Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin

Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology during in vitro maturation (IVM) of porcine oocytes cultured with 100mM trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) or 10mM forskolin at different time periods. Four groups were constituted: control, excipient, t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48h and the initial 22 to 24h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1±8.0%, control = 72.6±5.0%; exp. 2: forskolin =24.3±7.4%, control =71.6±5.6%) and cleavage (exp. 1: forskolin=0.0±0.0%, control=55.4±4.1%; exp. 2: forskolin=8.3±3.3%, control=54.5±3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P<0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented with t10,c12 CLA during 44 to 48h presented a lighter (P<0.04) colour tone cytoplasm than those of control and forskolin. In conclusion, t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner

Mode I fracture characterization of human bone using the DCB test

Purpose – Fracture characterization of human cortical bone under pure mode I loading was performed in this work. The purpose of this paper is to validate the proposed test and procedure concerning fracture characterization of human cortical bone under pure mode I loading. Design/methodology/approach – A miniaturized version of the double cantilever beam (DCB) test was used for the experimental tests. A data reduction scheme based on crack equivalent concept and Timoshenko beam theory is proposed to overcome difficulties inherent to crack length monitoring during the test. The application of the method propitiates an easy determination of the Resistance-curves (R-curves) that allow to define the fracture energy under mode I loading from the plateau region. The average value of fracture energy was subsequently used in a numerical analysis with element method involving cohesive zone modelling. Findings – The excellent agreement obtained reveals that the proposed test and associated methodology is quite effective concerning fracture characterization of human cortical bone under pure mode I loading. Originality/value – A miniaturized version of traditional DCB test was proposed for cortical human bone fracture characterization under mode I loading owing to size restrictions imposed by human femur. In fact, DCB specimen propitiates a longer length for self-similar crack propagation without undertaking spurious effects. As a consequence, a R-curve was obtained allowing an adequate characterization of cortical bone fracture under mode I loading

A utilização de sémen fresco na fertilização in vitro de embriões ovinos melhora a qualidade dos blastocistos na raça portuguesa Merino

