresnel zones of SS precursors and their interferences with other seismic phases. In this study, we calculate the finite-frequency sensitivities of SS waves and their precursors based on a single-scattering (Born) approximation in the framework of travelling-wave mode summation. The global discontinuity surface is parametrized using a set of triangular gridpoints with a lateral spacing of about 4°, and we solve the linear finite-frequency inverse problem (2-D tomography) based on singular value decomposition (SVD). The new global models start to show a number of features that were absent (or weak) in ray-theoretical back-projection models at spherical harmonic degree l > 6. The thickness of the mantle transition zone correlates well with wave speed perturbations at a global scale, suggesting dominantly thermal origins for the lateral variations in the mantle transition zone. However, an anticorrelation between the topography of the 410-km discontinuity and wave speed variations is not observed at a global scale. Overall, the mantle transition zone is about 2–3 km thicker beneath the continents than in oceanic regions. The new models of the 410- and 660-km discontinuities show better agreement with the finite-frequency study by Lawrence & Shearer than other global models obtained using SS precursors. However, significant discrepancies between the two models exist in the Pacific Ocean and major subduction zones at spherical harmonic degree >6. This indicates the importance of accounting for wave interactions in the calculations of sensitivity kernels as well as the use of finite-frequency sensitivities in data quality control.