bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Hepatocyte Lipid Metabolism and Inflammatory Response by 25‐Hydroxycholesterol and 25‐Hydroxycholesterol‐3‐sulfate
Authors: Xu Leyuan , Bai Qianming , Rodriguez‐Agudo Daniel , Hylemon Phillip B. , Heuman Douglas M. , Pandak William M. , Ren Shunlin
Published: August 2010

Dysregulation of lipid metabolism is frequently associated with inflammatory conditions. The mechanism of this association is still not clearly defined. Recently, we identified a nuclear oxysterol, 25‐hydroxycholesterol‐3‐sulfate (25HC3S), as an important regulatory molecule involved in lipid metabolism in hepatocytes. The present study shows that 25HC3S and its precursor, 25‐hydroxycholesterol (25HC), diametrically regulate lipid metabolism and inflammatory response via LXR/SREBP‐1 and IκBα/NFκB signaling in hepatocytes. Addition of 25HC3S to primary rat hepatocytes decreased nuclear LXR and SREBP‐1 protein levels, down‐regulated their target genes, acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and SREBP‐2 target gene HMG reductase, key enzymes involved in fatty acid and cholesterol biosynthesis. 25HC3S reduced TNFα‐induced inflammatory response by increasing cytoplasmic IκBα levels, decreasing NFκB nuclear translocation, and consequently repressing expression of NFκB‐dependent genes, IL‐1β, TNFα, and TRAF1. NFκB‐dependent promoter reporter gene assay showed that 25HC3S suppressed luciferase activity in the hepatocytes. In contrast, 25HC elicited opposite effects by increasing nuclear LXR and SREBP‐1 protein levels, and by increasing ACC1 and FAS mRNA levels. 25HC also decreased cytoplasmic IκBα levels and further increased TNFα‐induced NFκB activation. The current findings suggest that 25HC and 25HC3S serve as potent regulators in cross‐talk of lipid metabolism and inflammatory response in the hepatocytes.
Social enterprise in Europe: At the crossroads of market, public policies and third sector
Authors: Defourny Jacques , Nyssens Marthe
Published: August 2010

Over the last 15 years, the concept of social enterprise has been raising an increasing interest in various regions of the world. In the present paper, we describe, first, the European historical landscapes in which the concept of social enterprise took root. In the second part, we analyze the various public policies introduced in several European countries. In the third part, we analyse the logics of financing of social enterprises. Based on the EMES conception of social enterprise, we underline the “hybridization” of their resources. European social enterprises indeed, most often, combine income from sales with public subsidies linked to their social mission and private donations and/or volunteering. This clearly contrasts with a strong US tendency to define social enterprises as non-profit organizations more oriented towards the market and developing “earned income strategies” as a response to decreasing public subsidies and to the limits of private grants from foundations.
Holocene beach deposits for assessing coastal uplift of the northeastern Boso Peninsula, Pacific coast of Japan
Authors: Tamura Toru , Murakami Fumitoshi , Watanabe Kazuaki
Published: August 2010

This paper presents a case study that assessed spatial variations in the tectonic uplift rates of beach deposits in the relict Kujukuri strand plain, situated on the northeastern coast of the Boso Peninsula, eastern Japan. The southern Boso Peninsula is tilted downward to the northeast due to plate subduction along the Sagami Trough. However, the cause of the northeastern coast uplift creating the relict strand plain is unclear, due to the absence of a Holocene raised marine terrace sequence. Elevations and ages of beach deposits were collected from drilled cores and ground-penetrating radar profiles along three shore-normal sections in the southern Kujukuri strand plain. From this, alongshore variations in the relative sea level since the mid-Holocene could be seen. These corresponded to north-to-northeast downward tilting at a rate of 0.4 m/ka for an interval 10 km and are concordant with the longer term tilting of the last interglacial marine terrace surrounding the plain. Although it is difficult to assess shore-normal variations of uplift based on the present dataset, the recognized tilting apparently continues to the tilting of the southern Boso Peninsula, implying the Sagami Trough probably affects the uplift of the Kujukuri coast.
Encouraging appropriate, evidence-based use of oral nutritional supplements
Authors: Stratton Rebecca J. , Elia Marinos
Published: August 2010

With the considerable cost of disease-related malnutrition to individuals and to society (estimated to be >£13×109 for the UK, 2007 prices), there is a need for effective and evidence-based ways of preventing and treating this condition. The wide range of oral nutritional supplements that may be prescribed for the dietary management of malnutrition and other conditions account for only about 1% (about £99×106, 2007 data) of the prescribing budget in England. Systematic reviews and meta-analyses consistently suggest that ready-made, multi-nutrient liquids which may be prescribed can improve energy and nutritional intake, body weight and have a variety of clinical and functional benefits in a number of patient groups. Meta-analyses have repeatedly shown that oral nutritional supplements produce significant reductions in complications (e.g. infections) and mortality, and a recent meta-analysis shows a reduction in hospital admissions (OR 0·56 (95% CI 0·41, 0·77), six randomised controlled trials). Such benefits suggest that the appropriate use of oral nutritional supplements should form an integral part of the management of malnutrition, particularly as there is currently a lack of evidence for alternative oral nutrition strategies (e.g. food fortification and counselling). As with all therapies, compliance to oral nutritional supplements needs to be maximised and the use monitored. To make sure that those at risk of malnutrition are identified and treated appropriately, there is a need to embed national and local policies into routine clinical practice. In doing so, the economic burden of this costly condition can be curtailed. As recently suggested by the National Institute for Health and Clinical Excellence, substantial cost savings could be made if screening and treatment of malnourished patients was undertaken.
An information perspective on path dependence
Authors: HEIKKILA ERIC J.
Published: August 2010