A produção de embriões em ovinos é uma tarefa difícil, exigindo experiência e condições onerosas, principalmente na produção de embriões in vivo. A recolha sistemática de oócitos em animais de matadouro ou em animais vivos por ovum pick-up, permite a produção in vitro de embriões (IVP), em larga escala e menos dispendiosa, nos pequenos ruminantes. Esta possibilidade é importante não só como fonte de embriões mas também de oócitos e zigotos para fins comerciais ou de investigação, facilitando a sua disponibilidade em tecnologias emergentes tais como a clonagem ou a transgénese. Para IVP foram desenvolvidos vários protocolos de maturação, utilizando fertilização in vitro (IVF) com sémen fresco ou congelado. Em Portugal, a produção de embriões in vitro foi somente realizada com sémen congelado dada a sua disponibilidade em condições de rotina. Contudo, o sémen fresco poderá melhorar a produção de embriões frescos ou criopreservados. Este trabalho teve como objectivo comparar a eficiência da IVP em ovinos usando diferentes protocolos de maturação de oócitos e IVF com sémen fresco ou congelado. Oócitos (n=1768) recolhidos em matadouro foram maturados em meio TCM199 com 100 μM cisteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 e gentamicina (mat A, n=692) ou suplementada com 10 μg mL-1 FSH e 0,3 mM piruvato de sódio (mat B, n=707) a 39 ºC e 5% CO2 durante 22h. O sémen fresco (FS) e congelado/descongelado (TS) de carneiros de raça Merino Branco (n=3) foi lavado ou submetido a swim-up, respectivamente. Após a fertilização (18h p.i.), os presumíveis zigotos foram cultivados em meio de fluido sintético do oviducto (SOF) enriquecido com aminoácidos e BSA a 38,5 ºC, em atmosfera humidificada com 5% O2, 5% CO2 e 90% N2 até ao estadio de 2-4-8 células. Após clivagem, o desenvolvimento embrionário prosseguiu até ao estadio de blastocisto em meio SOF, BSA e 10% FCS. A qualidade foi avaliada no dia 6-7, classificando-se como bons, médios e maus, baseado nos parâmetros IETS. Os dados das taxas de produção embrionária foram analisados utilizando ANOVA. Foi utilizado o teste de Mann-Whitney U para avaliação da qualidade dos embriões. Os diferentes protocolos de maturação não interferiram (p>0,05) quer com as taxas de maturação quer com as taxas de produção de embriões. A qualidade embrionária foi superior (p=0,004) na fertilização com sémen fresco (bom: FS=40,1±8,0% vs TS=32,9±5,6%; média: FS=20,1±4,7% vs TS=35,7±5,8%; má: FS=39,8±9,8% vs TS=31,4±7,6%). Em conclusão, estes resultados preliminares mostram que o sémen fresco de carneiro pode ser facilmente utilizado para fertilização in vitro e melhora a qualidade dos embriões produzidos.#Embryo production in sheep is a difficult task demanding experience and expensive facilities, particularly when dealing with in vivo embryo production. Easy ways to obtain ovine embryos consist of collecting oocytes at slaughterhouses or systematically pick them up from live animals, allowing a large scale and cheaper in vitro embryo production (IVP) for small ruminants. Those are important sources of embryos, oocytes and zygotes for commercial, laboratorial and research proposes, making easier the availability of resources for emerging techniques like cloning or transgenesis. For IVP, several oocyte maturation protocols have been developed using fertilization (IVF) either with fresh or frozen-thawed semen. In Portugal, IVP has been done through IVF using cryopreserved semen because it is easily available for routine use. However, the use of fresh semen could improve embryo production and cryopreservation results. The aim of this work was to compare the efficiency of in vitro embryo production in ovine using different oocyte maturation protocols and fresh or frozen semen for IVF. Abattoir-derived oocytes (n=1768) were matured in TCM199, 10 μM cysteamine, 10 ng mL-1 EGF, 10 μg mL-1 E2 and gentamicin (mat A, n=692) or plus 10 μg mL-1 FSH and 0.3 mM sodium piruvate (mat B, n=707) at 39 ºC and 5% CO2 for 22h. Prior to fertilization, either fresh (FS) or frozen/thawed (TS) semen from Merino rams (n=3) was washed or submitted to swim-up respectively. Presumptive zygotes (18h p.i.) were cultured in synthetic oviductal fluid (SOF) enriched with aminoacids and 6 mg mL-1 BSA at 38.5 ºC, under 5% O2, 5% CO2 and 90% N2 in an humidified atmosphere until the stage of 2-4-8 cell embryos. After assessing cleavage, embryo development proceeded until the blastocyst stage in SOF+BSA and 10% FCS. Quality was evaluated on D6-7 by scoring embryos as good, fair and bad based on IETS guidelines. Data from embryo production rates were analysed using ANOVA. Mann-Whitney U test was used for embryo quality evaluation. Different maturation protocols did not interfere (P>0.05) either on maturation or on embryo quality or production rates. Embryo quality was higher (P=0.004) when fertilization was accomplished with fresh semen (good: FS=40.1±8.0% vs TS=32.9±5.6%; fair: FS=20.1±4.7% vs TS=35.7±5.8%; bad: FS=39.8±9.8% vs TS=31.4±7.6%). Preliminar results show that ram fresh semen can be easily used for in vitro fertilization and improves the quality of produced embryos

Aberrant p15, p16, p53, and DAPK Gene Methylation in Myelomagenesis: Clinical and Prognostic Implications