This paper proposes an information perspective on path dependence. From this perspective, historical paths are important insofar as knowledge about them shapes current decisions, for better or worse. A key consideration is the extent to which relevant information is fully inscribed in the existing configuration of state variables, including organizational structures and institutional norms. Using a chess analogy, path dependency arises whenever a decision maker's ‘move’ depends not only upon existing state variables, but also knowledge of the path by which this configuration came about. This chess analogy is then extended to various institutional contexts such as legal expungement of criminal records, patient privacy rights, and corporate executive succession strategies. A formal notation is introduced to specify this definition more precisely, and to compare it with other perspectives on path dependency, such as lock-in effects, increasing returns to scale, ergodic equilibria, and generalized notions that ‘history matters’.
Effect of soil water repellency on moisture distribution from a subsurface point source
Authors: Wallach Rony
Published: August 2010

Wetting and redistribution of water in air‐dried wettable, slightly repellent, and strongly repellent soils was investigated by tracking the spatial and temporal moisture‐content variation in a transparent flow chamber. Water was applied as a subsurface point source. The degree of water repellency had a substantial effect on the plume's shape, dimensions, and internal moisture‐content distribution. The high uniform moisture content in the repellent soil's plume surrounded by a narrow transition layer within which moisture content was sharply decreasing indicates that unstable flow shapes a finger‐like wetting front. Moisture redistribution in the repellent soils was limited and took place mainly in the vertical direction. The repellent soils were wetted immediately, very likely by local positive pressure buildup induced by the hydraulic resistance imposed by the initial >90° contact angle. The plume shape dimensions and internal moisture‐content distribution should be considered in the design and operation of subsurface drip irrigation in water‐repellent soils.
Munc18b regulates core SNARE complex assembly and constitutive exocytosis by interacting with the N-peptide and the closed-conformation C-terminus of syntaxin 3
Authors: Peng Ren-Wang , Guetg Claudio , Abellan Eric , Fussenegger Martin
Published: August 2010

The interaction between SM (Sec1/Munc18) and SNARE (soluble N-ethylmaleimide-sensitive factor-attachment receptor) proteins constitutes the core eukaryotic membrane fusion machinery which manages exocytosis by mediating fusion of constitutively exocytic vesicles with the plasma membrane. However, mechanistic details on the nature and the physiological impact of SM–SNARE interactions remain largely elusive. Detailed characterization of the interaction profiles between Munc18b and its cognate SNAREs, Stx3 (syntaxin 3), SNAP-23 (soluble N-ethylmaleimide-attachment protein 23) and VAMP8 (vesicle-associated membrane protein 8), revealed that Munc18b binds Stx3, VAMP8 and the assembled core SNARE complex consisting of Stx3, SNAP-23 and VAMP8. Dissection of the Munc18b–Stx3 heterodimer suggested that Munc18b interacts with Stx3's conserved N-peptide as well as with its closed-conformation C-terminus encompassing the Habc domain, a linker and the SNARE (H3) motif. Deletion of the Habc domain or mutations interrupting the intramolecular binding of the Habc and H3 domains abrogated the Munc18b–Stx3 interaction. Although only the N-peptide deletion mutant, but not the soluble wild-type Stx3, is assembled into the core SNARE complex in the presence of Munc18b in vitro, ectopic expression of this SM protein increases constitutive exocytosis in mammalian cells. Our results suggest that Munc18b is functionally coupled to the assembly of exocytic SNARE complexes and increases exocytosis by interacting with the N-peptide and closed-conformation C-terminus of Stx3, thereby neutralizing the secretion-inhibitory effect of this SNARE.
Multi-keV x-ray source development experiments on the National Ignition Facility
Authors: Fournier K. B. , May M. J. , Colvin J. D. , Kane J. O. , Schneider M. , Dewald E. , Thomas C. A. , Compton S. , Marrs R. E. , Moody J. , Bond E. , Michel P. , Fisher J. H. , Newlander C. D. , Davis J. F.
Published: August 2010

We report results from a five shot campaign carried out with Ar–Xe gas-filled targets at the National Ignition Facility (NIF). The targets were shot with ≈350 kJ of 3ω laser energy delivered with a 5 ns trapezoidal laser pulse. We report measured x-ray output from the target in different spectral bands both below and above 1.5 keV photon energies: We find yields of ≈20.5 kJ/sr with peak x-ray power approaching 4 TW/sr over all energies, as measured for the unique viewing angle of our detector, and ≈3.6 kJ/sr with peak x-ray power of 1 TW/sr for x-rays with energies >3 keV. This is a laser-to-x-ray conversion efficiency of 13±1.3% for isotropic x-rays with energies >3 keV. Laser energy reflected by the target plasma for both inner and outer-cone beams is measured and found to be small, between 1% and 4% of the drive energy. The energy emitted in hard x-rays (with energies >25 keV) is measured and found to be ≈1 J/sr. Two-dimensional imaging of the target plasma during the laser pulse confirms a fast, volumetric heating of the entire target, resulting in efficient laser-to-x-ray conversion. Postshot simulations with a two-dimensional radiation-hydrodynamics code reproduce well the observed x-ray flux and fluence, backscattered light, and bulk target motion.
Microfabrication of polystyrene microbead arrays by laser induced forward transfer
Authors: Palla-Papavlu Alexandra , Dinca Valentina , Paraico Iurie , Moldovan Antoniu , Shaw-Stewart James , Schneider Christof W. , Kovacs Eugenia , Lippert Thomas , Dinescu Maria
Published: August 2010