BACKGROUND: Aberrant DNA methylation is considered a crucial mechanism in the pathogenesis of monoclonal gammopathies. We aimed to investigate the contribution of hypermethylation of 4 tumor suppressor genes to the multistep process of myelomagenesis. METHODS: The methylation status of p15, p16, p53, and DAPK genes was evaluated in bone marrow samples from 94 patients at diagnosis: monoclonal gammopathy of uncertain significance (MGUS) (n = 48), smoldering multiple myeloma (SMM) (n = 8) and symptomatic multiple myeloma (MM) (n = 38), and from 8 healthy controls by methylation-specific polymerase chain reaction analysis. RESULTS: Overall, 63% of patients with MM and 39% of patients with MGUS presented at least 1 hypermethylated gene (P < .05). No aberrant methylation was detected in normal bone marrow. The frequency of methylation for individual genes in patients with MGUS, SMM, and MM was p15, 15%, 50%, 21%; p16, 15%, 13%, 32%; p53, 2%, 12,5%, 5%, and DAPK, 19%, 25%, 39%, respectively (P < .05). No correlation was found between aberrant methylation and immunophenotypic markers, cytogenetic features, progression-free survival, and overall survival in patients with MM. CONCLUSIONS: The current study supports a relevant role for p15, p16, and DAPK hypermethylation in the genesis of the plasma cell neoplasm. DAPK hypermethylation also might be an important step in the progression from MGUS to MM.info:eu-repo/semantics/publishedVersio

Interaction of paraoxonase-192 polymorphism with low HDL-cholesterol in coronary artery disease risk.

A doença coronária (DC) é a principal causa de mortalidade nos países desenvolvidos. O aumento da peroxidação lipídica está associado com a progressão acelerada da arteriosclerose. A Paraoxonase (PON1) é uma enzima antioxidante, que protege contra a peroxidação lipídica e a DC. A actividade da PON1 está sob controlo genético e a sua base molecular consiste num polimorfismo do gene da PON1 que apresenta duas isoformas comuns: a forma nativa, Q (192 Gln) com elevada capacidade de protecção das LDL da peroxidação lipídica in vitro, e a isoforma mutada R (192 Arg) com baixa capacidade de protecção. Objectivo: O objectivo deste trabalho foi investigar a interacção entre o alelo R do gene da PON 1 e os níveis plasmáticos baixos de colesterol HDL, no risco do aparecimento da DC. Métodos: Participaram no estudo 818 indivíduos, 298 doentes coronários com idade média 55.0±10.3 anos, 78.9% do sexo masculino, e 520 controlos, com uma idade média de 53.3±11, 7 anos, 72, 5% do sexo masculino, tendo casos e controlos sido emparelhados por idade e sexo. Foi considerado um valor <de 40 mg/dl (0,90 mmol/L), nos homens e <de 50 mg/dl (1,11 mmol/L), nas mulheres como um nível baixo de Colesterol HDL. As comparações genotípicas, entre casos e controlos, foram efectuadas pelo teste do Chi-quadrado. A significância estatística foi aceite para valores de p <0,05. Para determinar o risco relativo de DC, em relação ao genótipo RR e aos níveis baixos de colesterol HDL, foi usada uma análise univariada e foram utilizadas as tabelas epidemiológicas 4x2 e medidas de sinergismo (modelo aditivo - SI e multiplicativo - SIM) para determinar a interacção entre o genótipo RR e os níveis baixos de colesterol HDL. Foi finalmente calculado o excesso de risco relativo (RERI) e proporção atribuída à interacção (AP). Resultados: A PON 1 192 RR está associada à DC [OR=1,61; p=0,043] para toda a população. A associação de níveis baixos de HDL com o genótipo 192 RR mostrou um aumento do risco de DC (OR=17,38; p <0,0001) comparada aos níveis normais de HDL associados ao mesmo genótipo (OR=1,39; p=0,348) e aos níveis baixos de HDL sem o genótipo RR (OR=7,79; p <0,0001). Índices de Sinergismo: SI= 2,3; SIM = 1.6; RERI=9,2; AP=0,53. Conclusão: Estes dados sugerem a existência de um efeito sinérgico entre o genótipo 192 RR da PON1 e os valores baixos de colesterol HDL, na emergência de DC, pois este genótipo aumentou o risco de DC, em especial, na população com níveis plasmáticos baixos de colesterol HDL. A proporção de DC que pode ser atribuída a esta interacção (AP) foi de 0,53 significando que 53% da DC que surgiu nestes indivíduos, foi explicada por esta interacção.INTRODUCTION: Coronary artery disease (CAD) is the main cause of mortality in developed countries. Increased lipid peroxidation is associated with accelerated progression of atherosclerosis. Paraoxonase (PON1) is an antioxidant enzyme bound to high-density lipoprotein (HDL), which protects against lipid peroxidation and coronary artery disease. PON1 activity is under genetic control and its molecular basis is a polymorphism in the PON1 gene that shows two common isoforms: the wild Q form (192 Gln) with high ability to protect LDL from lipid peroxidation in vitro, and the mutated R (Arg) form with lower ability. AIM: To explore the interaction of the R allele of the paraoxonase gene and low HDL-cholesterol concentrations in CAD risk. METHODS: The study population consisted of 818 individuals, 298 coronary patients, aged 55.0 +/- 10.3 years, 78.9% male, and 520 age and gender matched healthy controls, aged 53.3 +/- 11.7 years, 72.5% male. Low HDL-cholesterol was defined as < 0.90 mmol/l in men and < 1.11 mmol/l in women. Comparisons of genotypes between cases and controls were performed by a chi-square test. Statistical significance was accepted at p < 0.05. Odds ratios and 95% confidence intervals for the RR genotypes and HDL-deficient subjects were computed using univariate analysis (2 x 2 tables). To determine the interaction between the RR paraoxonase genotype and HDL-deficient subjects, we used 4 x 2 epidemiologic tables and synergy measures: the additive model (Rothman's synergy index, SI) and multiplicative model (Khoury's synergy index, SIM). The relative excess risk due to interaction (RERI) and the attributable proportion (AP) due to interaction (Rothman) were calculated. RESULTS: The PON1 RR192 polymorphism was associated with coronary heart disease (OR = 1.61; p = 0.043) in the whole population. HDL-deficient subjects with the RR192 genotype showed increased risk for CAD (OR = 17.38; p < 0.0001) compared to those with normal HDL and RR192 (OR = 1.39; p = 0.348) and HDL-deficient subjects not carrying the RR genotype (OR = 7.79; p < 0.0001). Synergy measures were SI = 2.3, SIM = 1.6; RERI = 9.2. CONCLUSION: These data suggest the existence of a synergistic effect of the PON1 RR192 genotype (with lower antioxidant ability) and HDL-deficient subjects in risk for development of CAD. The AP due to this interaction was 0.53, meaning that 53% of CAD was explained by this interaction.info:eu-repo/semantics/publishedVersio