In this study we describe a simple method to fabricate microarrays of polystyrene microbeads (PS-μbeads) on Thermanox coverslip surfaces using laser induced forward transfer (LIFT). A triazene polymer layer which acts as a dynamic release layer and propels the closely packed microspheres on the receiving substrate was used for this approach. The deposited features were characterized by optical microscopy, scanning electron microscopy, atomic force microscopy, and Raman spectroscopy. Ultrasonication was used to test the adherence of the transferred beads. In addition, the laser ejection of the PS-μbead pixels was investigated by time resolved shadowgraphy. It was found that stable PS-μbeads micropatterns without any specific immobilization process could be realized by LIFT. These results highlight the increasing role of LIFT in the development of biomaterials, drug delivery, and tissue engineering.
Monte Carlo simulation of a single ring among linear chains: Structural and dynamic heterogeneity
Authors: Yang Yong-Biao , Sun Zhao-Yan , Fu Cui-Liu , An Li-Jia , Wang Zhen-Gang
Published: August 2010

We perform lattice Monte Carlo simulation using the bond-fluctuation model to examine the conformation and dynamic properties of a single small flexible ring polymer in the matrix of linear chains as functions of the degree of polymerization of the linear chains. The average conformation properties as gauged by the mean-square radius of gyration and asphericity parameter are insensitive to the chain length for all the chain lengths examined (30, 100, 300, and 1000). However, in the longer chain (300 and 1000) samples, there is an increased spread in the distribution of the value of these quantities, suggesting structural heterogeneity. The center-of-mass diffusion of the ring shows a rapid decrease with increasing chain length followed by a more gradual change for the two longer chain systems. In these longer chain systems, a wide spread in the value of the apparent self-diffusion coefficient is also observed, as well as qualitatively different square displacement trajectories among the different samples, suggesting heterogeneity in the dynamics. A primitive path analysis reveals that in these long chain systems, the ring can exist in topologically distinct states with respect to threading by the linear chains. Threading by the linear chain can dramatically slow down and in some cases stall the diffusive motion of the ring. We argue that the life times for these topological conformers can be longer than the disentanglement time of the linear chain matrix, so that the ring exhibits nonergodic behavior on time scales less or comparable to the life time of these conformers. Our results suggest a picture of the ring diffusion as one where the diffusion path consists of distinctive segments, each corresponding to a different conformer, with slow interconversion between the different conformers.
Parametrization of the Gay–Berne potential for conjugated oligomer with a high aspect ratio
Authors: Lee Cheng K. , Hua Chi C. , Chen Show A.
Published: August 2010

The Gay–Berne (GB) potential has been a popular semiempirical model for describing the short-range intermolecular forces for a wide variety of aspherical molecules, including liquid crystals and anisotropic colloids, with generally small molecular dimensions and low aspect ratios (<5). This study evaluates the parametrization of the GB potential for a high-aspect-ratio (=10) oligomer belonging to a model conjugated polymer. We elaborate that the semiflexibility associated with a large oligomer species demands a variant umbrella-sampling scheme in establishing the potentials of mean force (PMFs) for four pair ellipsoid arrangements typically utilized to parametrize the GB potential. The model ellipsoid so constructed is shown to capture the PMFs of essential intermediate arrangements as well, and, according to the results of simplex optimizations, recommendations are given for the minimum set of parameters to be included in the optimization of a large oligomer or particulate species. To further attest the parametrized GB potential, the coarse-grained (CG) Monte Carlo simulations employing the GB potential and the back-mapped, full-atom atomistic molecular dynamics (AMD) simulations were performed for a dense oligomer system at two representative system temperatures. The results indicated that the CG simulations can capture, with exceptional computational efficiency, the AMD predictions with good thermal transferability. In future perspectives, we remark on potential applications to construct efficient, parameter-free CG models for capturing fundamental material properties of large oligomer/particulate species as well as long-chain conjugated polymers.
HfO
2
–
GaAs
metal-oxide-semiconductor capacitor using dimethylaluminumhydride-derived aluminum oxynitride interfacial passivation layer
Authors: He G. , Zhang L. D. , Liu M. , Sun Z. Q.
Published: August 2010

In this letter, treatment of GaAs surface by using dimethylaluminumhydride-derived AlON passivation layer prior to HfO2 deposition is proposed to solve the issue of Fermi level pinning. It has been found that AlON passivation layer effectively suppresses the oxides formation and leads to the Fermi level unpinning at the interface between GaAs and HfO2. Based on analysis from metal-oxide-semiconductor capacitors of Au/HfO2/AlON/GaAs stack, excellent capacitance-voltage characteristics with saturated accumulation capacitance and reduced leakage current have been achieved, which may originate from the decrease in the interface state density and the increase in the conduction band offset.
Near room temperature continuous-wave laser operation from type-I interband transitions at wavelengths beyond 4 μm
Authors: Eibelhuber M. , Schwarzl T. , Pichler S. , Heiss W. , Springholz G.
Published: August 2010

Nonradiative Auger recombination has limited room temperature continuous-wave (cw) operation of type-I interband lasers to wavelengths shorter than 3.36 μm. Using IV–VI semiconductor quantum well microdisk structures, near room temperature laser operation at longer wavelengths is achieved. Their active region consists of type-I single quantum wells of PbSe embedded in PbSrSe barriers. Under optical excitation, single mode cw emission at 4.3 μm is demonstrated up to 2 °C. This proves the feasibility of cw-operation of long wavelength interband lasers up to room temperature.
Low-density InP quantum dots embedded in Ga0.51In0.49P with high optical quality realized by a strain inducing layer
Authors: Richter Daniel , Roßbach Robert , Schulz Wolfgang-Michael , Koroknay Elisabeth , Kessler Christian , Jetter Michael , Michler Peter
Published: August 2010

We present a method to reduce the intrinsically high InP quantum dot density embedded in a Ga0.51In0.49P barrier by introducing an InGaAs quantum dot seed layer. The additional strain reduces the total InP quantum dot density by around one order of magnitude from 2×1010 to 3×109 cm−2 but only ∼1% of the InP nanostructures seem to be optically active (107 cm−2). Therefore, microphotoluminescence measurements could be accomplished without masks. We found resolution-limited photoluminescence linewidths (ΔE<100 μeV), good signal-to-noise ratios (∼65), single-photon emission behavior [g(2)(τ=0)=0.3], and excitonic decay times of typically between 1 and 2 ns. Furthermore the structural quantum dot properties were investigated.
Quantification of trap densities at dielectric/III–V semiconductor interfaces
Authors: Engel-Herbert Roman , Hwang Yoontae , Stemmer Susanne
Published: August 2010

High-frequency capacitance-voltage curves for capacitors with high-k gate dielectrics and III–V semiconductor channels are modeled. The model takes into account the low conduction band density of states, the nonparabolicity of the Γ valley, and the population of higher lying conduction band valleys. The model is used to determine interface trap densities (Dit) and band bending of HfO2/In0.53Ga0.47As interfaces with different Dit and with pinned and unpinned Fermi levels, respectively. Potential sources of errors in extracting Dit are discussed and criteria that establish unpinned interfaces are developed.
A fast response and large electrically tunable-focusing imaging system based on switching of two modes of a liquid crystal lens
Authors: Lin Hung-Chun , Lin Yi-Hsin
Published: August 2010

We demonstrated a fast response and large tunable focusing imaging system consisting of a lens module and a liquid crystal (LC) lens based on the switching of two modes of a LC lens: the negative LC lens and positive LC lens. By discarding the conventional operation of a positive LC lens only in the imaging system, large tunable focusing range can be achieve from 300 to 10 cm owning to the phase change between the positive and the negative LC lens; meanwhile, the response time is fast (∼433 ms). The potential applications are autofocused cell phones, and cameras.
Synthesis and Solution-state Assembly or Bulk State Thiol-ene Crosslinking of Pyrrolidinone- and Alkene-functionalized Amphiphilic Block Fluorocopolymers: From Functional Nanoparticles to Anti-fouling Coatings
Authors: Ma Jun , Bartels Jeremy W. , Li Zhou , Zhang Ke , Cheng Chong , Wooley Karen L.
Published: August 2010

With an ever increasing interest in the combined functionality and versatility of materials, increasing demands are placed on synthetic methodologies by which to produce such materials. This work demonstrates the preparation of block copolymers having fluorocarbon content, pyrrolidinone units, and alkene groups as complex building blocks for the assembly of discrete nanoparticles in solution and, alternatively, transformation into sophisticated crosslinked networks. Reversible addition–fragmentation chain transfer (RAFT) polymerization is a facile tool for the synthesis of well-defined polymers containing imbedded side-chain functionalities. In this work, the synthesis of well-defined multifunctional fluorinated polymers bearing pendant pyrrolidinone groups, and block copolymers bearing both pyrrolidinone and alkenyl groups on different segments was achieved, by using RAFT polymerizations of unique bifunctional monomers. Upon micellization, the amphiphilic diblock copolymers were transformed into regioselectively-functionalized nanoparticles. Further transformations of pyrrolidinone- and alkene-dual functionalized-block copolymers into complex amphiphilic networks were accomplished by highly efficient UV-induced thiol-ene reactions. Whether as discrete nanoparticles or nanoscopically-segregated crosslinked networks, these materials have great potential for several diverse technologies, including as anti-fouling materials.
Biomaterials for Brain Tissue Engineering
Authors: Pettikiriarachchi Jerani T. S. , Parish Clare L. , Shoichet Molly S. , Forsythe John S. , Nisbet David R.
Published: August 2010