Realization of a Tunable Artificial Atom at a Supercritically Charged Vacancy in Graphene

The remarkable electronic properties of graphene have fueled the vision of a graphene-based platform for lighter, faster and smarter electronics and computing applications. One of the challenges is to devise ways to tailor its electronic properties and to control its charge carriers. Here we show that a single atom vacancy in graphene can stably host a local charge and that this charge can be gradually built up by applying voltage pulses with the tip of a scanning tunneling microscope (STM). The response of the conduction electrons in graphene to the local charge is monitored with scanning tunneling and Landau level spectroscopy, and compared to numerical simulations. As the charge is increased, its interaction with the conduction electrons undergoes a transition into a supercritical regime 6-11 where itinerant electrons are trapped in a sequence of quasi-bound states which resemble an artificial atom. The quasi-bound electron states are detected by a strong enhancement of the density of states (DOS) within a disc centered on the vacancy site which is surrounded by halo of hole states. We further show that the quasi-bound states at the vacancy site are gate tunable and that the trapping mechanism can be turned on and off, providing a new mechanism to control and guide electrons in grapheneComment: 18 pages and 5 figures plus 14 pages and 15 figures of supplementary information. Nature Physics advance online publication, Feb 22 (2016

Synthesis, Crystal Structure, and Infrared Spectroscopy of a novel hydronium trihydrate hybrid compound: (C6H22N4)2H9O4CdCl6CdCl5Cl2