Neurological disorders such as traumatic brain injuries or stroke result in neuronal loss and disruption of the brain parenchyma. Current treatment strategies are limited in that they can only mitigate the degeneration process or alleviate the symptoms but do not reverse the condition. In contrast, regenerative cell-based therapies offer long-term hope for many patients. Bioactive scaffolds are likely to reinforce the success of cell replacement therapies by providing a microenvironment that facilitates the survival, proliferation, differentiation, and connectivity of transplanted and/or endogenous cells. This Review outlines various biomaterials (including hydrogels, self-assembling peptides, and electrospun nanofibres) that have been investigated for the repair of brain tissue, and discusses strategies for the immobilization of biomolecules. An overview of the potential clinical applications of such scaffolds in neurodegenerative diseases is also provided.
Use of knowledge regarding LH receptors to improve superstimulatory treatments in cattle
Authors: Barros Ciro M. , Ereno Ronaldo L. , Simões Renato A. L. , Fernandes Paulo , Buratini José , Nogueira Marcelo F. G.
Published: August 2010

Embryo transfer is a biotechnology that has been used worldwide to increase the production of offspring from female bovines. Treatments to induce multiple ovulations (superovulation) have evolved from superstimulatory protocols that depended upon detection of oestrus to treatments that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-timed AI (FTAI). The protocols associated with FTAI facilitate animal handling and produce at least as many viably embryos as conventional treatment protocols that required detection of oestrus. Recent knowledge regarding LH receptors (LHR) and follicular development can be applied to improve embryo transfer protocols. In fact, improvements in the superstimulatory treatment called the ‘P-36 protocol’, which include hormones that stimulate LHR, indicate that adjustments related to LHR availability may increase bovine embryo yield compared with conventional protocols based on the detection of oestrus.
111 EFFECT OF ETHYLENE GLYCOL IN VITRIFICATION MEDIUM ON BOVINE OOCYTE ACTIVATION AND IN VITRO EMBRYO PRODUCTION
Authors: Rios G. L. , Kaiser G. G. , Mucci N. C. , Alberio R. H.
Published: August 2010

In this study the effect of increasing ethylene glycol (EG) concentrations in the vitrification media (VM) and its relation with oocyte activation and in vitro embryo production were evaluated. Cumulus-oocytes complexes (COC) were matured in vitro for 22 h and then partially denuded by gently pipetting with hyaluronidase, and randomly assigned to 4 treatments: T1 = control group; T2 = COC exposed to 10% EG + 10% DMSO (VM1), 20% EG + 20% DMSO (VM2); T3 = 15% EG + 5% DMSO (VM1), 30% EG + 10% DMSO (VM2); T4 = 20% EG + 0% DMSO (VM1), 40% EG + 0% DMSO (VM2). Exposition to VM1 and VM2 lasted 3 min and 30 s, respectively. After treatment COC were incubated in maturation medium up to 24 h. In Experiment 1 COC were cultured for 24 h in fertilization TALP supplemented with 50 μg mL-1 of heparin, then completely denuded and cultured 24 h in CR1-aa. After this, oocytes were stained with bisbenzimide (Hoechst 33342) and the number of nuclei and cells were recorded. Structures presenting 2 cells or 2 nuclei were considered as activated oocytes. In Experiment 2 matured COC exposed to cryoprotectants as in Experiment 1 were fertilized and cultured for 6 days as previously described (Mucci et al. 2006). Variables analyzed included oocyte activation, cleavage rate at 48 h, and percentage of viable embryos at Day 7. Data were analyzed by PROC GENMOD (SAS Institute Inc., Cary, NC, USA). Activated oocytes percentage did not differ between EG concentrations in VM and were higher (T2, 24.7%, n = 101; T3, 25.0%, n = 96; T4, 30.2%, n = 119) compared with controls (T1, 9.8%, n = 61). In Experiment 2 no differences were found in cleavage rates (T1, 81.9%, n = 68; T2, 87%, n = 67; T3, 85.9%, n = 61; T4, 84.2%, n = 64) and Day 7 percentage of viable embryos (T1, 34.9%, n = 29; T2, 28.6%, n = 22; T3, 26.8%, n = 19; T4, 27.6%, n = 21) in treated COC. The exposition of COC to cryoprotectants per se could trigger oocyte activation in the range of 10 to 40%.

Thanks toAdriana Cano (Instituto Nacional de Tecnología Agropecuaria) for contributions in statistical analysis.
118 ARE LIPID CONTENTS AND APOPTOSIS DETERMINANTS OF IN VITRO-PRODUCED BOVINE EMBRYO SUSCEPTIBILITY TO VITRIFICATION?
Authors: Sudano M. J. , Paschoal D. M. , Magalhães L. C. O. , Crocomo L. F. , Landim-Alvarenga F. D.
Published: August 2010

Supplementation of fetal calf serum (FCS) during culture of bovine in vitro-produced embryos (IVPE) has been correlated with lipid accumulation, representing a limiting factor on embryo cryotolerance. In the present experiment we studied lipid content and apoptosis in Nelore IVPE cultured in different concentrations of FCS. The experimental design was a 4 × 2 × 2 factorial, in which embryos, exposed or not to the metabolic regulator, phenazine ethosulfate (PES), were cultured in 4 concentrations of FCS. In 16 replicates, 560 IVPE were vitrified after cultured in SOFaa supplemented with 0, 2.5, 5 or 10% FCS. On Day 2.5 of culture, one-half of the embryos in each group were treated with 0.3 μM of PES and then returned to standard culture conditions (5% O2, 5% CO2 and 90% N2 atmosphere at 38.5°C). All embryos were vitrified on Day 7 (Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200-1214). After warming, embryos were placed into culture for 12 h under standard conditions. A sample of fresh embryos was stained with Sudan Black B for quantification of small (<2 μm), medium (2-6 μm), and large (>6 μm) cytoplasmic lipid droplets. Apoptosis was analyzed, before and after vitrification, using TUNEL. As a positive control (CV), in vivo-produced embryos were collected from a superovulated Nelore cow. Data were analyzed using ANOVA followed by Tukey’s test; and Pearson’s linear correlation (P < 0.05). The results indicate that FCS concentration and PES treatment had no influence (P > 0.05) on cleavage and blastocyst formation rates (mean of 85.7 ± 2 and 36.6 ± 5, respectively). The total number of cells in fresh embryos was also similar (P > 0.05) among groups (mean of 134.1 ± 28). Additional results are shown in Table 1. Elevated concentrations of FCS increased lipid accumulation (r = 0.9) and the percentage of apoptotic cells in fresh and vitrified embryos, reducing the re-expansion rate after warming. Moreover, the number of apoptotic cells observed in fresh embryos was strongly correlated with the apoptosis observed after vitrification (r = 0.95). Although PES was able to reduce lipid accumulation, it was not efficient at preventing apoptosis in vitrified embryos. In summary, the results of this experiment indicate that the concentration of FCS during culture did not influence cleavage and blastocyst formation in bovine IVPE. We conclude that the apoptosis rate in fresh embryos is a key factor affecting survival after vitrification.

Table 1.Lipid droplets, re-expansion, apoptosis of in vivo and IVPE

Acknowledgements: FAPESP (07/57766-4).
124 EXPRESSION OF PLURIPOTENCY MARKERS IN GENITAL RIDGES OF OVINE FOETUSES
Authors: Bebbere D. , Pirino S. , Bogliolo L. , Ariu F. , Pintus E. , Nieddu S. , Pau S. , Ledda S.
Published: August 2010

Fundamental problems of primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Historically, these cells can be identified using criteria as alkaline phosphatase, but also by the expression of other pluripotency markers suchas OCT4, Nanog, cKIT, SSEA1, SSEA4. Much less is known about PGC inother mammals. The objective ofthis study wasto describe, for the first time, the expression of multiple pluripotency markers in the genital ridges of ovine fetuses evaluating the binding of antibodies for stem cell surface markers (SSEA-1 and EMA-1) and the expression of Oct4, Nanog, and Sox2 genes. Ovine fetuses (24 and 27 days) were collected from slaughtered ewes and transported in a PBS solution at 4°C, to be processed as follows: Immunohistochemistry: 4 foetuses for each age were frozen in OCT TissueTek freezing compound and stored at -80°C. Tissue blocks were cut in 6-μm sections, placed on slides, and immediately prepared for indirect immunofluorescent staining. Section were fixed in 4% paraformaldehyde (10 min) and incubated overnight with SSEA-1 (1:100) and EMA-1 (1:50) antibodies (Hybridoma bank). Antibodies were detected with fluorescently-labelled goat anti-mouse secondary antibodies (1:100 dilution; Sigma) in 15% goat serum in DPBS for 1 h at room temperature. After washing, slides were mounted using ProLong antifade mounting medium (Molecular Probes). Gene expression: total RNA was extracted from fetal genital ridge samples with Trizol™ (Invitrogen). Any contaminating DNA was removed with RNase-free DNase I (Invitrogen). First strand cDNA synthesis was performed with SuperScript™ III RNase H-Reverse Transcriptase (Invitrogen) in the presence of RNase inhibitor (Promega). Real Time PCR amplification was performed in a BioRad iCycler™, with Platinum SYBR Green qPCR Super Mix UDG (Invitrogen). The relative quantification of the target genes was calculated with the 2-ddCt method (Livak and Schmittgen 2001 Methods 25, 402-408), after normalization against β-actin expression levels. Fragment identity was confirmed by sequencing. Our results indicate that the cortical region of the genital ridges is positive to stem cell surface markers. A larger number of positive cells was recorded in Day 24 fetuses with respect to Day 27 ones. The SSEA-1 and EMA-1 showed identical expression patterns. Similar levels of Oct4 and Nanog mRNA were detected in the genital ridges at both stages, whereas Sox2 displayed a lower abundance at Day 27. In conclusion, ovine PGC can be recognized by stem cell surface antigens and by the expression of Oct4, Nanog, and Sox2 pluripotency markers, as in mouse. The combined biomolecular and immunohistochemical approach provides a useful tool to follow the ovine PGC during in vitro culture to derive pluripotent stem cells in this species.
125 SWINE EMBRYOS WITH HIGH POTENTIAL TO DEVELOP IN VITRO HAVE LESS γH2A.X AND MORE NBS1 PROTEINS
Authors: Coutinho A. R. S. , Bordignon V.
Published: August 2010