A 0-dimensional (0D) hybrid compound, (C6H22N4)2H9O4CdCl6CdCl5Cl2 has been prepared by a facile conventional evaporation method. The crystal packing of discrete constituents of [Cd(1)Cl6] octahedra, [Cd(2)Cl5] trigonal bipyramids, Cl– ions, protonated tris(2-aminoethyl)amine molecules ([(C2H7N)3NH]4+) and H9O4+ ions, is stabilized by diverse hydrogen bonds of N-H···Cl, C-H···Cl and C-H···O. Uncommonly, an isolated chlorine ion (i.e. Cl(4)) is fixed at a special position at 12c(3.) by hydrogen bonds from four surrounding hydrogen atoms at a trigonal pyramidal configuration whereas other chlorine atoms Cl(1), Cl(2) and Cl(3) are stabilized by hydrogen bonds from 2, 2 and 3 hydrogen atoms at bifurcated, linear and trigonal configurations, respectively. The ordered arrangement of [Cl(4) [Cl(4)···H4] trigonal pyramidal configuration upward or downward is responsible for the long c-axis of the title compound. Additionally, a H9O4+ ion is entangled with symmetry restriction and half occupancy. All these features of the title compound add our new knowledge about hydrogen bonds

Chemical composition, and antioxidant and antimicrobial activities of three hazelnut (Corylus avellana L.) cultivars.

Hazelnut (Corylus avellana L.) is a very popular dry fruit in the world being consumed in different form and presentations. In the present work, three hazelnut cultivars (cv. Daviana, Fertille de Coutard and M. Bollwiller) produced in Portugal, were characterized in respect to their chemical composition, antioxidant potential and antimicrobial activity. The samples were analysed for proximate constituents (moisture, fat, crude protein, ash), nutritional value and fatty acids profile by GC/FID. Antioxidant potential was accessed by the reducing power assay, the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and b-carotene linoleate model system. Their antimicrobial capacity was also checked against Gram positive (Bacillus cereus, B. subtilis, Staphylococcus aureus) and Gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and fungi (Candida albicans, Cryptococcus neoformans). Results showed that the main constituent of fruits was fat ranging from 56% to 61%, being the nutritional value around 650 kcal per 100 g of fruits. Oleic was the major fatty acid varying between 80.67% in cv. F. Coutard and 82.63% in cv. Daviana, followed by linoleic, palmitic, and stearic acids. Aqueous hazelnut extract presented antioxidant activity in a concentration-dependent way, in general with similar behaviour for all cultivars. Hazelnut extracts revealed a high antimicrobial activity against Gram positive bacteria (MIC 0.1 mg/ mL) showing a good bioactivity of these fruits

Effects of hydroxyapatite and PDGF concentrations on osteoblast growth in a nanohydroxyapatite-polylactic acid composite for guided tissue regeneration

The technique of guided tissue regeneration (GTR) has evolved over recent years in an attempt to achieve periodontal tissue regeneration by the use of a barrier membrane. However, there are significant limitations in the currently available membranes and overall outcomes may be limited. A degradable composite material was investigated as a potential GTR membrane material. Polylactic acid (PLA) and nanohydroxyapatite (nHA) composite was analysed, its bioactive potential and suitability as a carrier system for growth factors were assessed. The effect of nHA concentrations and the addition of platelet derived growth factor (PDGF) on osteoblast proliferation and differentiation was investigated. The bioactivity was dependent on the nHA concentration in the films, with more apatite deposited on films containing higher nHA content. Osteoblasts proliferated well on samples containing low nHA content and differentiated on films with higher nHA content. The composite films were able to deliver PDGF and cell proliferation increased on samples that were pre absorbed with the growth factor. nHA–PLA composite films are able to deliver active PDGF. In addition the bioactivity and cell differentiation was higher on films containing more nHA. The use of a nHA–PLA composite material containing a high concentration of nHA may be a useful material for GTR membrane as it will not only act as a barrier, but may also be able to enhance bone regeneration by delivery of biologically active molecules