The developmental potential of embryos produced in vitro is lower than those produced in vivo. The artificial environment and the stressful conditions of culture may affect embryo development through various mechanisms including DNA damage and, consequently, cell death. We hypothesized that the developmental competence of in vitro-cultured embryos is influenced by mechanisms signalling DNA damage and repair processes. Therefore, the aim of the study was to assess these processes by systematic quantification of phosphorylated histone H2A.X (γH2A.X) and p95 or nibrin protein (NSB1) in early- and late-cleaved swine embryos cultured in vitro. Studies from several groups including ours have demonstrated superior in vitro development for early-cleaved (within 24 h of culture) compared with late-cleaved (between 24 and 48 h) embryos. The presence of γH2A.X is associated with the DNA double-strand breaks, and NBS1 is involved in the process of DNA damage repair. These proteins were detected by both immunofluorescence and western blotting. Swine embryos were produced by parthenogenetic activation using in vitro-matured oocytes. Oocyte maturation, activation, and embryo culture were conducted as previously described (Che L et al. 2007 Theriogenology 67 1297-1304). At 24 and 48 h after activation, embryos were categorized as early- and late-cleaved, and were collected for protein detection on D2-3, D4-5, or D6-7 of culture. A minimum of 3 replicates were performed per treatment. The amount of protein in relation to the β-actin at D2-3, D4-5, and D6-7 as revealed by western blotting was 76.4% ± 1.3, 63.3% ± 10.5, and 43.2% ± 11.2 for γH2A.X and 60.2% ± 4.2, 67.3% ± 13.2, and 61.3% ± 6.2 for NBS1, respectively. Comparisons between early and late-cleaved groups were then performed by immunoflorescence detection of both proteins. Differences between groups were verified using Student’s t-test. The average proportion of cells that were positively stained for γH2AX at D2-3, D4-5, and D6-7 of culture was 64.4% ± 2.6 (n = 178) v. 65.92% ± 3.7 (n = 114; P = 0.7), 55.7% ± 2.4 (n = 121) v. 59.8% ± 4.7 (n = 62; P = 0.4) and 29.1% ± 2.1 (n = 137) v. 43.5% ± 3.4 (n = 41; P = 0.001), for early v. late-cleaved embryos. The values for NSB1 staining were 13.9% ± 3.8 (75) v. 3.9% ± 3.0 (34; P = 0.09), 50.5% ± 4.2 (66) v. 35.8% ± 6.0 (33; P = 0.05), and 51.0% ± 4.5 (n = 54) v. 38.2% ± 5.5 (n = 24; P = 0.1). These findings confirm the presence of γH2A.X and NBS1 proteins in swine embryos during all stages of in vitro culture. We further show that early cleaved embryos have a lower proportion of γH2A.X and a higher proportion of NSB1-positive cells compared with late-cleaved embryos. Together, these findings suggest that early cleaved embryos that have a superior capacity for in vitro development are better prepared to repair DNA damage during in vitro culture.

Supported by NSERC.
129 THE RELATIONSHIP OF BODY CONDITION SCORE AT TIME OF AI AND SUBSEQUENT GENDER RATIO OF OFFSPRING
Authors: McFarlane M. M. , McFarlane B. J. , Pursley J. R.
Published: August 2010

The chance of a female calf being born is less than that of a male calf from lactating dairy cows (M:F ratio from two large data sets was 54:46; Ryan and Boland 1991 Theriogenology 36, 1-10, and Berry et al. 1995 J. Anim. Sci. 78, 76 abst). Data from this study were analyzed to determine if there was a relationship in body condition of the dam at time of artificial insemination and subsequent gender ratio of offspring from lactating Holstein dairy cows. Lactating dairy cows from n = 3 farms were timed-AI at approximately 8 h before or 16 h after a GnRH induced LH surge and were body condition scored (BCS; 1 to 5, with 1 representing least amount of condition) at time of AI. Previous data from our laboratory indicated that cows in the -8 and +16 h groups received AI approximately 36 and 12 h prior to ovulation, respectively, and the longer sperm incubated in vivo the greater the subsequent percentage of female calves were born. In the study, the aforementioned data were analyzed in SPSS for the relationship between gender ratio and BCS from n = 333 cows that carried calves to term utilizing correlation analysis, and for pregnancy losses in cows (n = 1501) that received AI and had been evaluated for BCS utilizing binary logistic regression analysis. Cows were classed into 3 body condition groups at time of AI: <2.5 (low), 2.5 to 3.5 (medium), and >3.5 (high) for analysis. There was a significant negative correlation in BCS with M:F gender ratio (P < 0.05). Cows with low, medium, or high BCS had 40:60 (n = 82), 52:48 (n = 141), and 56:44 (n = 110) M:F ratio, respectively. The AI approximately 36 h prior to ovulation tended (P = 0.10) to attenuate M:F ratio in the high BCS group. Cows in the high BCS group that received AI approximately 36 h prior to ovulation had 46:54 compared with 62:38 M:F ratio in cows receiving AI approximately 12 h prior to ovulation. Pregnancy loss between 28 and 56 d post-AI was not different (P > 0.10) between the three BCS groups suggesting that differences in gender ratio were not the result of sex-specific losses. In summary, BCS at time of AI altered subsequent gender ratio of offspring and was attenuated in the high BCS group by time of AI relative to ovulation. We speculate that differences in gender ratio in cows with different BCS may be due to alterations in the uterine and/or oviductal environment that in turn alters the ratio of X and Y chromosome-bearing sperm at time of fertilization.
130 EXPRESSION AND LOCALIZATION OF µ OPIOID RECEPTOR IN PORCINE PRE-IMPLANTATION EMBRYOS
Authors: Minoia R. , Dang-Nguyen T. Q. , Matsukawa K. , Kaneda M. , Dell'Aquila M. E. , Akagi S. , Sassone F. , Palermo G. , Kikuchi K. , Nakai M. , Nagai T.
Published: August 2010

Embryonic stem cells can become any tissue in the body, excluding a placenta. Growth factors, hormones, and neurotransmitters have been implicated in the regulation of their fate. Because various neural precursors express functional neurotransmitter receptors, as G-protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. Moreover, a high level of endogenous opioids linked to G-protein-coupled receptor above all μ opioid receptors (MOR) has been shown to interfere with normal calcium metabolism and with the activity of the mitogen-activated protein kinase (MAPK). Thus it is very important to understand the possible influence of opioid activities in the regulation of stem cell fate. In this study we investigated the presence of MOR on porcine in vitro-produced embryos at one-cell, 4-cell, morula, and blastocyst stages by immunostaining. The COC were collected by aspiration, cultured in NCSU-37 medium supplemented with hormones for 20 to 22 h, and then in maturation medium without hormones for 24 h. After this time, COC were inseminated with frozen-thawed epididymal spermatozoa at the concentration of 10 × 5 sperm cells mL-1 for 3 h. After removal of cumulus cells, putative zygotes were cultured in IVC Pyr-Lac medium for the first 2 days and in IVC Glu medium until Day 6 (the day of IVF was defined as Day 0). Embryos at different stages were collected at 12, 36, 120, and 144 h post fertilization, and kept in 4% (v/v) paraformaldehyde until examination. All samples were washed and incubated for 30 min in PBS-1%BSA. Controls were incubated in PBS-1% BSA for 90 min, whereas embryos were incubated with a 1 : 2500 dilution of the primary rabbit antibody against the third extracellular loop of MOR. Prior to examination, all samples were washed in PBS and incubated with a FITC-conjugated anti rabbit IgG-secondary antibody diluted 1:200 in Evans Blue/PBS1x. Samples were visualized by laser scanning confocal microscope (Nikon). The immunofluorescence localize, by intense brilliant green, the presence of MOR on blastomers of all stage embryos examined, whereas the embryos of negative control did not show any fluorescent region or spotted coloring. Our results support specific implication of the opioid receptors in developmental process of porcine embryos. Their presence suggests a possible role of MOR in embryonic development. Thus it can be speculated that there is a role for MOR in controlling key events of the stem cell life. However, these primary results must be confirmed by the demonstration of protein expression (by Western blot) of MOR in the embryos and deeply studied to understand the exact functional role of MOR in them at this level.

JSPS short-term scholarship.
145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE
Authors: Choe C. Y. , Cho S. R. , Son J. K. , Choi S. H. , Cho C. Y. , Kim J. B. , Kim S. J. , Kang D. , Son D. S.
Published: August 2010

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.
146 CO-CULTURE OF EARLY CLEAVAGE STAGE IVP EMBRYOS WITH BOVINE OVIDUCT EPITHELIAL CELLS DOES NOT IMPROVE EMBRYO DEVELOPMENT OR PREGNANCY RATES
Authors: Fry R. C. , Fry K. L. , Lan W.
Published: August 2010

Pregnancy rates after the transfer of bovine IVP embryos are lower than that achieved after the transfer of MOET embryos. One reason may be that the relatively defined IVC (SOFaaBSA) culture system used in vitro is suboptimal for embryo development. We investigated whether the co-culture of early stage IVP embryos with bovine oviduct epithelial cell (BOEC) to Day 3 could provide some of the missing substrates and improve both embryo production and subsequent pregnancy rates after embryo transfer. COCs were collected by ovum pickup (OPU) from donor Brahman females, transported overnight at 38.5°C to the laboratory in HEPES-IVM media, then fertilized and cultured by our standard IVP methodology (Fry et al. 2003 Theriogenology 59, 446). Briefly, the IVC was carried out at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2 in 4-well Nunc dishes in 500 μL of SOFaaBSA media overlayed by 500 μL of mineral oil. After 3 days of culture, the embryos were transferred to fresh IVC media and after 6 days placed in 5-mL Falcon tubes (Becton Dickinson Labware, Lincoln, NJ, USA) in fresh IVC media containing 2% FCS for overnight shipment. All grade 1 and 2 embryos were transferred to synchronized recipients on Day 7. Pregnancy diagnosis was between Days 50-90. In the BOEC treatment group, frozen aliquots of BOEC were thawed, seeded at 300,000 cells/mL, and grown for 2 days to 60-80% confluence in the Nunc wells in 500μL of DMEM/F12 media containing 10% FCS. On the first day of embryo culture, the media was removed and replaced by IVC media prior to the introduction of presumptive zygotes. After 3 days of co-culture, the embryos were transferred to fresh IVC media and thereafter cultured and transferred as for the Control group. In the Control group, 80 OPU sessions produced 1277 COCs (mean 16.0) of which 1064 (81.6%) cleaved producing 385 (33.7%) transferable embryos. Of the 337 embryos transferred to recipients (48 were vitrified), 141 (40.1%) resulted in pregnancies. In the BOEC group, 73 OPU sessions produced 1111 COCs (mean 15.2) of which 891 (80.2%) cleaved producing 388 (35%) transferable embryos that resulted in 161 (41.5%) pregnant recipients after transfer. Chi-square analysis showed no difference in either IVP embryo production or subsequent pregnancy rate between the Control group or the group where the IVP embryo was co-cultured for the first 3 days with BOEC.